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Volume 3A
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A great deal of additional information on the European Union is available on the Internet.
It can be accessed through the Europa server (http://europa.eu.int).
Cataloguing data can be found at the end of this publication.
Luxembourg: Office for Official Publications of the European Communities, 1998
ISBN 92-828-2437-3
European Communities, 1998
Reproduction is authorised, provided the source is acknowledged.
Printed in Belgium
Volume 3A
Guidelines
Medicinal products for human use
Quality and biotechnology
1998 Edition
EUROPEAN COMMISSION
Directorate General III - Industry
Pharmaceuticals and cosmetics
Volume 1
Pharmaceutical legislation
Medicinal products for human use
Volume 2
Notice to applicants
Medicinal products for human use
Volume 3
Guidelines
Medicinal products for human use
Volume 4
Volume 5
Pharmaceutical legislation
Veterinary medicinal products
Volume 6
Notice to applicants
Veterinary medicinal products
Volume 7
Guidelines
Veterinary medicinal products
Volume 8
Volume 9
Pharmacovigilance
Medicinal products for human use and veterinary medicinal products
FOREWORD
Directive 75/318/EEC describes the requirements for the demonstration of the quality, safety
and efficacy of medicinal products. The conduct of tests and studies for such demonstration
has been harmonised, both within the European Union and internationally.
Volume 3 of "The Rules Governing Medicinal Products in the European Union" incorporates
testing guidelines prepared within the European Union including those which have been
developed in the International Conference on Harmonisation (ICH) process. It is presented
in three parts:
These guidelines serve a two-fold objective. Firstly, they are intended to provide a basis for a
practical harmonisation of the manner in which the Member States and the European
Agency for the Evaluation of Medicinal Products interpret and apply the detailed
requirements for the demonstration of quality, safety and efficacy contained in the
Community directives. Secondly, they are intended to facilitate the preparation of
applications for marketing authorization which will be recognized as valid by all Member
States and the European Agency for the Evaluation of Medicinal Products.
The use of guidelines, which are not legally binding, rather than a formal legal instrument,
such as a directive, has been preferred in order to maintain an element of flexibility and not
to place undue legislative restraints on scientific progress. It is recognized that in some
cases, as a result of scientific developments, an alternative approach may be appropriate.
However, where an applicant chooses not to apply a guideline, that decision must be
explained and justified in the Expert Reports submitted by the company in support of the
application.
By their very nature, the guidelines must be updated in the light of scientific and technical
progress. Moreover, further guidelines are currently under discussion, thus it is intended
that this volume should be updated and revised as necessary.
These Notes for Guidance, which have no legal force, have been prepared by the Committee
for Proprietary Medicinal Products of the European Agency for the Evaluation of Medicinal
Products, in consultation with the competent authorities of the Member States, to assist
applicants for a marketing authorization for a medicinal product. In case of doubt, reference
should be made to the text of the relevant EEC Directives.
Ill
TABLE OF CONTENTS
QUALITY GUIDELINES
DEVELOPMENT PHARMACEUTICS AND PROCESS VALIDATION
11
19
23
31
39
47
57
67
75
83
95
107
119
PRODUCTS *)
127
143
153
157
167
RADIOPHARMACEUTICALS
175
185
195
Table of contents
BIOTECHNOLOGY GUIDELINES
203
205
217
223
237
263
275
287
295
311
315
323
333
351
355
ALLERGEN PRODUCTS
373
381
393
INDEX
VI
405
QUALITY GUIDELINES
3AQ1a
CONTENTS
DEVELOPMENT PHARMACEUTICS
II
1.
INTRODUCTION
2.
CONSTITUENTS
3.
COMPOSITION
4.
CONTAINER
PROCESS VALIDATION
3AQ1a
DEVELOPMENT PHARMACEUTICS
INTRODUCTION
Pharmaceutical development studies may need to be carried out to establish that the type of
dosage form selected and the formulation proposed are satisfactory for the purpose specified
in the application. They also aim to identify those formulation and processing aspects that
are crucial for batch reproducibility and which therefore need to be monitored routinely.
Because of the great variety in active substances and dosage forms, this note for guidance is
only an illustration of the type of information which has been found useful in establishing
the factors which affect quality of a finished product.
2.
CONSTITUENTS
characteristics
It may be necessary to study the effect of such parameters as e.g. crystal form, moisture
content and particle size of the active substance on the formulation. The latter may be of
importance in bioavailability, content uniformity, suspension properties, stability and for
eye irritation studies. Having identified a parameter as being critical, its control should
then be reflected in the active substance specification, or dealt with by other appropriate
means.
2.1.3 Overage
Overages are primarily employed to cover losses during manufacture, i.e. manufacturing
overage, and/or during shelf life, stability overage. The inclusion of any overage should be
justified.
2.2 Excipients
2.2.1 An explanation should be provided with regard to the function of the excipients in the
formulation.
2.2.2
2.2.3 Where unusual excipients are used in the manufacture of the product, e.g. the matrix
of a slow release preparation, full information on the composition and function of the
3AQ1a
excipient in the formulation of the product should be furnished together with any
documentation which may be available to demonstrate safety of the raw material. A new
substance introduced as an excipient will be regarded in the same way as a new active
substance, unless it is already approved for use in food by the same route of administration.
3.
COMPOSITION
parameters
pH
Evidence should be presented to show that the effect of pH within the specified range of the
formulation has been investigated. Consideration should be given to the effect of pH on active
constituent(s), and, where relevant, on the antimicrobial efficacy. Should such a study show
positive results any long-term effects would need to be investigated during stability studies.
b)
others
Additives
preservatives,
antioxidants,
others.
with other
products
3AQ1a
3.2
3.2.1 Physical
a)
parameters
pH
Evidence should be presented to show that the effect of pH within the specified range of the
formulation, where relevant, including preservative activity, has been investigated. Where
a significant effect is observed, any long-term effects would need to be investigated during
stability studies.
b)
others
Where the active substance is suspended rather than dissolved, particle size control and
particle size aggregation should be taken into consideration during development studies..
Rheology studies may also need to be carried out during the development of semi-solids.
3.2.2
Additives
preservatives,
antioxidants,
others.
3.3 S o l i d d o s a g e f o r m s
3.3.1
Dissolution
The dissolution apparatus used in the testing of both unmodified and modified release
preparations should be either of those described in the European Pharmacopoeia. Where these
prove unsuitable, dissolution test equipment described in the National Pharmacopoeia of the
Member State should be adopted as second choice, or, failing this, any other method.
However, justification for the use of a method other than European Pharmacopoeia must be
put forward.
a)
Dissolution tests must be performed during development and stability studies in order to
establish whether such testing would need to be done during stability studies and routinely as
part of the finished product specification.
b)
The choice of dissolution test conditions and release rates adopted for assessing batch
reproducibility needs to be justified. This should take account of in vivo studies carried out to
establish the release and absorption profile of the product and would, if feasible, consist of a
study correlating in vitro release rates to in vivo results to allow meaningful batch
reproducibility evaluation. Such a correlation would be of particular importance for
3AQ1a
3.3.2 Homogeneity
The European Pharmacopoeia includes a requirement for uniformity of content of singledose preparations where the amount of active constituent is less than 2 mg per dose or less
than 2% by mass of the total mass. Notwithstanding this requirement, the adequacy of the
mixing process in obtaining the required homogeneity of the mixture ought to be considered
for all solid dose forms i.e., tablets, powders, etc.
4.
CONTAINER
Appropriate studies should be performed to demonstrate the integrity of the container and
closure. A possible interaction between product and container may need to be considered.
4.2 Leaching
Data should be presented to show that there is no significant leaching of any pack
component, including label adhesive, into liquid or finely divided solid preparations over
the shelf life period, where relevant.
3AQla
II.
PROCESS VALIDATION
the manufacturing process for a modified release system will only vary to an extent
that will still yield a product of the desired quality and not have any effect on product
efficacy or safety.
3AQ2a
Date of entry i n t o
force
Status
P r e v i o u s titles/other
references
Additional Notes
May 1996
This version re-issued in April 1996
N o n e /CPMP/QWP/486/95
This note for guidance concerns the application of Part
2, section D of t h e Annex to Directive 75/318/EEC as
amended, with a view to the g r a n t i n g of a m a r k e t i n g
a u t h o r i s a t i o n for a new medicinal product.
CONTENTS
1.
INTRODUCTION
2.
3.
MANUFACTURING FORMULA
4.
5.
6.
7.
SPECIAL ITEMS
11
3AQ2a
INTRODUCTION
2.
Medicinal products on the market in the EC should be produced under the EC Rules for Good
Manufacturing Practice (GMP), see Directive 91/356/EEC.
Many general elements of GMP and quality assurance do not need to be described in the
application for marketing authorisation. Examples are qualifications of key personnel,
13
3AQ2a
cleaning procedures for the production equipment and production areas, final packaging
and labelling procedures etc.
In general, the dossier for marketing authorisation should contain only those elements of the
quality assurance which are specific for the medicinal product, whereas non product related
elements of the quality assurance fall within the field of GMP, consequently, no description
is necessary in the application for a marketing authorisation.
This note for guidance addresses the items that should be presented in the application for a
marketing authorisation. For items not to be covered by the application for a marketing
authorisation, the obligation for adherence to the EC GMP principles is implicit.
3.
MANUFACTURING FORMULA
4.
14
3AQ2a
The various steps in the manufacturing process and corresponding in-process controls
should also be shown in a flow-chart.
The presented data on the manufacturing process, apparatus and in-process controls are
binding for the future manufacturing of the medicinal product, unless authorisation for
changes is given by the Competent Authority.
It is in the interest of both the applicant and the regulatory authorities to avoid unnecessary
applications for variations. Very detailed descriptions of the manufacturing process,
apparatus and in-process controls should therefore be avoided.
In selecting the necessary level of detail the following should be considered:
the testing at release of the finished product,
the description of the manufacturing process and apparatus,
the in-process controls and validated acceptance limits.
Together these should provide a high degree of probability that each unit of every batch of the
finished product, will be in conformity with the specifications.
So, if the consistent quality of a medicinal product can be fully safeguarded by the "implicit"
production under GMP and testing of the finished product at release, the description of the
manufacturing process need not be comprehensive, and apparatus and in-process controls
need not to be described.
However, many quality parameters that are tested at release do not provide sufficient
certainty of the quality of the whole batch from a statistical point of view, because the quality
parameter may not necessarily be homogeneous within the batch.
An example is the homogeneous distribution of the active ingredient in solid and semi-solid
dosage forms, i.e. content uniformity. Testing at release alone does not provide sufficient
certainty for the content uniformity of the whole batch from a statistical point of view.
So, the apparatus to be used and the appropriate in-process controls (i.e. mixing time, mixing
speed etc.) and the validated acceptance limits for these in-process controls (see below) must
be proposed in the application file.
Another example is sterilisation. For all sterilisation processes, appropriate in-process
controls and their acceptance limits are to be described in the application file, see below.
5.
An account shall be given of the sites at which each stage of the manufacturing and
assembly operations takes place. Different manufacturing sites belonging to the same
company shall be mentioned as separate units. The company responsible for the final
approval of the release of the product onto the market shall be specified.
6.
Validation studies that are used to identify critical steps in non-standard manufacturing
processes are part of the Development Pharmaceutics, and should be described in Part IIA of
the application file.
15
3AQ2a
7.
SPECIAL ITEMS
should
be justified
under
Development
According to the text of the Ph. Eur.: "Methods of preparation of sterile products", terminal
sterilisation in the final container is to be preferred. Refraining from terminal sterilisation
in the final container should be justified in the application file.
In Part IIB the actual sterilisation process to be applied should be described.
All sterilisation processes should be carried out according to the instructions of the Ph. Eur.
In the application file, an explicit statement should be made that the instructions of the Ph.
Eur. are followed.
According to the Ph. Eur., all sterilisation procedures should be validated and be carried out
under the EC GMP-rules. However, in the application file for marketing authorisation for
some sterilisation procedures no, or only limited validation data and data on the bioburden
of the product prior to the sterilisation need to be presented, see below.
In the case of terminal sterilisation in the final container by heat using a reference
condition of the Ph. Eur., only the time and temperature of the cycle and the acceptance
limits of the corresponding in-process controls need to be provided in the application file.
So, this holds for sterilisation by saturated steam at a minimum of 121 C for 15 min. and by
dry heat at a minimum of 160 C for at least 2 hours.
16
3AQ2a
In accordance with the Ph. Eur., these conditions should be met within all units. However,
the validation data showing that all units are subjected to these conditions are normally not
required in the application file. They may be requested by the competent authorities in
certain circumstances.
For terminal sterilisation cycles in the final container by heat with a time and/or
temperature below the values of the reference conditions of the Ph. Eur., not only the
acceptance limits for the in-process controls for time and temperature should be stated, but
also the maximum acceptable bioburden before sterilisation.
The results of the validation of the sterilisation cycle with regard to the effectiveness i n
terms of the Sterility Assurance Level (SAL) obtained should be presented in the application
file.
For sterilisation by filtration the maximum acceptable bioburden prior to the filtration must
be stated in the application. In most situations NMT 10 CFU's/100 ml will be acceptable,
depending on the volume to be filtered in relation to the diameter of the filter. If this
requirement is not met, it is necessary to use a pre-filtration through a bacteria-retaining
filter to obtain a sufficiently low bioburden.
The type of bacteria-retentive filter, and its pore size should also be described in the
application. Pore sizes of 0.22 um or less are acceptable without further justification, in
accordance with the Ph. Eur. A proposal to use a larger pore size in combination with an
additional sterilisation step has to be validated and justified in the application file.
Results of media filling fall within the field of GMP and need not be presented routinely i n
the application for marketing authorisation but may be requested by the competent authorities
in certain circumstances.
For sterilisation by gamma and electron radiation, see the note for guidance The Use of
Ionisation Radiation in the Manufacture of Medicinal Products.
For sterilisation by ethylene oxide, the provisions laid down in the note for guidance
Limitations to the use of Ethylene Oxide in the Manufacture of Medicinal Products should be
followed, i.e. its use as a sterilisation method is only acceptable if no other method of
sterilisation is available.
The application for marketing authorisation should contain a description of the apparatus,
quantitative data on the mixture of gases to be used, data on the bioburden prior to
sterilisation, the time of exposure of the gas, the temperature and humidity prior to and
during the sterilisation cycle, and the conditions for the removal of ethylene oxide. All these
conditions should be monitored by suitable in-process controls that are to be described
together with the acceptance limits for these in-process controls.
Results of the process validation should be presented to justify these acceptance limits for the
in-process controls. The results should demonstrate both an acceptable assurance, of sterility
and removal of ethylene oxide to an acceptable level.
Limits of NMT 1 ppm of ethylene oxide (if applicable, measured by means of a simulated use
extraction method) and NMT 50 ppm of ethylene chlorhydrin (or any other halogenated
ethylenehydrin) are acceptable without further justification, once sterilisation by ethylene
oxide has been justified.
Notwithstanding successful process validation, a limit for residual ethylene oxide, and the
corresponding validated analytical method, should be included in the product release- and
end of shelf life specifications.
17
3AQ2a
18
3AQ3a
CONTENTS
1
TOXICOLOGICAL BACKGROUND
SPECIFICATIONS/TEST PROCEDURES
19
3AQ3a
raw
TOXICOLOGICAL BACKGROUND
Ethylene oxide is a substance which, due to its epoxide structure, is counted among the very
reactive compounds. This reactivity also includes organic structures within cells and cell
nuclei. In this case, alkylation and reactions with DNA, RNA and proteins occur.
Cytotoxicity, carcinogenicity and mutagenicity of ethylene oxide, which have been
demonstrated by many in vitro and in vivo tests, are attributed to these properties.
Epidemiological data from many sources indicated that workers exposed to ethylene oxide at
their working place had an increased incidence of leukaemia and other tumours.
In view of the known positive potential of ethylene oxide for genotoxic carcinogenicity, it is
recommended that use is acceptable only when pharmaceutically absolutely necessary, and
then at a limit of 1 ppm. This limit is based on the current limit of detection for ethylene
oxide.
Any deviation upwards from this limit must be justified and defended, taking into account
the clinical risk/benefit assessment for the particular products under consideration.
2.
Ethylene oxide is used in the synthesis of pharmaceutical raw materials and as a sterilant.
Since it is effective only as a surface sterialnt it should be used only for sterilising justified
and validated on an individual basis.
Ethylene oxide sterilisation should be used only where safer alternatives cannot be used.
3.
SPECIFICATIONS/TEST PROCEDURES
Due to the above mentioned considerations, the limits are fixed on a mass/mass basis and
not on a daily intake basis. If no official test procedure (e.g. Pharmacopoeia) is available a
validated test procedure must be proposed by the applicant (see also note for guidance on
Validation of Analytical Procedures: Methodology).
21
3AQ3a
3.3 Containers
Specification (based on simulated use):
Ethylene oxide: 1 /1 (container volume)
Ethylene chlorhydrin (or any other halogenated ethylenehydrine): 50 g/ml
volume).
22
(container
3AQ4a
CONTENTS
1.
INTRODUCTION
2.
ADMINISTRATIVE DATA
3.
MANUFACTURING PROCESS
4.
GLOSSARY
23
3AQ4a
INTRODUCTION
This note for guidance is intended for applicants wishing to use ionising radiation in the
manufacture of medicinal products. Irradiation may be used for microbial decontamination,
sterilisation or other treatments. Different materials or products may be irradiated: starting
materials, packaging materials, intermediate products, bulk products and finished products.
Information should be given in sufficient detail to enable the competent authority to evaluate
whether or not the manufacturing subprocess is effective and the product is safe for the
patient.
Manufacturers using ionising radiation in the manufacture of medicinal products should
refer to the Guide to Good Manufacturing Practice (Volume IV of "The Rules Governing
Medicinal Products in the European Union") and in particular to the annex on ionising
radiation used in the manufacture of medicinal products and, where relevant, to the annex
on manufacture of sterile medicinal products.
2.
ADMINISTRATIVE DATA
a)
The name and description of the product (including its packaging material) to be
irradiated should be given. Its shape, size and composition (type and quantity of
substances) should be described in detail. Furthermore, it should be made clear whether
starting materials, packaging materials, intermediate products, bulk products or the
finished product are irradiated. Sizes of production batches and of irradiation batches
should be defined. In the case of a continuous process, a batch comprises all the units
processed in a given period of time.
b)
The purpose of the irradiation should be stated. Both the minimum dose to achieve this
purpose and the maximum permissible dose should be stated.
c)
d)
3.
MANUFACTURING PROCESS
and
Irradiation of a medicinal product is part of its manufacturing process and the description of
that part of processing should be sufficiently detailed. The application should include the
following information:
25
3AQ4a
b)
c)
authorised and actual activity of the radionuclide in the radiation source (GBq), or the
maximum and minimum electron energy (MeV) of the generator as appropriate;
d)
concise description of the plant including drawings, showing clearly the course of the
product within the plant, the position and geometry of the irradiation source and the
conveyor system including the source pass mechanism.
b)
the number and positions of the irradiation containers in relation to the position of the
source during the whole dwelling time, and the method of moving them through the
chamber, should be described;
c)
d)
the maximum total irradiation time and the maximum dwelling time of the product i n
the irradiation chamber should be stated;
e)
f)
the loading pattern of the product must be stated for each irradiation container. If the
load consists of mixed products, the composition of the load m u s t be described
including their stated position in the irradiation container. The m e a n density of the
load and the acceptable m a x i m u m density should be given. A modification of the
loading p a t t e r n may be acceptable provided a new dose mapping is performed, showing
that the stated minimum and maximum doses are not exceeded;
g)
when the loading pattern of the product within the irradiation container has been
defined, dose mapping should be performed with a sufficient number of appropriate
dosimeters to show the distribution of the absorbed dose within the loaded i r r a d i a t i o n
container and to show the places of m i n i m u m and m a x i m u m doses. This dose
mapping should be carried out for a representative number of irradiation containers to
determine the variability of the absorbed dose in the load of one container and the
differences between several containers.
Note: Separate dose mapping exercises should be carried out for each product or distinct
category of products and each pathway to be used for processing products.
h)
26
the type, number and location of routine dosimeters within one irradiation batch
or within a stated period of time in the case of a continuous process;
3AQ4a
whether or not repeated treatment is acceptable; for the product concerned, the
circumstances in which such repeated treatment is allowed, and the number of
occasions on which it is allowed for a particular batch;
in the case of electron beam irradiators electron energy, average beam current,
beam width and conveyor speed should be stated with acceptable limits.
experimentally
Note: The stated minimum dose is that required for the intended purpose, the stated
maximum dose is limited by unacceptable changes induced by irradiation in the product
and/or the packaging, or imposed by official restrictions.
A minimum absorbed dose of 25 kGy may be regarded as adequate for the purpose of
sterilising pharmaceutical components or products which have a low initial bioburden and
no radioresistant spores. Other doses may be used provided that a biological validation has
been performed.
4.
with electron irradiation, if the maximum electron energy exceeds 10 MeV, it should be
demonstrated that no radionuclides develop in the product;
b)
c)
information should be included on the errors due to the type of dosimeters used and on
the influence of their position;
d)
information on the relationship between the absorbed doses in the extreme positions
within the load and the positions of routine dosimeters should be given.
b)
c)
an inactivation curve derived from the above data should be submitted. If the test
specimen itself has a low bioburden, it should be artificially contaminated with
> 107 cfu/single unit preferably with a microorganism originally occurring in the
product and with a minimum D-Value of 3 kGy;
doses,
27
3AQ4a
d)
the bioburden limit on the product prior to irradiation should be based on data derived
from a) - c).
In other cases, experimental results should show that the purpose of irradiation has been
achieved.
Information should be given about any qualitative and quantitative changes in the
product, including its packaging, as a result of irradiation;
Note:
Methods used for quantitative determinations should be validated in
accordance with the note for guidance Validation of Analytical
Procedures:
Methodology.
b)
c)
the results of the studies carried out with high doses of radiation to determine the
maximum dose should be given;
d)
e)
information should be given about the effect of irradiation on the stability of the product
and therefore stability studies should be performed on products which have received the
maximum absorbed dose.
Note:
The relevance of any changes in the product induced by irradiation as
regards quality of the product as well as health and safety of the patient should be
discussed. The toxicological risks caused by products of irradiation (see section 3.3.b)
should be evaluated. Safety of the irradiated product for the patient should be discussed
in the expert report.
28
3AQ4a
GLOSSARY
Absorbed
Dose
The quantity of radiation energy imparted per unit mass of material. The unit of absorbed
dose is the Gray (Gy) where 1 Gray is equivalent to absorption of 1 Joule per kilogram (J.kg1).
Batch
A defined quantity of starting material, packaging material or product processed in one
process or series of processes so that.it could be expected to be homogeneous.
Note: To complete certain stages of manufacture, it may be necessary to divide a batch into
a number of subbatches, which are later brought together to form a final homogeneous batch.
In the case of continuous manufacture, the batch must correspond to a defined fraction of the
production, characterised by its intended homogeneity.
For control of the finished product, the following definition has been given in Directive
75/318/EEC as amended: 'For the control of the finished product, a batch of a proprietary
medicinal product comprises all the units of a pharmaceutical form which are made from
the same initial mass of material and have undergone a single series of manufacturing
operations or a single sterilisation operation or, in the case of a continuous production
process, all the units manufactured in a given period of time'.
Bioburden
The total number of all viable aerobic bacteria, yeasts and moulds expressed as colony
forming units (cfu) per unit or gram of product.
Bulk
Product
Any product which has completed all processing stages up to, but not including,
packaging.
Dose
final
Mapping
Product
Homogeneous material of known density for filling the irradiation container for the purpose
of carrying out dose distribution experiments with ionising radiation.
29
3AQ4a
Finished
Product
A medicinal product which has undergone all stages of production including packaging.
Intermediate
Product
Partly processed material which must undergo further manufacturing steps before it becomes
a bulk product.
Irradiation
Container
Material
Any material employed in the packaging of a product, excluding any outer packaging used
for transportation or shipment. Packaging materials are referred to as primary or
secondary according to whether or not they are intended to be in direct contact with the
product.
Starting
Material
Any substance used in the production of a product, but excluding packaging materials.
30
3AQ5a
CONTENTS
1.
INTRODUCTION
2.
IDENTITY OF MATERIALS
3.
MANUFACTURE
4.
DEVELOPMENT CHEMISTRY
5.
IMPURITIES
6.
7.
BATCH ANALYSIS
8.
REFERENCE STANDARDS
9.
RADIOLABELLED PRODUCT
31
3AQ5a
INTRODUCTION
The purpose of this note for guidance is to set out the type of information required for the
control of new active substances used for the first time in a medicinal product, which are not
described in the European Pharmacopoeia or a pharmacopoeia of a Member State.
2.
IDENTITY OF MATERIAL
This section deals with the identity, nomenclature and chemical structure of the active
substance which is the subject of the application for marketing authorisation. Only brief
details of physical characteristics should be stated, as full details and proof of structure are
required later.
2.1
Nomenclature
Laboratory Code(s),
2.2
Description
physical form,
structural formula,
molecular formula,
A brief description should be given of the appearance of the material. Where possible, the
structural formula should be given diagrammatically with all known stereochemistry
indicated conventionally, with molecular formula and relative molecular mass; otherwise a
detailed description of the nature of the substance should be given.
The relative molecular mass of the therapeutically active moiety should also be included,
where appropriate.
33
3AQ5a
3.
MANUFACTURE
A concise but comprehensive account of the manufacture of the active substance should be
provided. The headings given below should be followed where the active substance concerned
is a totally synthetic product. Some modification may be required where the molecule is only
partially synthetic e.g. penicillin-derivatives.
catalysts used,
The description of the process should indicate the scale of manufacture. It is often helpful if
an indication of the yield produced at each stage is given.
The description must normally fully define the method of synthesis. However, if alternative
steps or solvents are proposed these should be justified and show that the final quality of
material obtained does not differ significantly.
materials
Describe the analytical controls which are applied to ensure that the starting materials,
which make a significant contribution to the molecular formula, and any reagents are
correctly identified and are shown to be of a satisfactory quality. An indication of the content
of significant impurities in starting materials should be given. Specifications for solvents
used in the final stages of synthesis, crystallisation and/or washing should be submitted.
The criteria for accepting or rejecting batches of these materials should be indicated. The
control of starting materials should be designed to detect isomeric or other impurities which
are potentially reactive and could be carried through to the final product of the synthesis.
3.3.2 Intermediate
control
The quality control checks which are carried out at each stage of the process and on the
isolated intermediates should be described. A statement of the test procedure(s) and criteria
for acceptance should be given for each stage, where appropriate.
34
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4.
DEVELOPMENT CHEMISTRY
This section should indicate the research and development programme which h a s been
undertaken on the new active substances to investigate the evidence of structure and the
chemical and physico-chemical properties.
The findings described in this section should be reflected in the control tests on the active
substance by which batch-to-batch uniformity is controlled.
evidence of structure of key intermediates of synthesis (e.g. using IR, NMR, etc.),
characteristic chemical reactions which are diagnostic of the structure of the molecule,
35
3AQ5a
coefficient,
5.
IMPURHTES
A broad outline should be given of the research programme which has been undertaken to
demonstrate that the test procedures used for impurity control in the active substance
specification are valid including limit of detection and limit of quantification. Negative
information can sometimes be important.
5.1 Impurities
-
by-products of the synthesis arising from side reactions, impurities in the starting
materials or isomerisation,
trace elements arising from the use of catalysts or from other sources,
36
3AQ5a
degradation products (see note for guidance Stability Tests on Active Substances and
Finished Products).
A list and brief description of the products which have been considered as potential
impurities arising from the synthesis should be given. In each case, it should be stated
whether actual samples of such impurities have been synthesised for test purposes and which
of the analytical methods described under 5.2 have been used to detect those impurities.
Possible routes of degradation should also be discussed on the basis of results of
investigations on exposure of the substances to stress conditions (such as heat, light, pH,
moisture and other relevant factors).
6.
6.1
The tests applied and the limits thereby imposed should be stated for:
-
physical characteristics,
6.2
7.
BATCH ANALYSIS
Data should be provided in this section to illustrate the actual results which have been
obtained from routine quality control of the active substance. Results should be given, if
possible, for:
-
batches of material used in the toxicity tests and clinical trials reported in support of
the application,
recent consecutive batches (5) which are representative of the product which will be
supplied for the purposes covered by the marketing authorisation to show that the
proposed methods will give routine production material which falls within the
37
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date of manufacture,
place of manufacture,
use of batches.
As far as possible, the results should give actual figures for tests on, for example, impurity
levels. Results which merely state that the material "complies" with the test are not
sufficiently informative, especially where a relatively wide limit is allowed in the
specification.
The batch analyses should include all the tests set out in the specification. There may,
however, be cases where earlier batches of material were tested using a slightly different
specification. In these cases, a brief explanatory note should be included.
8.
REFERENCE STANDARDS
The criteria for establishing the reference substances (primary and secondary) for routine
analysis should be given with full analytical profiles.
9.
RADIOLABELLED PRODUCT
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R e q u i r e m e n t s in relation to Active S u b s t a n c e s
Directive 75/318/EEC as a m e n d e d
October 1991
April 1992
Last revised 1991
None/ III/8315/89
This note for guidance clarifies the r e q u i r e m e n t s to b e
included in a m a r k e t i n g a u t h o r i s a t i o n for an active
substance d e p e n d i n g on the described classification.
CONTENTS
1.
2.
3.
4.
39
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existing active substances not included in the Ph. Eur. or the pharmacopoeia of a
Member State;
2.
For new chemical active substances, the requirements are set out in the note for guidance
Chemistry of the Active Substance.
For biotechnologically derived new active substances, the requirements are described in the
specific "biotechnology" guidelines.
The information may be supplied either as part of the marketing authorisation (MA)
application or using the European Drug Master File procedure.
3.
The requirements are as set out as above for new chemical active substances.
Where appropriate, information may be omitted in relation to proof of structure (e.g. where
this can be carried out by specific identification tests in relation to a reference substance
fully described in the dossier, where necessary).
Evidence of the stability of the active substance may be provided from the literature (see note
for guidance on Stability Tests on Active Substances and Finished Products).
The information may be supplied either as part of the MA application or using the European
Drug Master File procedure.
4.
inorganic substances;
41
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Each batch of substances must comply with the current requirements of the Ph. Eur. or
pharmacopoeia of a Member State.
In each case, evidence should be presented to the competent authorities to demonstrate the
suitability of the pharmacopoeial monograph for material from the named manufacturer.
In relation to solid-state properties, the official pharmacopoeial monographs are intended to
control the general suitability of an active substance for any of the likely intended uses. In
cases where it is necessary for the particular intended use to control the bulk substance with
respect to solid-state properties, a suitable specification must be proposed with details of test
methods, batch analyses, validation data, etc.
42
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of the monograph
established
by the
European
The manufacturer of the active substance may submit documentation to the Secretariat of the
Commission of the European Pharmacopoeia with a view to evaluating the suitability of the
Ph. Eur. monograph in relation to the manufacturing method used (in this context,
appropriate procedures are in place within the Commission of the European Pharmacopoeia).
The applicant should include a copy of the certificate of compliance with the quality defined
by the European Pharmacopoeia, together with a written assurance that no significant
changes in the manufacturing method has taken place since the date of certification.
monograph
The applicant may supply other evidence obtained from the active substance manufacturer
(ASM). This might include the following evidence:
a)
information as to the length of time that the particular named source has been on sale
in the European Community and elsewhere; and
b)
a statement that, in the above period, there had been no significant change in the
method of manufacture leading to a change in the impurity profile of the substance;
and
c)
a statement that samples from the named source had been supplied to the Ph. Eur.
Commission or national Commission and had been taken into account in the
development of their monograph; and
d)
a statement that no additional tests were necessary arising from the use of the
manufacturing route to identify and limit additional impurities (particularly toxic
impurities) not specifically controlled by the pharmacopoeial monograph.
The above is one possible approach to providing reassurance to the authorities of the
suitability of a well-defined active substance from the named source with long and safe
patient exposure. It is noted, however, that even where a monograph has been in force for
many years, it will not necessarily be sufficient in relation to a new route of manufacture.
4.5.3 Full details
of
manufacture
The applicant may submit full details on the active substance, its manufacture, control etc.
as outlined in the guideline on "Chemistry of active substances" but omitting, where
appropriate, information on proof of structure (where this can be shown by specific
identification tests in relation to reference substances fully described in the dossier, where
necessary) and stability (where adequate literature evidence can be cited and summarised
as indicated in the note for guidance Stability Tests on Active Substances and Finished
Products).
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44
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Substances
Note: Reference to Ph. Eur. means the current edition of the European Pharmacopoeia, to
Ph. Eur./EC means the current edition of the European Pharmacopoeia or the pharmacopoeia
of a Member State.
Type of active ingredient?
Non-pharmacopeial
(i.e. not in Ph. Eur.)
^ = L
Ph. Eur
Monograph
1
Type of active
Biotech derived
drug
Vegetable
drug
Organic active
(human or animal
origin)
Inorganic
drug
Pharmacopeia monograph +
necessary additional tests e.g. for
pesticides
Oganic,
synthetic or
semi-synthet
^ZT
No
Active in long use,
NO changes in the manufacture,
NO uncontrolled toxic impurities etc.
No
Yes
Use transparent
Ph. Eur./EC monograph
Information on manufacture
confidential, made by another
firm?
-i*.
<Yes
Information in EuroDMF
'31
1 .
No
Information in MA Application
45
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CONTENTS
A.
B.
1.
INTRODUCTION
2.
3.
4.
5.
GLOSSARY OF TERMS
47
3AQ7a
This compilation has been prepared in order to help applicants for marketing authorisations
and manufacturers of active substances when using the European Drug Master File
procedure in the preparation of dossiers for application for marketing authorisations. It does
not have any legal force and in case of doubt, applicants should refer to the original texts of
the directives.
The words "medicinal products" cover both products for use in humans and veterinary
medicinal products
Contents:
1.
and
2.
Note for guidance Use of the European Drug Master File Procedure, adopted by the
Committee for Proprietary Medicinal Products (CPMP) on 12.5.1993 and revised on
16.6.1993, and by the Committee for Veterinary Medicinal Products (CVMP) on
23.3.1994, with immediate entry into operation.
in
or in
the
( *)
(**>
49
3AQ7a
manufacturer shall confirm in writing to the applicant that he shall ensure batch to
batch consistency and not modify the manufacturing process or specifications without
informing the applicant. Documents and particulars supporting the application for such
a change shall be supplied to the competent authorities.
B.
Note for guidance concerning a possible way of presenting the documentation required in
Part 2 C and F of the Annex to Directives 75/318/EEC as amended and 81/852/EEC as
amended. The documentation needed is described in detail in the "Notice to Applicants for
marketing authorisations for medicinal products for human use in the Member States of the
European Union" Part II, C 1 and F 1 and in the same document dealing with veterinary
medicinal products and in the note for guidance Chemistry of Active Substances.
INTRODUCTION
The European Drug Master File (DMF) procedure may be used when the active substance
manufacturer (ASM) is not the applicant for a product marketing authorisation (applicant),
with a view to protecting valuable know-how on the manufacture of the active substance.
A DMF is a document containing the information required to demonstrate that the quality of
the active substance is adequately controlled by the specification proposed by the applicant.
The applicant must, therefore, collaborate with the person submitting a separate DMF to
ensure that all relevant information required is supplied. Furthermore it must be ensured
that the applicant's part of the DMF (cf. below) contains all information needed for the
applicant to take full responsibility for the preparation, including the suitability of the active
substance (as supplied) for the intended route of administration.
It is not a requirement to present information on the active substance in the form of a DMF.
The information may also form part of the application for authorisation to place a medicinal
product on the market.
Three types of active substances may be described in a European DMF.
A.
New active substances still covered by a patent, not described in the European
Pharmacopoeia or in the pharmacopoeia of a Member State.
B.
C.
50
of
in
to
as
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2.
The applicant must be supplied by the ASM with sufficient information to be able to take
responsibility for an evaluation of the suitability of the active substance specification to
control the quality of the substance. This normally includes a brief outline of the
manufacturing
method, information
on potential impurities originating from
the
manufacturing method, from the isolation procedure (natural products) or from degradation
and, where applicable, information on the toxicity of specific impurities.
b)
ASM R e s t r i c t e d P a r t of DMF
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TABLE
Applicant's p a r t
+
+
+
+
+
- potential isomerism
- physico-chemical characterisation
- analytical validation
Impurities (C. 1.2.6.)
Batch analysis (incl. impurities) (C. 1.2.7.)
Stability (where necessary) (F.l.)
+
+
+
+
+
+
+
3.
The ASM should give permission to the competent authorities to assess the data in his DMF
on behalf of a specified applicant in the form of a 'Letter of access'. Such a letter should
include the following information:
-
name and address of the importer of the active substance, where applicable;
A written assurance that there is a formal agreement between the ASM and the applicant
which ensures that if there is a significant change in the manufacturing method (likely to
alter the quality or safety of the product) or in active substance specification, this
information will be communicated to the applicant and to the authorities. It is the
responsibility of the applicant to consider any necessary changes in his specification and to
inform the competent authorities accordingly.
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A 'letter of access' must be lodged by the ASM for every application for marketing
authorisation.
4.
The applicant for the product marketing authorisation has the full responsibility for the
inclusion in the Expert Report of the required critical evaluation of the dossier. However, as
information in the ASM Restricted Part of the DMF is not available to the applicant, a
critical appraisal should be included in the ASM Restricted Part of the DMF on specific
items important for the quality of the active substance. In particular this section should
consider:
-
the manufacturing method and discuss how it will consistently guarantee material of
the desired quality;
demonstration of how the specification and routine tests in the applicant's part is
justified in relation to the ASM Restricted Part.
5.
GLOSSARY OF TERMS
European Drug Master File: EC procedure where information can be provided to the
authorities and the applicant, where the active substance manufacturer is not the applicant
for a product marketing authorisation, with a view to protecting valuable manufacturing
know-how.
ASM: Active Substance Manufacturer.
Applicant's part of a DMF: Section of the European DMF given to the applicant to include
in the application for a product marketing authorisation.
ASM Restricted Part of DMF: Section of the European DMF given by the ASM only to the
authorities.
Letter of Access: Letter of authority from the ASM to allow the competent authorities to
assess the European DMF on behalf of a specified applicant.
The possibility of submitting confidential data to the competent authorities directly from an
Active Substance Manufacturer (ASM) who is different from the applicant for the marketing
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Is t h e p r o c e d u r e m a n d a t o r y ?
W h e n t o s u b m i t a n EDMF?
Who c a n s u b m i t an EDMF ?
Only ASMs and their authorised representatives (e.g. importers) may submit an EDMF.
If an applicant for a m a r k e t i n g authorisation wants to rely on alternative suppliers of the
active substance, he should refer to this situation in the application.
5.
Where a n d h o w to s u b m i t an EDMF?
5.1
One copy of the complete EDMF, comprising the 2 parts (applicant's part and A S M ' s
restricted part) should be sent by the ASM directly to each of the competent authorities
concerned.
5.2
A copy of the applicant's part should be supplied in advance by the ASM to the
applicant. This applicant's part should be included in the application for m a r k e t i n g
authorisation.
6.
H o w t o i n c l u d e a d d i t i o n a l s p e c i f i c a t i o n s for c e r t a i n d o s a g e forms?
Specific quality requirements for certain dosage forms may be included in the application
for m a r k e t i n g authorisation. Such requirements will not form part of the EDMF. T h e y
should be described in a formal contract between the ASM and the applicant and included i n
the application for marketing authorisation.
7.
If a DMF h a s b e e n p r e v i o u s l y a s s e s s e d by t h e c o m p e t e n t authority, w h a t
i n f o r m a t i o n s h o u l d b e g i v e n w i t h a n e w DMF o n t h e s a m e a c t i v e s u b s t a n c e ?
7.1
If an EDMF has been updated since the previous assessment, the changes should be
clearly identified (particularly those in the method of manufacture and in the specifications
of the active substance).
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3AQ7a
7.2
If a DMF was not presented in accordance with the current guideline on EDMFs
(prior to 1992 for human medicinal products and prior to May 1993 for veterinary medicinal
products), it should be re-arranged into the 2-part format, and the different parts sent to the
authorities and to the applicant as described above.
8.
An EDMF will never be approved as such, it may only be accepted in relation to a specific
application for marketing authorisation. No claims should therefore be made by any ASM as
to his EDMF having been "approved".
9.
One of the major objectives of the EDMF procedure is to ensure that the applicant is always
fully aware of any aspects of the active substance which could impact on its safety, quality or
efficacy. Thus, all deficiencies in the EDMF will normally be addressed by the competent
authorities to the applicant for the marketing authorisation. When asking such questions,
rather general terms will be used to avoid any disclosure of confidential data from the ASM
restricted part. The applicant will then ask the ASM to supply the further data to the
concerned competent authorities and also to ensure that his part of the EDMF is completed.
10.
The competent authority will not inform the Active Substance Manufacturer that his EDMF
has been accepted in relation to a particular application for marketing authorisation. This
information should be sought by the ASM from the applicant.
11.
Need for identical specifications between the application and the EDMF
The specification of the active substance should be identical in the application and in the
applicant's part of the EDMF. Any proposal from the applicant to use a different test
procedure for the purity tests must be fully validated in relation to the procedure used by the
active substance manufacturer, in particular as regards the precision and accuracy of the
results of the tests, including the limits of detection for all possible impurities.
12.
How to inform the interested parties when an ASM wants to modify the ASM
restricted part or the applicant's part?
12.1
If the only change to be made is in relation to the contents of the ASM restricted part
of the file, this information should only be given to the authorities. A prerequisite for such
procedure is that there is no change in specification and no change in impurity profile.
12.2
If changes are to be made in the applicant's part of the EDMF, this information must
also be given to any other applicant or holder of a marketing authorisation referring to this
EDMF. All the applicants involved will then need to seek to change their marketing
authorisation dossier (using the appropriate variation procedures).
55
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13.
13.1
Fees, if any, for the EDMF assessment are to be paid by the applicant for a marketing
authorisation according to the instructions given by the relevant competent authority.
13.2
Variations to marketing authorisations because of changes to the EDMF attract the
relevant fee, to be paid by the holder of the authorisation.
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CONTENTS
1.
PREAMBLE
2.
CLASSIFICATION OF IMPURITIES
3.
4.
ANALYTICAL PROCEDURES
5.
6.
7.
QUALIFICATION OF IMPURITIES
8.
NEW IMPURITIES
9.
GLOSSARY
DECISION TREE FOR SAFETY STUDHiS
57
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PREAMBLE
This document is intended to provide guidance for applicants on the content and
qualification of impurities in new active substances produced by chemical syntheses and not
previously authorised in a Member State. It is not intended to apply to the regulation of new
substances
used
during
the
clinical
research
stage
of
development.
Biological/biotechnological, peptide, oligonucleotide, radiopharmaceutical, fermentation and
semi-synthetic products derived therefrom, herbal products, and crude products of animal or
plant origin are not covered.
Impurities in new substances are addressed from two perspectives:
Chemistry Aspects includes classification and identification of impurities, report
generation, setting specifications, and a brief discussion of analytical procedures; and,
Safety Aspects includes specific guidance for qualifying impurities which were not present
in batches of new substance used in safety and clinical studies and/or impurity levels
substantially higher than in those batches. Threshold limits are defined, below which,
qualification is not needed.
2.
CLASSIFICATION OF IMPURITIES
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Solvents are organic or inorganic liquids used during the manufacturing process. Since
these are generally of known toxicity, the selection of appropriate controls is easily
accomplished.
Excluded from this document are: extraneous contaminants which should not occur in new
substances and are more appropriately addressed as GMP issues; polymorphic forms, a solid
state property of the new substance; and, enantiomeric impurities.
3.
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3.3 Solvents
The control of residues of the solvents used in the manufacturing process for the new
substance should be discussed. Any solvents which may appear in the substance should be
quantified using analytical procedures with an appropriate level of sensitivity.
Pharmacopoeial or other appropriate procedures should be utilised. Limits should be based on
pharmacopoeial standards or known safety data taking into consideration dose, duration of
treatment, and route of administration. Particular attention should be given to quantitation
of toxic solvents used in the manufacturing process
4.
ANALYTICAL PROCEDURES
The application should include documented evidence that the analytical procedures are
validated and suitable for the detection and quantitation of impurities. Differences in the
analytical procedures used during development and proposed for the commercial product
should be discussed in the marketing authorisation application.
Organic impurity levels can be measured by a variety of techniques, including those which
compare an analytical response for an impurity to that of an appropriate reference standard
or to the response of the new substance itself. Reference standards used in the analytical
procedures for control of impurities should be evaluated and characterised according to their
intended uses. It is considered acceptable to use the substance to estimate the levels of
impurities. In cases where the response factors are not close, this practice may still be
acceptable, provided a correction factor is applied or the impurities are, in fact, being
overestimated. Specifications and analytical procedures used to estimate identified or
unidentified impurities are often based on analytical assumptions (e.g., equivalent detector
response, etc.). These assumptions should b discussed in the marketing authorisation
application.
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batches, from method validation studies showing separation and detectability of impurities
(e.g., on spiked samples), along with any other impurity tests routinely performed, can serve
as the representative impurity profiles. The applicant should ensure that complete impurity
profiles (i.e., chromatograms) of individual batches are available if requested.
A tabulation should be provided which links the specific new substance batch to each safety
study and each clinical study in which it has been used.
For each batch of the new substance, the report should include:
Batch Identity and Size
Date of Manufacture
Site of Manufacture
Manufacturing Process
Impurity Content, Individual and Total
Use of Batches
Reference to Analytical Procedure Used
6.
The specifications for a new substance should include limits for impurities. Stability
studies, chemical development studies, and routine batch analyses can be used to predict
those impurities likely to occur in the commercial product. The selection of impurities to
include in the new substance specifications should be based on the impurities found in
batches manufactured by the proposed commercial process. Those impurities selected for
inclusion in the specifications for the new substance are referred to as "specified
impurities" in these guidelines. Specified impurities may be identified or unidentified and
should be individually listed in the new substance specifications.
A rationale for the inclusion or exclusion of impurities in the specifications should be
presented. This rationale should include a discussion of the impurity profiles observed in the
safety and clinical development batches, together with a consideration of the impurity profile
of material manufactured by the proposed commercial process. Specific identified impurities
should be included along with recurring unidentified impurities estimated to be at or above
0.1%. For impurities known to be unusually potent or to produce toxic or unexpected
pharmacological effects, the quantitation/detection limit of the analytical methods should be
commensurate with the level at which the impurities must be controlled. For unidentified
impurities, the procedure used and assumptions made in establishing the level of the
impurity should be clearly stated. Unidentified impurities included in the specifications
should be referred to by some appropriate qualitative analytical descriptive label (e.g.,
"unidentified A" unidentified with relative retention of 0.9", etc.). Finally, a general
specification limit of not more than 0.1% for any unspecified impurity should be included.
Limits should be set no higher than the level which can be justified by safety data, and,
unless safety data indicate otherwise, no lower than the level achievable by the
manufacturing process and the analytical capability. In other words, where there is no
safety concern, impurity specifications should be based on data generated on actual batches
of the new substance allowing sufficient latitude to deal with normal manufacturing and
analytical variation and, the stability characteristics of the new substance. Although
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7.
QUALIFICATION OF IMPURITIES
Qualification is the process of acquiring and evaluating data which establishes the
biological safety of an individual impurity or a given impurity profile at the level(s)
specified. The applicant should provide a rationale for selecting impurity limits based on
safety considerations. The level of any impurity present in a new substance which has been
adequately tested in safety and/or clinical studies is considered qualified. Impurities which
are also significant metabolites present in animal and/or human studies do not need further
qualification. A level of a qualified impurity higher than that present in a new substance
can also be justified based on an analysis of the actual amount of impurity administered i n
previous safety studies.
If data are not available to qualify the proposed specification level of an impurity, studies to
obtain such data may be needed when the usual qualification threshold limits given below
are exceeded:
Maximum Daily Dose
Qualification Threshold
< 2g/day
> 2g/day
0.05%
Higher or lower threshold limits for qualification of impurities may be appropriate for some
individual substances based on scientific rationale and level of concern, including product
class effects and clinical experience. For example, qualification may be especially
important when there is evidence that such impurities in certain substances or therapeutic
classes have previously been associated with adverse reactions in patients. In these
instances, a lower qualification threshold limit may be appropriate. Conversely, a higher
qualification threshold limit may be appropriate for individual substances when the level of
concern for safety is less than usual based on similar considerations (patient population,
substance class effects, clinical considerations, etc.). Technical factors (manufacturing
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3AQ8a
capability and control methodology) may be considered as part of the justification for
selection of alternative threshold limits. Proposals for alternative threshold limits are
considered on a case by case basis.
The "Decision Tree for Safety Studies" (Attachment I) describes considerations for the
qualification of impurities when thresholds are exceeded. In some cases, decreasing the
level of impurity below the threshold may be simpler than providing safety data.
Alternatively, adequate data may be available in the scientific literature to qualify an
impurity. If neither is the case, additional safety testing should be considered. The studies
desired to qualify an impurity will depend on a number of factors, including the patient
population, daily dose, route and duration of medicinal product administration. Such studies
are normally conducted on the new substance containing the impurities to be controlled,
although studies using isolated impurities are acceptable.
8.
NEW IMPURITIES
During the course of a substance development program, the qualitative impurity profile of the
new substance may change, or a new impurity may appear as a result of synthetic route
changes, process optimisation, scale-up, etc. New impurities may be identified or
unidentified. Such changes call for consideration of the need for qualification of the level of
the impurity unless it is below the threshold values as noted above. When a new impurity
exceeds the threshold, the "Decision Tree for Safety Studies" should be consulted. Safety
studies should compare the new substance containing a representative level of the new
impurity with previously qualified material, although studies using the isolated impurity
are also acceptable (these studies may not always have clinical relevance).
9.
GLOSSARY
An impurity arising
from
any
Impurity: Any component of the new substance which is not the chemical entity defined as
the new substance.
Impurity Profile: A description of the identified and unidentified impurities present in a
new substance.
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Intermediate: A material produced during steps of the synthesis of a new substance which
must undergo further molecular change before it becomes a new substance.
Ligand: An agent with a strong affinity to a metal ion.
New Substance: The designated therapeutic moiety which has not been previously
registered in a region or member state (also referred to as a new molecular entity or new
chemical entity). It may be a complex, simple ester, or salt of a previously approved
substance.
Polymorphism:
Potential Impurity: An impurity which, from theoretical considerations, may arise from
or during manufacture. It may or may not actually appear in the new substance.
Qualification: The process of acquiring and evaluating data which establishes the
biological safety of an individual impurity or a given impurity profile at the level(s)
specified.
Reagent: A substance, other than a starting material or solvent, which is used in the
manufacture of a new substance.
Safety Information: The body of information that establishes the biological safety of an
individual impurity or a given impurity profile at the level(s) specified.
Solvent: An inorganic or an organic liquid used as a vehicle for the preparation of
solutions or suspensions in the synthesis of a new substance.
Specified Impurity: Identified or unidentified impurity that is selected for inclusion i n
the new substance specifications and is individually listed and limited in order to assure
the safety and quality of the new substance.
Starting Material: A material used in the synthesis of a new substance which is
incorporated as an element into the structure of an intermediate and/or of the new
substance. Starting materials are normally commercially available and of defined
chemical and physical properties and structure.
Toxic Impurity: Impurities having significant undesirable biological activity.
Unidentified Impurity: An impurity which is defined solely by qualitative analytical
properties (e.g., chromatographic retention time).
Validated Limit of Quantitation: For impurities at a level of 0.1%, the validated limit of
quantitation should be less than or equal to 0.05%. Impurities limited at higher levels may
have higher limits of quantitation.
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Decrease impurity
level below
threshold
Above threshold?
NO
Qualified
4, YES
Structure elucidated
J, YES
YES
J, NO
NO
Acceptable
justification?
i NO
VES
Qualified
I
Consider need for:
1. Genotoxicity studies (point mutation, chromosonal abberation)
2. General toxicity studies (one species, min. 14 days, max90 days)
3. Other specific toxicity endpoints as appropiate
Adverse Effects
YES
NO
Qualified
If considered desirable, a minimum screen for genotoxic potential should be conducted. A study to detect point mutations and
one to detect chromosomal aberrations, both in vitro, are seen as an acceptable minimum screen.
If general toxicity studies are desirable, study(ies) should be designed to allow comparison of unqualified to qualified
material. The study duration should be based on available relevant information and performed in the species most likely to
maximise the potential to detect the toxicity of an impurity. In general, a minimum duration of 14 days and a maximum
duration of 90 days will be acceptable.
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CONTENTS
INTRODUCTION
2 COMPOSITION OF THE MEDICINAL PRODUCT
2.A.4 DEVELOPMENT PHARMACEUTICS
2.C.2 EXCIPIENTS
2.E
ANNEX
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INTRODUCTION
This note for guidance is concerned with the application to excipients of Part 2, sections A, C,
E, F of the annex to Directive 75/318/EEC as amended with a view to the granting of a
marketing authorisation for a new medicinal product.
The data should be presented according to the standard format described in the Notice to
Applicants (Volume II of "The Rules Governing Medicinal Products in the European Union"
series), parts II A, C, E and F.
PART 2.A.1
Excipients must be listed, specifying their common name, their quantity and the use and
reference to any relevant standard. When the common name is not sufficient to indicate
functional specifications, the brand name with commercial grade should be specified. In the
case of excipients presented as a mixture of compounds, details as to the composition should
be provided in qualitative and quantitative terms. However, for flavouring agents and
aromatic substances, it is permitted to give the qualitative composition only.
PART 2.A.4
DEVELOPMENT PHARMACEUTICS
This section should comprise an explanation of the choice of the excipient (and grade where
necessary) according to the note for guidance Development Pharmaceutics and Process
Validation.
PART 2.C.2
EXCIPIENTS
2.1
or, failing
this, in
the
The routine tests which are to be carried out on each batch of starting materials must be
stated in the application for marketing authorisation. If tests other than those mentioned i n
the pharmacopoeia are used, proof must be supplied that the test methods used are suitable to
establish that the starting materials meet the quality requirements of that pharmacopoeia.
When the monograph covers a family of related products, the particular
specifications
chosen for the excipients must be submitted. In addition and when necessary, the test used to
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determine the quality of the excipient should be shown to be in relation to the function that it
fulfils in the medicinal product.
Data on microbiological contamination of the excipients used in the manufacture of sterile
products should always be given where membrane filtration is used to achieve sterility.
2.1.2 Excipients not described in the European Pharmacopoeia or in the pharmacopoeia of a
Member State
The entire routine test procedure and storage conditions must follow on from the scientific
data (2.2.2): an appropriate specification of the excipient must be established, based on the
following types of tests:
Physical characteristics
Identification tests
'
Purity tests, including limits for total or individual impurities, which should be
named. Purity tests may be physical, chemical, biological and, if appropriate,
immunological.
Where sterile filtration is used in the manufacture of a parenteral medicinal product, data
and routine tests on microbiological contamination of excipients should always be given.
Other relevant tests including, e.g. the tests on parameters which may influence the
performance of the dosage form.
Assay or limit tests if necessary.
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2.2.2 Other excipients: a dossier should be established containing the same data as
required for new active substances:
a)
A strict definition of the excipient, its function and its conditions of use. If the excipient
is complex or is made of a mixture of compounds, the composition must be specified i n
qualitative and quantitative terms.
b)
For new excipients and for excipients presented as a mixture of compounds the
following should be taken into consideration:
i.
Any bibliographical data on the chemistry and on the toxicology and the field i n
which the product is already used.
ii.
The quality specifications which have been laid down in the directives are satisfactory
as long as the routine control tests used are validated.
c)
iii.
iv.
For medicinal products for topical use, data on the starting material in cosmetic
products (Directive 76/768/EEC).
v.
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1.
For new excipients, stability data should be provided as required for new active substances.
2.
The maintenance of the physico-chemical properties of the finished product are dependent
upon the properties and the stability of the excipients (see note for guidance Specifications and
Control Tests on the Finished Product).
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ANNEX
Examples of requirements concerning different kinds of excipients
1.
Excipients which are a single chemical entity include, for example, organic and
inorganic acids and their salts, sugars and alcohols.
They may have undergone physical treatments which gave them special technological
characteristics (e.g. micronisation).
2.
3.
4.
Mixed excipients are ready-for-use preparations, for example for direct compression or
film coating.
The qualitative and quantitative composition of the mixed excipient should be
submitted, the specifications of the product as a whole and of each component
must be stated.
5.
Excipients of natural origin, so called "natural" products have often undergone some
kind of chemical treatment.
In general and if relevant for the quality control of the product, data should give a n
outline of the operations carried out to obtain and to purify the product, and any special
characteristics: decomposition products, specific impurities, chemical substances used
during the treatment with residual limits, methods of sterilisation or decontamination,
with a description of the effect of these processes on the excipient (e.g. modification of
the physical structure).
6.
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7.
Flavouring agents (flavours and aromatic substances) are either natural products
and/or products obtained by chemical synthesis. Because of the complexity of their
composition, it is only necessary to describe the general qualitative composition
mentioning the main constituents with an appropriate process of identification to
ensure the consistency of the composition (in particular, identification of the m a i n
constituents and if necessary carriers).
Most constituents of artificial flavours have internationally accepted purity criteria i n
food use (FAO/WHO). Reference to these standards is acceptable for medicinal
products.
8.
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Guideline Title
Legislative basis
Date of first adoption
Date of entry
into
force
Status
P r e v i o u s titles/ o t h e r
references
Additional Notes
Plastic P r i m a r y P a c k a g i n g Materials
Directive 75/318/EEC a s a m e n d e d
F e b r u a r y 1994
August 1994
Last revised F e b r u a r y 1994
Plastic Containers and Packaging Material
(Immediate
Packaging)
/9090/90
This note for guidance concerns the application to p l a s t i c
p r i m a r y packaging materials of P a r t 2, sections A, C, and F
of t h e Annex to Directive 75/318/EEC as amended, w i t h a
view to t h e granting of a m a r k e t i n g authorisation for a
new m e d i c i n a l p r o d u c t . The p r o v i s i o n s of C o m m u n i t y
legislation r e l a t i n g to plastic materials i n t e n d e d to c o m e
into contact w i t h foodstuffs, in p a r t i c u l a r D i r e c t i v e
90/218/EEC should be t a k e n into account.
CONTENTS
INTRODUCTION
2.A.2 IMMEDIATE PACKAGING
2.A.4 DEVELOPMENT PHARMACEUTICS
2.C.3 PACKAGING MATERIAL (PRIMARY OR IMMEDIATE PACKAGING)
2.F.2 STABILITY - STUDIES OF MIGRATIONS AND INTERACTIONS
SUMMARY PRESENTATION
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INTRODUCTION
This note for guidance is concerned with the application to plastic primary packaging
materials of Part 2, sections A, C, F of the Annex to Directive 75/318/EEC as amended, with a
view to the granting of a marketing authorisation for a new medicinal product.
The present note is limited to plastic primary (or immediate) packaging materials i.e.
packaging materials intended to be in direct contact with the medicinal product. They
comprise containers, closures, seals and other parts which come into contact with the
medicinal product.
The data should be presented according to the standard format described in the Notice to
Applicants (Volume II of The Rules governing Medicinal Products in the European Union),
parts 2 A2, A4, C3 and F2.
Two other notes for guidance refer to containers: development pharmaceutics and process
validation and stability tests on active substances and finished products.
The importance and characteristics of data to be given are related to the pharmaceutical
dosage form (see appended summary).
When establishing the specifications for packaging materials for non-parenteral
preparations, the provisions of Community legislation relating to plastic materials intended
to come into contact with foodstuffs in particular Directive 90/128/EEC as amended, should be
taken into account.
For packaging materials used for ophthalmic and parenteral products, appropriate
development studies should be carried out if they are not described in the European
Pharmacopoeia or in the pharmacopoeia of a Member State.
Reference should be made to monographs of the European Pharmacopoeia or of the national
pharmacopoeia of a Member State.
PART 2 A.2
IMMEDIATE PACKAGING
This part of the dossier should contain a brief description of the container. It should include:
-
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PART 2 A.4
DEVELOPMENT PHARMACEUTICS
A justification of the choice of the container should be given in relation to data on stability of
the active substance(s) and of the finished product, to the method of administration and to
any sterilisation procedures.
The data collected during the development should be presented to justify the choice of the
plastic material(s) and of the container(s). These data should include details on:
-
tightness of closure
PART 2 C.3
PACKAGING MATERIAL (PRIMARY OR
IMMEDIATE PACKAGING)
3.1 Specifications and routine test
Information should be provided on:
-
The type of materials used in the different parts, and nature of polymers
The specifications: the nature, the extent and the frequency of routine tests of
packaging materials vary significantly according to the type of material, the type of
immediate packaging and the use of the product. Due account should be taken of the
route of administration, e.g. for parenteral and ophthalmic products.
visual inspection
dimensional tests
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packaging
General information
The following information should be provided for plastic materials used in the container,
including those already described in the pharmacopoeia where the monographs authorise the
use of several additives from which the manufacturer may choose one or several (within
certain limits).
-
For ophthalmic and parenteral preparations, the name of the plastic manufacturer.
B.
Technical information
Characteristics
Description of the material, its solubility in various solvents.
Identification of dyes
by using chromatographic or any other appropriate method.
Tests
General tests
Mechanical tests
Physical tests: an extraction test should be performed where the plastic material
is used as primary packaging material for liquid and semi-solid preparations.
The choice of solvent for this test depends on the composition of the product. The
test should investigate the level of extractives (antioxidants, plasticisers...).
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3.2.2 Container
The name of the manufacturer (also called converter) of the container should be specified for
ophthalmic and parental preparations.
The converter should ensure that the manufacturing process is reproducible and that there i s
no change in the composition of the material as defined for the type-sample.
Any significant change in the composition of the plastic material needs new specifications
and verification by the pharmaceutical manufacturer that the new packaging is suitable for
the intended use.
Dosage forms
1.1
Solid
forms
For oral or topical solid dosage forms, the risk of migration is low and generally does not
require a content/container interaction study. Solid forms intended for parenteral use may
need interaction studies between the elastomer closure and the components of the
formulation.
1.2
Semi-solid
forms
The study should consist mainly of technological controls of the container and study of the
eventual migration of additives or dyes. The risk of migration into aqueous or non-aqueous
semi-solid requires suitable specific studies for each formulation.
The study should be performed under normal and accelerated conditions.
1.3
Liquid
forms
The risks of migration require suitable specific studies for each formulation.
In the case of parenteral and ophthalmic products, determination of the active substance and
preservative contents should be performed under simulated conditions of use.
Levels of extractives (e.g. antioxidants, plasticisers, catalysts processing aids etc..) should
be investigated mainly for parenteral and ophthalmic products.
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2.
2.1
General scheme
Samples
During the development stage, migration studies on initial formulations often allow the
choice of a suitable packaging material for the finished product to be chosen.
The study should be performed on at least one batch of finished product.
2.2
Study
condition
Studies should be performed under normal and accelerated conditions according to the
current notes for guidance on stability.
2.3
Study
methods
Simulation studies performed with extraction solvents (as in the case of food) can only be
considered as predictive tests and do not preclude the need to perform a study on the finished
product.
-
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Plastic material
Name of material
Chemical name
Manufacturer
Complete composition
including additives
Container/closure
Manufacturer
(converter)
Description
Identification* polymer
Dimensional
Solid
preparations
Semi-solid/liquid
preparations
Ophthalmic and
Parenteral preparations
+
+
+
+
+
+
if necessary
+
+
+
+
+
+
+
+
+
+
if necessary
if necessary
+
if necessary
+
if necessary
properties
Technical properties
Suitability**
. . **
Toxicity
Compatibility,
4=
migration
Routine tests
** The studies of suitability toxicity and compatibility content-container and migrations are carried out during
the development and not for routine test.
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Product!
III/3324/89
CONTENTS
1.
SPECIFICATIONS
2.
TEST PROCEDURES
3.
BATCH ANALYSIS
APPENDIX
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Note for guidance concerning the application of Part 2, section E of the Annex to Directive
75/318/EEC as amended, for the purposes of granting a marketing authorisation.
A glossary of terms used is included in an appendix to this note.
SPECIFICATIONS
LI
assay of active substances (and also for herbal medicines, quantitative determination
of the constituents with known therapeutic activity);
purity tests (if necessary, the investigation of breakdown products, residual solvents or
other process related impurities, microbial contamination);
In order to determine the specifications of the finished product, the quality characteristics
related to the manufacturing process should be taken into account.
An appropriate specification for each aspect of quality studied during the phase of
development and during the validation of the manufacturing process should be determined.
At least those aspects considered to be critical should be the object of specifications routinely
verified.
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Directive 75/318/EEC as amended recapitulates those aspects of quality which must be the
object of appropriate specifications by referring to the general monographs of the European
Pharmacopoeia or, failing this, to pharmacopoeias of Member States.
Concerning monographs on medicinal products, the European or national pharmacopoeiae
define the reference quality level. In the marketing authorisation application, the applicant
should determine the most appropriate means for reaching the stated objective, and supply the
appropriate analytical validation data. In all cases, routine tests on the finished product at
release follow the stipulations of section 1.4.
In addition, as a result of the fact that these monographs correspond to regulatory
specifications, they represent the limit values of medicinal product at the end of their shelf
life (except for those provisions referring to production). The specifications for release of the
finished product must comply with the criteria defined by Directive 75/318/EEC as amended,
i.e. 5% for the assay of active substance(s) except when otherwise justified.
The aim of the application dossier for a marketing authorisation is to set the quality
level of the medicinal product as intended for marketing. It establishes specifications,
i.e. qualitative and quantitative characteristics, with test procedures and acceptance
limits, with which the medicinal product must comply during its intended shelf life.
In Part II F of the dossier, the applicant proposes a shelf life for the medicinal product
mainly on the basis of the level of active constituents (efficacy) and the admissible
level of any breakdown products or impurities (safety) and consistent
pharmacotechnical properties. From the behaviour of the medicinal product the
applicant deduces the appropriate storage conditions which will maintain compliance
with the specifications of the medicinal product.
b)
The specification limits of the finished product at the time of batch release are set by the
marketing authorisation applicant such that the specifications proposed at the end of
shelf life are guaranteed; they are established on the basis of a critical detailed review
of the data gathered from the batches analysed. Nevertheless, acquired experience is a
factor recognised to be very important in terms of good manufacturing practice. One of
the basic requirements of GMP (see the Guide to GMP) is the systematic review of all
manufacturing processes in the light of experience. Thus, the applicant, in compliance
with Directive 65/65/EEC as amended, Article 9. a, shall adapt or refine the
specifications at release as a function of experience acquired by the manufacturer(s) of
the medicinal product.
c)
The specifications of the finished product at manufacture may be different from those
of the medicinal product at expiry.
In certain cases, for characteristics of the medicinal product which may change during
storage under the approved conditions, the quality required at the end of shelf life
should be taken into account in determining appropriate specifications at the time of
manufacture, for example in the case of overages for reasons of stability.
It is desirable that all specifications (characteristics and acceptance limits) of the
medicinal product and the finished product at the time of release be presented in the
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form f a summary table. In this table, the limits of any likely breakdown products
which may form under the approved conditions of storage should be stated.
The summary table could be presented as follows:
1. Characteristics of the
pharmaceutical form
product at
1.4
Specifications a n d r o u t i n e t e s t s for t h e r e l e a s e of b a t c h e s of
finished p r o d u c t at t h e t i m e of m a n u f a c t u r e (at r e l e a s e )
The compliance of each batch of finished product with its specifications at manufacture
should be guaranteed by GMP (see Guide to GMP). Nevertheless, those features of the batch
documentation required by GMP may not be included in the marketing authorisation
dossier.
In the marketing authorisation dossier, it must be shown that the manufacturing process
used in compliance with GMP is capable of producing the finished product consistently in
compliance with the specifications chosen; these specifications take into account:
-
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the validation of critical steps in the manufacturing process described in Part III 3 of
the marketing authorisation dossier which enable scale up and batch reproducibility to
be monitored.
1.4.2 Routine
tests
tests to be carried out batch by batch on the finished product or, possibly, on the bulk
product;
b)
tests whose performance during a manufacturing step (intermediate products or inprocess controls) will contribute a greater guarantee of finished product compliance
than their performance on the finished product or on the bulk product;
c)
periodic tests (not carried out batch by batch on the finished product or, possibly on the
intermediate product or bulk product), where the frequency of performance is highly
dependent on other parameters of GMP (e.g. microbiological quality);
d)
tests whose performance on the finished product or possibly on the bulk product at
manufacture can be replaced by the verification of another highly dependent
specification (for example replacement of the test for uniformity of mass with the test
for uniformity of content);
e)
tests which are not carried out routinely once the guarantees of compliance are
furnished by the manufacturer; these specific cases are exceptional (e.g. identification
of colorants);
f)
specifications
As a function of the conclusions of parts II A 4 and II 3, the dossier must clearly indicate
the individual tests to be carried out for the release of the finished product and state whether
they are done routinely on each batch of finished product, or indicate the periodicity of testing
(batch by batch, every V batches, the first "n" batches etc.).
Two cases are possible depending upon whether or not the products are known and for which
the manufacturer has available substantial production experience.
In the first case, for example a new dosage strength of an already authorised product, or a
new conventional formulation (solution, compressed tablet etc.) of a well known
pharmacopoeial active substance, tests carried out routinely and periodically can be stated at
the time of application.
In the second case, for example with formulations of a new active substance or new dose
delivery system such as modified release tablets, adjustments as a result of product
experience should also be described, where possible, and the regulatory authorities informed
of appropriate developments based on the results obtained.
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The tests proposed in both cases should be presented in the form of summary tables as
indicated below (Tables 1 and 2). Whenever necessary, the tests proposed during the first "n"
batches (Table 1) should be distinguished from those envisaged in a routine industrial
situation (Table 2).
Table 1
Proposed scheme for testing during the initial period of industrial
production
Frequency of Testing
(Surveillance of the first "n" batches)
Tests
The applicant should state the number ("n") of batches involved where applicable.
Table 2
Testing scheme confirmed
following
Tests
stabilised
industrial
production
Frequency of Testing
Each batch of medicinal product marketed must comply with all the specifications defining
its expected quality level, regardless of the testing plans envisaged, or confirmed after
experience.
Testing may be chemical, physical, pharmaceutical, microbiological or biological.
limits
of pharmacotechnical
parameters
For most pharmacotechnical specifications, the European Pharmacopoeia or failing this the
national pharmacopoeias of the Member States, describes general test procedures with, in
some cases, standards or maximal limits.
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1.5.2 Maximum
acceptable deviation
substances
Release limits wider than 5% would need to be justified in the part "Development
Pharmaceutics" with experimental results which are normally based on a confidence
level of 95%. The wider limits also include both, the variation of the production and of
the test procedure for the assay.
Exceptionally, for certain products with a well known degradation process and which
pose no safety problems, an overage at release can be tolerated (vitamins, etc.). This
overage, the aim of which is to guarantee a sufficient level at the end of shelf life, must
not cause an excessive level at release, and release limits must be adapted
accordingly.
Release limits 5% primarily concern the manufacturers of the product and not any
outside laboratory. At recontrol by official laboratories, the release limits of the
manufacturer will be taken into consideration despite the fact that the limits used at
recontrol may not be identical to the release limits of the manufacturer (due to
interlaboratory variability). Where recontrol tests are carried out by another
laboratory, the test procedures must be validated in their hands.
Overfilling to guarantee the delivery of the theoretical unit dose must be justified i
Part II A 4 "Development Pharmaceutics". The acceptance limits for determination of
unit content are adapted in consequence.
1.5.3 Acceptance
-
90
limits for
excipients
Excipients which affect the bioavailability of an active substance must be the object of a
quantitative determination in each batch, unless bioavailability is guaranteed by other
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2.
TEST PROCEDURES
Test procedures must be described in a sufficiently detailed manner to enable any official
laboratory to verify the compliance of the medicinal product up to the end of shelf life.
Control methods must be validated in accordance with the note for guidance Validation of
Analytical Procedures: Methodology. The results of such analytical validation must be
included in the dossier.
Thus, the description of the test procedure(s) should, if necessary, include the description of
the reference substance, including its specifications and the description of the calculation
formula or an example of calculation (whether or not the calculations are performed by an
automatic instrument), chromatograms and spectra (etc.) furnishing the proof of the results
obtained.
Except for those officially included in the European Pharmacopoeia, e.g. sterility tests, it is
not necessary to describe the sampling procedures, which are the domain of GMP and depend
on inspection services.
A test procedure may use either an official reference substance (European Pharmacopoeia,
national pharmacopoeias, WHO) or a working standard, providing the latter is
standardised against the official reference substance (see note for guidance Validation of
Analytical Procedures: Methodology ).
It is to be remembered that an analytical result cannot be dissociated from the method used.
Thus in a pharmacopoeial monograph, it is assumed that the specifications adopted for the
tests and assay are established from the methods described in the Pharmacopoeia.
Methods other than the methods described in the Pharmacopoeia may be used for control
purposes providing that these methods are validated with reference to the official method and
providing that these methods used enable an unequivocal decision to be made as to whether
compliance with the standards of the monograph would be achieved if the official methods
were used (see general provisions of the European Pharmacopoeia).
In addition, the general methods described in the Pharmacopoeia may be used for products
not described in the Pharmacopoeia or for specifications not described in a monograph.
Utilisation of these methods requires validation for the specific case envisaged.
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3.
BATCH ANALYSIS
The data which must be produced are summarised in the Notice to Applicants. The section
"batch analysis" must include the results obtained for all specifications at release, whether
or not they are intended to be verified batch to batch.
Where possible, the consecutive batches should correspond to production scale batches
manufactured by all manufacturers and at all manufacturing sites declared in the
marketing authorisation, according to the process described in Part 1 A of the dossier. If
these data are not available for industrial scale batches, they should be supplied to the
competent authorities as soon as possible after the marketing authorisation is granted.
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APPENDIX
Glossary
The following definitions are applicable to the terms used in this note for guidance.
Analytical Validation (Refer to note for guidance Validation of Analytical
Methodology
Procedures:
final
Factorisation
Adjustment by calculation of the mass of an substance (usually the active substance) added
for the manufacture of a batch of a product on the actual potency as determined by assay of
that particular batch of substance. For example, if the theoretical batch quantity of an active
substance in a batch of product is 100 gram, and the assay "as is" of the batch of substance
being used is 92.0% m/m, then the factorised mass to be added would be 100 g 100/92.0 =
108.7 g.
Finished Product
A medicinal product which has undergone all stages of production including
(refer to Guide to GMP).
packaging
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Overage
An additional amount of an substance (usually the active substance) over and above the
amount stated in the unit formula, added to prepare a batch of product in order to compensate
for losses incurred during manufacture and/or storage in the finished pack. It is usually
expressed as a percentage.
Quality Assurance (Refer to Guide to GMP)
For the medicinal product, it is the sum total of the organised arrangements made with the
object of ensuring that manufactured medicinal products are of the quality required for their
intended use.
Routine Tests and Periodic Tests
Routine tests are carried out on each industrial batch of the intermediate product, the bulk
product or the finished product.
If necessary, these tests are extended by special tests called "periodic tests" which are carried
out according to a certain periodicity defined in each case; they are mentioned separately.
Specification
Qualitative and/or quantitative characteristics with test procedures and acceptance limits,
with which a given product must comply.
Specifications of the Finished Product (At Release)
Monograph defining qualitative and quantitative characteristics with test procedures and
their acceptance limits, with which the finished product must comply at the time of the
manufacture (at its release).
Specifications of the Finished Product (Up to the End of Shelf life)
Monograph defining qualitative and quantitative characteristics with test procedures and
their acceptance limits, with which a medicinal product (on the market) must comply
throughout its valid shelf life.
Validation of Manufacturing Process (Refer to Guide to GMP)
Action of proving, in accordance with the principles of Good Manufacturing Practice, that
any procedure, process, equipment, material, activity or system actually leads to the expected
results.
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CONTENTS
1.
INTRODUCTION
2.
GUIDELINES
3.
GLOSSARY
ATTACHMENT I
ATTACHMENT II
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INTRODUCTION
LI
1.2
Background
This Guideline is an annex to the Impurities in New Active Substances Guideline which
should be consulted for basic principles.
2.
GUIDELINES
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characterised according to their intended uses. The active substance may be used to estimate
the levels of degradation products. In cases where the response factors are not close, this
practice may still be used if a correction factor is applied or the degradation products are, in
fact, being overestimated. Specifications and analytical procedures used to estimate
identified or unidentified degradation products are often based on analytical assumptions
(e.g., equivalent detector response). These assumptions should be discussed in the application
for marketing authorisation. Differences in the analytical procedures used during
development and those proposed for the commercial product should be discussed.
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origin of these impurities should be discussed. Chromatograms, or equivalent data (if other
methods are used), from representative batches including long-term and accelerated stability
conditions should be provided. The procedure should be capable of quantifying at least at the
reporting threshold and the chromatograms should show the location of the observed
degradation products and impurities from the new active substance.
The following information should be provided:
Batch identity, strength and size
Date of manufacture
Site of manufacture
Manufacturing process, where applicable
Immediate container/closure
Degradation product content, individual and total
Use of batch
Reference to analytical procedure(s) used
Batch number of the drug substance used in the drug product
Storage conditions
2.5 Q u a l i f i c a t i o n of I m p u r i t i e s
Qualification is the process of acquiring and evaluating data which establishes the
biological safety of an individual degradation product or a given degradation profile at the
level(s) specified. The applicant should provide a rationale for selecting degradation product
limits based on safety considerations. The level of any degradation product present in a new
medicinal product which has been adequately tested and found safe in safety and/or clinical
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studies using the isolated degradation products are also considered acceptable (these studies
may not always have clinical significance).
3.
GLOSSARY
Degradation Product
A molecule resulting from a chemical change in the substance brought about over time
and/or by the action of, e.g., light, temperature, pH, or water or by reaction with an excipient
and/or the immediate container/closure system (also called decomposition product).
Degradation Profile
A description of the degradation products observed in the active substance or medicinal
product.
Development Studies
Studies conducted to scale-up, optimise and validate the manufacturing
medicinal product.
process for a
Identified Impurity
An impurity for which a structural characterisation has been achieved.
Impurity
Any component of the medicinal product which is not the chemical entity defined as the
active substance or an excipient in the product.
Impurity Profile
A description of the identified and unidentified impurities present in a medicinal product.
New Active Substance
The designated therapeutic moiety which has not been previously registered in a member
state (also referred to as a new molecular entity or new chemical entity). It may be a
complex, simple ester, or salt of a previously approved substance.
Potential Degradation Product
An impurity which, from theoretical considerations, may arise during or after manufacture
or storage of the medicinal product. It may or may not actually appear in the active substance
or medicinal product.
Qualification
The process of acquiring and evaluating data which establishes the biological safety of an
individual impurity or a given impurity profile at the level(s) specified.
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Reaction Product
Product arising from the reaction of a substance with an excipient in the medicinal product
or immediate container/closure system.
Safety Information
The body of information that establishes the biological safety of an individual impurity or a
given impurity profile at the level(s) specified.
Specified Degradation Product
Identified or unidentified degradation product that is selected for inclusion in the new
medicinal product specifications and is individually listed and limited in order to assure
the safety and quality of the new medicinal product.
Toxic Impurity
An impurity having significant undesirable biological activity.
Unidentified Degradation Product
An impurity which is defined
chromatographic retention time.
solely
by
qualitative
analytical
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properties,
e.g.,
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ATTACHMENT I
THRESHOLDS FOR REPORTING OF DEGRADATION PRODUCTS IN NEW
MEDICINAL PRODUCTS
Maximum Daily Dose1
>lg
Threshold
0.1%
0.05%
Threshold 2
1.0% or 5 ug TDI3 whichever is lower
0.5% or 20 ug TDI whichever is lower
0.2% or 2 mg TDI whichever is lower
0.1%
Threshold 2
1.0% or 50 ug TDI whichever is lower
0.5% or 200 ug TDI whichever is lower
0.2% or 2 mg TDI whichever is lower
0.1%
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1
OS
I de i t fica tio
""" Qualification
Reporting
2000
Maximum LbflyDose expressed in mg of Aclive Substance
Expanded scale:
mm mm
mm
10
15
104
20
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Above threshold?
YES
Qualified
NO
*
YES
Structure elucidated?
YES
YES
Toxicity documented
and sufficient?
NO
Related to others
with known toxicity?
NO
YES
Acceptable
justification?
J, NO
NO
YES
Qualified
i
Adverse Effects
YES
Consider additional testing or removal/
lowering level of degradation products
NO
Qualified
If considered desirable, a minimum screen e.g., genotoxic potential, should be conducted. A study to detect
point mutations and one to detect chromosomal aberrations, both in vitro, are seen as an acceptable
minimum screen, as discussed in the ICH-Guidelines: Specific Aspects of Regulatory Genotoxicity Tests
(included in Volume 3B ) and Genotoxicity: Definition of Core Battery Tests.
If general toxicity studies are desirable, study(ies) should be designed to allow comparison of unqualified to
qualified material. The study duration should be based on available relevant information and performed in
the species most likely to maximise the potential to detect the toxicity of an impurity. In general, a minimum
duration of 14 days and a maximum duration of 90 days would be acceptable.
On a case by case basis, single dose studies may be acceptable, especially for single dose drugs, and when
such studies are conducted using an isolated impurity. If repeat-dose studies are desirable, a maximum
duration of 90 days would be acceptable.
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Guideline Title
Legislative basis
Date of first adoption
Date of entry into
force
Status
Previous titles/other
references
Additional Notes
CONTENTS
INTRODUCTION
1.
SPECIFICITY
2.
LINEARITY
3.
RANGE
4.
ACCURACY
5.
PRECISION
6.
DETECTION LIMIT
7.
QUANTITATION LIMIT
8.
ROBUSTNESS
9.
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INTRODUCTION
This guideline is complementary to the parent guideline* which presents a discussion of the
characteristics that should be considered during the validation of analytical procedures. Its
purpose is to provide some guidance and recommendations on how to consider the various
validation characteristics for each analytical procedure. In some cases (for example,
demonstration of specificity), the overall capabilities of a number of analytical procedures in
combination may be investigated in order to ensure the quality of the active substance or
medicinal product. In addition, the document provides an indication of the data which should
be presented in an application for marketing authorisation.
All relevant data collected during validation and formulae used for calculating validation
characteristics should be submitted and discussed as appropriate.
Approaches other than those set forth in this guideline may be applicable and acceptable. It i s
the responsibility of the applicant to choose the validation procedure and protocol most
suitable for the product. However it is important to remember that the main objective of
validation of an analytical procedure is to demonstrate that the procedure is suitable for its
intended purpose. Due to their complex nature, analytical procedures for biological and
biotechnological products in some cases may be approached differently than in this
document.
Well-characterised reference materials, with documented purity, should be used throughout
the validation study. The degree of purity necessary depends on the intended use.
In accordance with the parent guideline*, and for the sake of clarity, this document
considers the various validation characteristics in distinct sections. The arrangement of
these sections reflects the process by which an analytical procedure may be developed and
evaluated.
In practice, it is usually possible to design the experimental work so that the appropriate
validation characteristics can be considered simultaneously to provide a sound, overall
knowledge of the capabilities of the analytical procedure, for instance: specificity, linearity,
range, accuracy and precision.
SPECIFICITY
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LI
Identification
1.2
Assay a n d I m p u r i t y Test(s)
1.2.1 Discrimination
ofanalytes
where impurities
are
available
For the assay, this should involve demonstration of the discrimination of the analyte in the
presence of impurities and/or excipients; practically, this can be done by spiking pure
substances (active substance or product) with appropriate levels of impurities and/or
excipients and demonstrating that the assay result is unaffected by the presence of these
materials (by comparison with the assay result obtained on unspiked samples).
For the impurity test, the discrimination may be established by spiking active substance or
product with appropriate levels of impurities and demonstrating the separation of these
impurities individually and/or from other components in the sample matrix.
1.2.2 Discrimination
are not
available
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Peak purity tests may be useful to show that the analyte chromatographic peak is not
attributable to more than one component (e.g., diode array, mass spectrometry).
2.
LINEARITY
A linear relationship should be evaluated across the range (see section 3) of the analytical
procedure. It may be demonstrated directly on the active substance (by dilution of a standard
stock solution) and/or on separate weighings of synthetic mixtures of the product
components, using the proposed procedure. The latter aspect can be studied during
investigation of the range.
Linearity should be evaluated by visual inspection of a plot of signals as a function' of
analyte concentration or content. If there is a linear relationship, test results should be
evaluated by appropriate statistical methods, for example, by calculation of a regression line
by the method of least squares. In some cases, to obtain linearity between assays and sample
concentrations, the test data may need to be subjected to a mathematical transformation prior
to the regression analysis. Data from the regression line itself may be helpful to provide
mathematical estimates of the degree of linearity.
The correlation coefficient, y-intercept, slope of the regression line and residual sum of
squares should be submitted. A plot of the data should be included. In addition, an analysis
of the deviation of the actual data points from the regression line may also be helpful for
evaluating linearity.
Some analytical procedures, such as immunoassays, do not demonstrate linearity after any
transformation. In this case, the analytical response should be described by an appropriate
function of the concentration (amount) of an analyte in a sample.
For the establishment of linearity, a minimum of 5 concentrations is recommended. Other
approaches should be justified.
3.
RANGE
The specified range is normally derived from linearity studies and depends on the intended
application of the procedure. It is established by confirming that the analytical procedure
provides an acceptable degree of linearity, accuracy and precision when applied to samples
containing amounts of analyte within or at the extremes of the specified range of the
analytical procedure.
The following minimum specified ranges should be considered:
for the assay of an active substance or a finished product: normally from 80 to 120
percent of the test concentration;
for content uniformity, covering a minimum of 70 to 130 percent of the test
concentration, unless a wider more appropriate range, based on the nature of the
dosage form (e.g., metered dose inhalers), is justified;
for dissolution testing: +/-20 % over the specified range; e.g., if the specifications for a
controlled released product cover a region from 20%, after 1 hour, up to 90%, after 24
hours, the validated range would be 0-110% of the label claim.
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for the determination of an impurity: from the reporting level of an impurity* to 120%
of the specification; for impurities known to be unusually potent or to produce toxic or
unexpected pharmacological effects, the detection/ quantitation limit should be
commensurate with the level at which the impurities must be controlled.
Note: for validation of impurity test procedures carried out during development, it may
be necessary to consider the range around a suggested (probable) limit;
if assay and purity are performed together as one test and only a 100% standard is
used, linearity should cover the range from the reporting level of the impurities 1 to
120% of the assay specification.
4.
ACCURACY
Accuracy should be established across the specified range of the analytical procedure.
4.1
Assay
4.1.1 Active
Substance
b)
comparison of the results of the proposed analytical procedure with those of a second
well-characterised procedure, the accuracy of which is stated and/or defined
(independent procedure, see 1.2.2);
c)
4.1.2 Medicinal
and specificity
have been
Product
4.2 I m p u r i t i e s (Quantitation)
Accuracy should be assessed on samples (substance/ product) spiked with known amounts of
impurities.
see chapters "Reporting Impurity Content of Batches" of the corresponding Guidelines: "Impurities in New
Active Substances" and "Impurities in New Medicinal
Products"
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e.g.,
4.3 R e c o m m e n d e d D a t a
Accuracy should be assessed using a minimum of 9 determinations over a minimum of 3
concentration levels covering the specified range (e.g. 3 concentrations/ 3 replicates each of
the total analytical procedure).
Accuracy should be reported as percent recovery by the assay of known added amount of
analyte in the sample or as the difference between the mean and the accepted true value
together with the confidence intervals.
5.
PRECISION
Validation of tests for assay and for quantitative determination of impurities includes an
investigation of precision.
5.1
Repeatability
a minimum of 9 determinations covering the specified range for the procedure (e.g.
3 concentrations/3 replicates each)
or
b)
5.2 I n t e r m e d i a t e P r e c i s i o n
The extent to which intermediate precision should be established depends on the
circumstances under which the procedure is intended to be used. The applicant should
establish the effects of random events on the precision of the analytical procedure. Typical
variations to be studied include days, analysts, equipment, etc. It is not considered necessary
to study these effects individually. The use of an experimental design (matrix) is
encouraged.
5.3
Reproducibility
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5.4 R e c o m m e n d e d D a t a
The standard deviation, relative standard deviation (coefficient of variation)
confidence interval should be reported for each type of precision investigated.
6.
and
DETECTION LIMIT
Several approaches for determining the detection limit are possible, depending on whether
the procedure is a non-instrumental or instrumental. Approaches other than those listed
below may be acceptable.
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6.4 R e c o m m e n d e d D a t a
The detection limit and the method used for determining the detection limit should be
presented. If DL is determined based on visual evaluation or based on signal to noise ratio,
the presentation of the relevant chromatograms is considered acceptable for justification.
In cases where an estimated value for the detection limit is obtained by calculation or
extrapolation, this estimate may subsequently be validated by the independent analysis of a
suitable number of samples known to be near or prepared at the detection limit.
7.
QUANTITATION LIMIT
Several approaches for determining the quantitation limit are possible, depending on
whether the procedure is a non-instrumental or instrumental. Approaches other than those
listed below may be acceptable.
7.1 B a s e d on Visual E v a l u a t i o n
Visual evaluation may be used for non-instrumental methods but may also be used with
instrumental methods.
The quantitation limit is generally determined by the analysis of samples with known
concentrations of analyte and by establishing the minimum level at which the analyte can
be quantified with acceptable accuracy and precision.
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3AQ13a
Curve
A specific calibration curve should be studied using samples, containing an analyte in the
range of QL. The residual standard deviation of a regression line or the standard deviation
of y-intercepts of regression lines may be used as the standard deviation.
7.4
Recommended
Data
The quantitation limit and the method used for determining the quantitation limit should be
presented.
The limit should be subsequently validated by the analysis of a suitable number of samples
known to be near or prepared at the quantitation limit.
8.
ROBUSTNESS
The evaluation of robustness should be considered during the development phase and
depends on the type of procedure under study. It should show the reliability of an analysis
with respect to deliberate variations in method parameters.
If measurements are susceptible to variations in analytical conditions, the analytical
conditions should be suitably controlled or a precautionary statement should be included in
the procedure. One consequence of the evaluation of robustness should be that a series of
system suitability parameters (e.g., resolution test) is established to ensure that the validity
of the analytical procedure is maintained whenever used.
Examples of typical variations are:
stability of analytical solutions,
extraction time
In the case of liquid chromatography, examples of typical variations are
influence of variations of pH in a mobile phase,
influence of variations in mobile phase composition,
different columns (different lots and/or suppliers),
temperature,
flow rate.
In the case of gas-chromatography, examples of typical variations are
different columns (different lots and/or suppliers),
temperature,
flow rate.
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9.
System suitability testing is an integral part of many analytical procedures. The tests are
based on the concept that the equipment, electronics, analytical operations and samples to be
analysed constitute an integral system that can be evaluated as such. System suitability test
parameters to be established for a particular procedure depend on the type of procedure being
validated. See Pharmacopoeias for additional information.
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Guideline Title
Legislative basis
Date of first adoption
Date of entry into
force
Status
Previous titles/other
references
Additional Notes
CONTENTS
1.
INTRODUCTION
2.
GLOSSARY
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INTRODUCTION
This document presents a discussion of the characteristics for consideration during the
validation of the analytical procedures included as part of applications submitted within the
EC, Japan and USA. This document does not necessarily seek to cover the testing that may be
required for registration in, or export to, other areas of the world. Furthermore, this text
serves as a collection of terms, and their definitions, and is not intended to provide direction
on how to accomplish validation. These terms and definitions are meant to bridge the
differences that often exist between various compendia and regulators of the EC, Japan and
USA.
The objective of validation of an analytical procedure is to demonstrate that it is suitable for
its intended purpose. A tabular summation of the characteristics applicable to identification,
control of impurities and assay procedures is included. Other analytical procedures may be
considered in future additions to this document.
2.
The discussion of the validation of analytical procedures is directed to the four most common
types of analytical procedures:
-
Identification tests.
A brief description of the types of tests considered in this document is provided below.
-
Identification tests are intended to ensure the identity of an analyte in a sample. This
is normally achieved by comparison of a property of the sample (e.g., spectrum,
chromatographic behaviour, chemical reactivity, etc.) to that of a reference standard.
Testing for impurities can be either a quantitative test or a limit test for the impurity
in a sample. Either test is intended to accurately reflect the purity characteristics of the
sample. Different validation characteristics are required for a quantitative test than
for a limit test.
Assay procedures are intended to measure the analyte present in a given sample. In
the context of this document, the assay represents a quantitative measurement of the
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major component(s) in the substance. For the medicinal product, similar validation
characteristics also apply when assaying for the active or other selected component(s).
The same validation characteristics may also apply to assays associated with other
analytical procedures (e.g., dissolution).
The objective of the analytical procedure should be clearly understood since this will govern
the validation characteristics which need to be evaluated. Typical validation characteristics
which should be considered are listed below:
-
Accuracy
Precision
Repeatability
Intermediate Precision
Specificity
Detection Limit
Quantitation Limit
Linearity
Range
Each of these validation characteristics is defined in the attached Glossary. The table lists
those validation characteristics regarded as the most important for the validation of different
types of analytical procedures. This list should be considered typical for the analytical
procedures cited but occasional exceptions should be dealt with on a case by case basis. It
should be noted that robustness is not listed in the table but should be considered at an
appropriate stage in the development of the analytical procedure.
Furthermore revalidation may be necessary in the following circumstances:
-
The degree of revalidation required depends on the nature of the changes. Certain other
changes may require validation as well.
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TABLE
Type of analytical
procedure
Identification
ASSAY
- dissolution
(measurement only)
- content/potency
Testing for
impurities
quantitat. limit
characteristics
-
Repeatability
Interra. Precision
+ (D
+
Accuracy
Precision
Specificity (2)
+ (1)
+
Detection Limit
-(3)
Quantitation Limit
Linearity
Range
(1)
intermediate
(2)
(3)
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GLOSSARY
1.
ANALYTICAL PROCEDURE
The analytical procedure refers to the way of performing the analysis. It should describe i n
detail the steps necessary to perform each analytical test. This may include but is not limited
to: the sample, the reference standard and the reagents preparations, use of the apparatus,
generation of the calibration curve, use of the formulae for the calculation, etc.
2.
SPECIFICITY
Specificity is the ability to assess unequivocally the analyte in the presence of components
which may be expected to be present. Typically these might include impurities, dgradants,
matrix, etc.
Lack of specificity of an individual analytical procedure may be compensated by other
supporting analytical procedure(s).
This definition has the following implications:
Identification:
Purity Tests:
Assay (content
or potency):
3.
on the
ACCURACY
The accuracy of an analytical procedure expresses the closeness of agreement between the
value which is accepted either as a conventional true value or an accepted reference value
and the value found.
This is sometimes termed trueness.
4.
PRECISION
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4.1.
Repeatability
Repeatability expresses the precision under the same operating conditions over a short
interval of time. Repeatability is also termed intra-assay precision.
4.2
Intermediate
precision
days, different
Reproducibility
5.
DETECTION LIMIT
The detection limit of an individual analytical procedure is the lowest amount of analyte i n
a sample which can be detected but not necessarily quantitated as an exact value.
6.
QUANTITATION LIMIT
The quantitation limit of an individual analytical procedure is the lowest amount of analyte
in a sample which can be quantitatively determined with suitable precision and accuracy.
The quantitation limit is a parameter of quantitative assays for low levels of compounds i n
sample matrices, and is used particularly for the determination of impurities and/or
degradation products.
7.
LINEARITY
The linearity of an analytical procedure is its ability (within a given range) to obtain test
results which are directly proportional to the concentration (amount) of analyte in the
sample.
8.
RANGE
The range of an analytical procedure is the interval between the upper and lower
concentration (amounts) of analyte in the sample (including these concentrations) for which
it has been demonstrated that the analytical procedure has a suitable level of precision,
accuracy and linearity.
9.
ROBUSTNESS
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CONTENTS
PREAMBLE
OBJECTIVE
SCOPE
ACTrVE SUBSTANCE
PRODUCT
ANNEX I
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OBJECTRHE
The purpose of stability testing is to provide evidence on how the quality of a an active
substance or medicinal product varies with time under the influence of a variety of
environmental factors such as temperature, humidity and light, and enables recommended
storage conditions, re-test periods and shelf lives to be established.
SCOPE
The guideline primarily addresses the information required in marketing authorisations
for new active substances and associated medicinal products.
This guideline does not currently seek to cover the information required for abbreviated or
abridged applications, variations, clinical trial applications, etc.
The choice of test conditions defined in this guideline is based on an analysis of the effects
of climatic conditions in the three areas of the EC, Japan and the USA. The mean kinetic
temperature in any region of the world can be derived from climatic data (Grimm, W.
Drugs Made in Germany, 28, 196-202, 1985 and 29, 39-47, 1986).
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ACTrVE SUBSTANCE
General
Information on the stability of the active substance is an integral part of the systematic
approach to stability evaluation.
Stress Testing
Stress testing helps to determine the intrinsic stability of the molecule by establishing
degradation pathways in order to identify the likely degradation products and to validate the
stability indicating power of the analytical procedures used.
Formal Studies
Primary stability studies are intended to show that the active substance will remain within
specification during the retest period if stored under recommended storage conditions.
Selection of Batches
Stability information from accelerated and long term testing is to be provided on at least
three batches. The long term testing should cover a minimum of 12 months duration on at
least three batches at the time of submission.
The batches manufactured to the minimum of pilot plant scale should be by the same
synthetic route and use a method of manufacture and procedure that simulates the final
process to be used on a manufacturing scale.
The overall quality of the batches of active substance placed on stability should be
representative of both the quality of the material used in preclinical and clinical studies
and the quality of material to be made on a manufacturing scale.
Supporting information may be provided using stability data on batches of active substance
made on a laboratory scale.
The first three production batches of active substance manufactured post approval, if not
submitted in the original marketing authorisation application, should be placed on long term
stability studies using the same stability protocol as in the approved marketing authorisation
application.
Specification
Limits of acceptability should be derived from the profile of the material as used in the pre
clinical and degradation products, the justification for which should be influenced by the
levels observed in material used in preclinical studies and clinical trials.
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Storage Conditions
The length of the studies and the storage conditions should be sufficient to cover storage
shipment and subsequent use. Application of the same storage conditions as applied to the
medicinal product will facilitate comparative review and assessment. Other storage
conditions are allowable if justified. In particular, temperature sensitive active substances
should be stored under an alternative, lower temperature condition which will then become
the designated long term testing storage temperature. The six months accelerated testing
should then be carried out at a temperature at least 15C above this designated long term
storage temperature (together with the appropriate relative humidity conditions for that
temperature). The designated long term testing conditions will be reflected in the labelling
and re-test date.
Conditions
12 months
Accelerated Testing
6 months
Where 'significant change' occurs during six months storage under conditions of
accelerated testing at 40C2C/75 percent RH5 percent, additional testing at an
intermediate condition (such as 30C2C/60 percent RH5 percent) should be conducted for
active substances to be used in the manufacture of dosage forms tested long term at 25C/60
percent RH and this information included in the marketing authorisation application. The
initial marketing authorisation application should include a minimum of 6 months data
from a 12 months study.
'Significant change' at 40C/75 percent RH or 30C/60 percent RH is defined as failure to
meet the specification.
The long term testing will be continued for a sufficient period of time beyond 12 months to
cover all appropriate re-test periods, and the further accumulated data can be submitted to the
Authorities during the assessment period of the marketing authorisation application.
The data (from accelerated testing at an intermediate condition) may be used to evaluate the
impact of short term excursions outside the label storage conditions such as might occur
during shipping.
Testing FrequencyFrequency of testing should be sufficient to establish the stability characteristics of the active
substance. Testing under the defined long term conditions will normally be every three
months, over the first year, every six months over the second year and then annually.
Packaging/Containers
The containers to be used in the long term, real time stability evaluation should be the same
as or simulate the actual packaging used for storage and distribution
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Evaluation
The design of the stability study is to establish, based on testing a minimum of three batches
of the active substance and evaluating the stability information (covering as necessary the
physical, chemical and microbiological test characteristics), a retest period applicable to all
future batches of the bulk substance manufactured under similar circumstances. The degree
of variability of individual batches affects the confidence that a future production batch will
remain within specification until the retest date.
An acceptable approach for quantitative characteristics that are expected to decrease with
time is to determine the time at which the 95% one-sided confidence limit for the mean
degradation curve intersects the acceptable lower specification limit. If analysis shows that
the batch to batch variability is small, it is advantageous to combine the data into one overall
estimate and this can be done by first applying appropriate statistical tests (for example,
values for level of significance of rejection of more than 0.25) to the slopes of the regression
lines and zero time intercepts for the individual batches. If it is inappropriate to combine
data from several batches, the overall retest period may depend on the minimum time a
batch may be expected to remain within acceptable and justified limits.
The nature of any degradation relationship will determine the need for transformation of
the data for linear regression analysis. Usually the relationship can be represented by a
linear, quadratic or cubic function on an arithmetic or logarithmic scale. Statistical methods
should be employed to test the goodness of fit of the data on all batches and combined batches
(where appropriate) to the assumed degradation line or curve.
The data may show so little degradation and so little variability that it is apparent from
looking at the data that the requested retest period will be granted. Under the circumstances,
it is normally unnecessary to go through the formal statistical analysis but merely to
provide a full justification for the omission.
Limited extrapolation of the real time data beyond the observed range to extend expiration
dating at approval time, particularly where the accelerated data supports this, may be
undertaken. However, this assumes that the same degradation relationship will continue to
apply beyond the observed data and hence the use of extrapolation must be justified in each
application in terms of what is known about the mechanism of degradation, the goodness of
fit of any mathematical model, batch size, existence of supportive data etc.
Any evaluation should cover not only the assay, but the levels of degradation products and
other appropriate attributes.
Statements/Labelling
A storage temperature range may be used in accordance with relevant national/regional
requirements. The range should be based on the stability evaluation of the active substance.
Where applicable, specific requirements should be stated, particularly for active substances
that cannot tolerate freezing. The use of terms such as 'ambient conditions' or 'room
temperature' is unacceptable.
A re-test period should be derived from the stability information.
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PRODUCT
General
The design of the stability program for the finished product should be based on the knowledge
of the behaviour and properties of the active substance and the experience gained from
clinical formulation studies and from the stability studies on the active substance. The
likely changes on storage and the rationale for the selection of product variables to include
in the testing programme should be stated.
Selection of Batches
Stability information from accelerated and long term testing is to be provided on three
batches of the same formulation and dosage form in the containers and closure proposed for
marketing. Two of these three batches should be at least pilot scale. The third one may be
smaller (e.g., 25,000 to 50,000 tablets or capsules for solid oral dosage forms). The long term
testing should cover at least 12 months duration at the time of submission. The
manufacturing process to be used should meaningfully simulate that which would be applied
to large scale batches for marketing. The process should provide medicinal product of the
same quality intended for marketing, and meeting the same quality specification as to be
applied for release of material. Where possible, batches of the finished product should be
manufactured using identifiably different batches of active substance.
Data on the laboratory scale batches is not acceptable as primary stability information. Data
on associated formulations or packaging may be submitted as supportive information. The
first three production batches manufactured post approval, if not submitted in the original
marketing authorisation application, should be placed on accelerated and long term stability
studies using the same stability protocols as in the approved marketing authorisation.
Specifications
Limits of acceptance should relate to the release limits (where applicable), to be derived from
consideration of all the available stability information. The shelf life specification could
allow acceptable and justifiable derivations from the release specification based on the
stability evaluation and the changes observed on storage. It will need to include specific
upper limits for degradation products, the justification for which should be influenced by the
levels observed in material used in pre-clinical studies and clinical trials. The justification
for the limits proposed for certain other tests such as particle size and/or dissolution rate will
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require reference to the results observed for batch(es) used in bioavailability and/or clinical
studies. Any differences between the release and shelf life specifications for antimicrobial
preservatives should be supported by preservative efficacy testing.
25C2C/60% RH5%
12 months
Accelerated Testing
40C2C/75% RH5%
6 months
2.
3.
4.
5.
Failure to meet specifications for appearance and physical properties e.g., colour, phase
separation, resuspendibility, delivery per actuation, caking, hardness, etc.
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Should significant change occur at 40C/75 percent RH then the initial marketing
authorisation application should include a minimum of 6 months data from an ongoing one
year study at 30C/60 percent RH; the same significant change criteria shall then apply.
The long term testing will be continued for a sufficient time beyond 12 months to cover shelf
life at appropriate test periods. The further accumulated data should be submitted to the
authorities during the assessment period of the marketing authorisation application.
The first three production batches manufactured post approval, if not submitted in the
original marketing authorisation application, should be placed on accelerated and long term
stability studies using the same stability protocol as in the approved marketing
authorisation.
Testing Frequency
Frequency of testing should be sufficient to establish the stability characteristics of the
medicinal product. Testing will normally be every three months over the first year, every
six months over the second year and then annually.
The use of matrixing or bracketing can be applied, if justified.(See Glossary).
Packaging Materials
The testing should be carried out in the final packaging proposed for marketing. Additional
testing of unprotected finished product can form a useful part of the stress testing and pack
evaluation, as can studies carried out in other related packaging materials in supporting the
definitive pack(s).
Evaluation
A systematic approach should be adopted in the presentation and evaluation of the stability
information which should cover as necessary physical, chemical, biological, microbiological
quality characteristics, including particular properties of the dosage form (for example
dissolution rate for oral solid dose forms).
The design of the stability study is to establish, based on testing a minimum of three batches
of the finished product, a shelf life and label storage instructions applicable to all future
batches of the dosage form manufactured and packed under similar circumstances. The
degree of variability of individual batches affects the confidence that a future production
batch will remain within specification until the expiration date.
An acceptable approach for quantitative characteristics that are expected to decrease with
time is to determine the time at which the 95% one-sided confidence limit for the mean
degradation curve intersects the acceptable lower specification limit. If analysis shows that
the batch to batch variability is small, it is advantageous to combine the data into one overall
estimate and this can be done by first applying appropriate statistical tests (for example,
values for level of significance of rejection of more than 0.25) to the slopes of the regression
lines and zero time intercepts for the individual batches. If it is inappropriate to combine
data from several batches, the overall shelf life may depend on the minimum time a batch
may be expected to remain within acceptable and justified limits.
The nature of the degradation relationship will determine the need for transformation of the
data for linear regression analysis. Usually the relationship can be represented by a linear,
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Statements/Labelling
A storage temperature range may be used in accordance with relevant national/regional
requirements. The range should be based on the stability evaluation of the medicinal
product. Where applicable, specific requirements should be stated particularly for medicinal
products that cannot tolerate freezing.
The use of terms such as 'ambient conditions' or 'room temperature' is unacceptable.
There should be a direct linkage between the label statement and the demonstrated stability
of the medicinal product.
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ANNEX 1
Glossary and Information
The following terms have been in general use and the following definitions are provided to
facilitate interpretation of the guideline.
Accelerated Testing
Studies designed to increase the rate of chemical degradation or physical change of an active
substance or medicinal product by using exaggerated storage conditions as part of the
formal, definitive, storage programme.
These data, in addition to long term stability studies, may also be used to assess longer term
chemical effects at non-accelerated conditions and to evaluate the impact of short term
excursions outside the label storage conditions such as might occur during shipping. Results
from accelerated testing studies are not always predictive of physical changes.
Bracketing
The design of a stability schedule so that at any time point only the samples on the extremes,
for example of container size and/or dosage strengths, are tested. The design assumes that
the stability of the intermediate condition samples are represented by those at the extremes.
Where a range of dosage strengths is to be tested, bracketing designs may be particularly
applicable if the strengths are very closely related in composition (e.g., for a tablet range
made with different compression weights of a similar basic granulation, or a capsule range
made by filling different plug fill weights of the same basic composition into different size
capsule shells). Where a range of sizes of immediate containers are to be evaluated,
bracketing designs may be applicable if the material of composition of the container and the
type of closure are the same throughout the range.
Climatic Zones
The concept of dividing the world into four zones based on defining the prevalent annual
climatic conditions.
Dosage Form; Preparation
A pharmaceutical product type, for example tablet, capsule, solution, cream etc. that contains
an active substance generally, but not necessarily, in association with excipients.
Medicinal Product; Finished Product
The dosage form in the final immediate packaging intended for marketing.
Excipient
Anything other than the active substance in the dosage form.
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Expiry/Expiration Date
The date placed on the container/labels of a product designating the time during which a
batch of the product is expected to remain within the approved shelf life specification if stored
under defined conditions, and after which it must not be used.
Formal (Systematic) Studies
Formal studies are those undertaken to a pre-approval stability protocol which embraces the
principles of these guidelines.
Long Term (Real Time) Testing
Stability evaluation of the physical, chemical, biological and microbiological characteristics
of a product and an active substance, covering the expected duration of the shelf life and retest period, which are claimed in the submission and will appear on the labelling.
Mass Balance; Material Balance
The process of adding together the assay value and levels of degradation products to see how
closely these add up to 100 per cent of the initial value, with due consideration of the margin
of analytical precision.
This concept is a useful scientific guide for evaluating data but it is not achievable in all
circumstances. The focus may instead be on assuring the specificity of the assay, the
completeness of the investigation of routes of degradation, and the use, if necessary, of
identified dgradants as indicators of the extent of degradation via particular mechanisms.
Matrixing
The statistical design of a stability schedule so that only a fraction of the total number of
samples are tested at any specified sampling point. At a subsequent sampling point, different
sets of samples of the total number would be tested. The design assumes that the stability of
the samples tested represents the stability of all samples. The differences in the samples for
the same product should be identified as, for example, covering different batches, different
strengths, different sizes of the same container and closure and possibly in some cases
different container/closure systems.
Matrixing can cover reduced testing when more than one variable is being evaluated. Thus
the design of the matrix will be dictated by the factors needing to be covered and evaluated.
This potential complexity precludes inclusion of specific details and examples, and it may
be desirable to discuss design in advance with the Regulatory Authority, where this is
possible. In every case it is essential that all batches are tested initially and at the end of the
long term testing.
Mean Kinetic Temperature
When establishing the mean value of the temperature, the formula of J. D. Haynes (J.
Pharm. Sci. 60, 927-929, 1971) can be used to calculate the mean kinetic temperature. It is
higher than the arithmetic mean temperature and takes into account the Arrhenius equation
from which Haynes derived his formula.
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Re-Test Period
The period of time during which the active substance can be considered to remain within the
specification and therefore acceptable for use in the manufacture of a given
product,
provided that it has been stored under the defined conditions; after this period, the batch
should be retested for compliance with specification and then used immediately.
Shelf life; Expiration Dating Period
The time interval that a product is expected to remain within the approved shelf life
specification provided that it is stored under the conditions defined on the label in the
proposed containers and closure.
Specification - Release
The combination of physical, chemical, biological and microbiological test requirements
that determine a product is suitable for release at the time of its manufacture.
Specification - Check/Shelf life
The combination of physical, chemical, biological and microbiological test requirements
that a active substance must meet up to its re-test date or a product must meet throughout its
shelf life.
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provided to
Light testing should be an integral part of stress testing. [The standard conditions for light
testing are discussed in the note for guidance Photostability Testing of New Active Substances
and Medicinal Products.]
It is recognised that some degradation pathways can be complex and that under forcing
conditions decomposition products may be observed which are unlikely to be formed under
accelerated or long term testing. This information may be useful in developing and
validating suitable analytical methods, but it may not always be necessary to examine
specifically for all degradation products, if it has been demonstrated that in practice these
are not formed.
Stress Testing (Product)
Light testing should be an integral part of stress testing (see note for guidance
Photostability Testing of New Active Substances and Medicinal Products).
on
Special test conditions for specific products (e.g., metered dose inhalations and creams and
emulsions) may require additional stress studies.
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Guideline Title
Legislative basis
Date of first adoption
Date of entry into
force
Status
Previous titles/other
references
Additional Notes
CONTENTS
1.
INTRODUCTION
2.
3.
ANNEX
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Note for guidance concerning the application of Part 2, section F of the Annex to Directive
75/318/EEC, as amended, with a view to the granting of a marketing authorisation for a new
medicinal product.
INTRODUCTION
This note concerns research enabling the applicant to determine what shelf-life to propose.
The purpose of stability tests is to obtain information which enables proposals to be made for
the shelf-life of the medicinal product and to recommend storage conditions.
The quality of a medicinal product is determined by its content of active substance(s), its
purity (limitation or absence of decomposition products of the active substance(s)) and its
organoleptic, physico-chemical and microbiological properties.
The purpose of the stability studies is to ascertain how the quality of a medicinal product
varies as a function of time and under the influence of a variety of environmental factors.
On the basis of the information thus obtained, storage conditions are recommended (the
purpose of these studies is to produce recommendations) which will guarantee maintenance
of the quality of the medicinal product, in relation to its safety, efficacy and acceptability,
throughout the proposed shelf-life (i.e. during storage, distribution, dispensing and use).
The design of the finished product stability studies for a medicinal product is based on the
knowledge obtained from the studies on the active substance and from the Development
Pharmaceutics studies.
2.
Where there are several possible active ingredient manufacturers and/or methods of
obtaining the active ingredient, consideration may need to be given to the examination of
studies on material from each source.
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the number of batches (minimum of two), together with the batch identification number,
date of manufacture and size of each batch and the name of the manufacturer.
details of the accelerated and proposed storage conditions used, taking into account the
physical and chemical properties of the substance: temperature, humidity and light;
exposure to air and chemical agents
(particularly in solution in water and/or other
solvents);
the assay techniques used, normally with evidence from the analytical validation
studies of their specificity
(i.e. that they separate the active ingredient from its
degradation products);
2.4 Results
-
2.5 Interpretation
-
the conclusions as to the most appropriate storage conditions for the active ingredient,
and the duration of storage before the substance needs retesting to check for compliance
with specification;
as
2.6 Conclusions
The stability data on the active ingredient and the development studies enable a preliminary
choice of the formulation and packing material to be made. They also enable some
consideration to be given to the choice of analytical methods and test conditions for the
studies on the pharmaceutical form.
3.1 Objective
The analysis of the results of the stability studies on the finished product should allow the
determination of the shelf-life, the recommendations for storage conditions (where relevant
before and after opening the container), and the justification of any overage of the active
ingredient applied to guarantee the potency at the end of the shelf-life.
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The design of the stability tests is based on the known properties of the active ingredient (see
section 2), the results of the Development Pharmaceutics studies, the properties of the chosen
formulation and the recommendations for use of the product.
The specifications proposed at the time of manufacture and to the end of the proposed shelflife must reflect, as far as possible, the results of the stability studies, particularly in relation
to any parameters which could have a bearing on efficacy and safety and product
acceptability.
Specific in-use stability tests must be carried out where the product is labile once the
container is opened, or where the product is to be diluted or reconstituted before use.
studies
These studies should be carried out under a range of controlled test conditions which will
enable the shelf-life and the product container label/package insert storage requirements to
be defined. This will normally include studies which will allow the properties of the product
at temperatures between 20C and 30C to be evaluated. However, 25C should be used as the
mean kinetic testing temperature for products in the European Union.
For each study, the mean temperature, the ranges of temperature and the mean humidity
should be stated.
conditions
Such studies should be carried out to provide essential additional information. They can
fulfil a number of objectives such as:
-
to support the initial shelf-life request, by complementing the limited results of the
early real-time studies as the decomposition, if it is occurring, is likely to be
accelerated,
to demonstrate the effects of adverse storage on the packaging and product, and to
enable storage conditions and suitable labelling to be provided,
to support major
preparation.
changes
in
formulation,
packaging
materials
or method of
The various test conditions should be stated. Depending on the nature and objectives of the
stability study, the following may need to be considered:
-
various test temperatures: three or more (see 3.7) particularly if long term real-time
data is unavailable. The effect of low temperatures may, in addition, need to be
considered such as below -15C (freezer), 2-8C (refrigerator) and freeze-thaw recycling,
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tested
The number of batches tested must be stated, with the batch number, details of the
composition, date of manufacture, the size of the batch and the batch number and name of the
manufacturer of the active ingredient(s) used. These should include where possible
production scale batches of the product.
Normally at least 3 batches of the dosage form must be studied; however in circumstances
where the active substance is an existing one (already used in licensed products), and it is
found that the active substance is stable in the product and no significant decomposition
products are formed, the stability study can be limited to 2 batches.
3.3.2 Immediate
packaging
The batches of product must be packaged in the way proposed for marketing. However,
supplementary evidence from batches of product in related packs must be used to augment
this data.
Details of the packaging should be stated as:
-
3.4 Characteristics
The characteristics studied should be:
-
those in the finished product specification that are likely to be affected by storage, and
those not monitored routinely at the time of manufacture, but which may be indicative
of the stability/instability of the particular dosage form, e.g. dissolution of tablets.
and microbiological
aspects of the
finished
organoleptic properties,
important quality parameters such as the in vitro dissolution test, moisture content
(e.g. in relation to any dessicant used in the packaging and particle size,
efficacy of preservatives at the end of the reported storage test period or at the end of the
shelf-life, except where otherwise justified,
any other physical characteristics of the finished product that must be known in order
to assess the product stability.
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3.4.3 Characteristics
-
of the packaging
to be considered
study of the container and closure interaction with the contents in any case where this
is a risk.
3.5 E v a l u a t i o n m e t h o d s
The test methods used must be fully described. It must be shown that they are capable of
detecting decomposition of the active ingredient in the medicinal product, and unless
justified, of quantifying any decomposition products. If possible, the assay method for the
active substance in the finished product should be stability indicating.
The test procedures applied to the stability tests on the finished product must be validated.
3.6 P r e s e n t a t i o n of r e s u l t s
The results should summarised (e.g. as tables and graphs). For each batch of product, the
initial results (at the time of manufacture), the results during storage and at the end of the
proposed shelf -line should be given. However, results of real time data should be supplied as
they become available.
3.7 D i s c u s s i o n , i n t e r p r e t a t i o n a n d c o n c l u s i o n s
The discussion in the Expert Report should provide a critical evaluation of the suitability of
the test methods used, the results obtained and proposed shelf-life specification.
If necessary to carry out any further studies due to significant changes in physical
properties, an explanation should be given, together with the results of these studies.
Studies under accelerated test conditions will increase the decomposition and may permit
some extrapolation of the room temperature shelf-life from that which would otherwise be
acceptable. However, such studies would always need to be supplemented by long-term real
time studies, and normally at least
6 month real time data should be presented in the
application for marketing authorisation.
If batches of the product demonstrate a different stability profile, the shelf-life proposed and
any overage should be based on the stability of the least stable, unless an explanation can be
given.
The shelf-life should be proposed for the product as packaged for sale. If necessary, a
recommendation should also be given regarding the shelf-life before and after opening the
container, and after dilution or reconstitution, and storage during marketing and use.
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If there is evidence that batches of the stored product as packed for sale are stable at
temperatures up to 30C, the product need bear no special temperature storage instructions.
However, if there is evidence that the product must be stored under defined conditions of
storage, this must be stated on the container label and the package insert (if included). The
maximum (or minimum) storage temperature should be stated in Celsius (e.g. store below
25C, store in a refrigerator at 2-8C, do not refrigerate - store above 8C). These
label/package insert storage recommendations must fully reflect conditions found in the
Member State for which marketing authorisation is sought (see Annex), and those in any
other Member State to which the product is likely to be supplied (whether by the person
responsible for placing the product on the market or another person).
would normally necessitate the results of comparative accelerated or long term stability
studies being provided to the competent authorities before such changes are introduced.
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ANNEX
International Climatic Zones and Climatic Conditions
Zone I
Temperate
Zone II
Mediterranean
(sub-tropical)
Zone III
Hot/dry or
Hot/moderate RH
Mean Annual
Temperature
<20C
20.5-24C
>24C
>24C
Kinetic Mean
Temperature
(Virtual
temperature)
21C
26C
31C
31C
45%
60%
40%
70%
Climatic
Condition
Mean Annual
Relative
Humidity
Zone rV
Vey hot/humid
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Guideline Title
Legislative basis
Date of first adoption
Date of entry into
force
Status
Previous titles/other
references
Additional Notes
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GENERAL
This document is an annex to the note for guidance: Stability Testing of New Active
Substances and Medicinal Products and addresses the recommendations on the data which
should be submitted regarding stability of new dosage forms by the owner of the original
application, after the original submission for new active substances and medicinal products.
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Guideline Title
Legislative basis
Date of first adoption
Date of entry into
force
Status
Previous titles/other
references
Additional Notes
CONTENTS
1.
GENERAL
2.
SUBSTANCES
3.
MEDICINAL PRODUCT
4.
ANNEX
5.
GLOSSARY
6.
REFERENCES
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GENERAL
The note for guidance on Stability Testing of New Active Substances and Medicinal Products,
originally published as an ICH Harmonised Tripartite Guideline (hereafter referred to as
the Parent Guideline) notes that light testing should be an integral part of stress testing. This
document is an annex to the Parent Guideline and addresses the recommendations for
photostability testing.
II
Preamble
The intrinsic photostability characteristics of new active substances and medicinal products
should be evaluated to demonstrate that, as appropriate, light exposure does not result i n
unacceptable change. Normally, photostability testing is carried out on a single batch of
material selected as described under Selection of Batches in the Parent Guideline. Under
some circumstances these studies should be repeated if certain changes are made to the
product (e.g., formulation, packaging). Whether these studies should be repeated depends on
the photostability characteristics determined at the time of first submission of an application
and the type of change made.
The guideline primarily addresses the generation of photostability information for
submission in applications for marketing authorisations for new active substances and
associated medicinal products. The guideline does not cover the photostability of medicinal
products after administration (i.e. under conditions of use) and those applications not
covered by the Parent Guideline. Alternative approaches may be used if they are
scientifically sound and justification is provided.
A systematic approach to photostability testing is recommended covering, as appropriate,
studies such as:
i)
ii)
iii)
iv)
The extent of product testing should be established by assessing whether or not acceptable
change has occurred at the end of the light exposure testing as described in the Decision Flow
Chart for Photostability Testing of Medicinal Products. Acceptable change is change within
limits justified by the applicant.
The formal labelling requirements for photolabile active substances and products are
established by national requirements.
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1.2
Light sources
The light sources described below may be used for photostability testing. The applicant should
either maintain an appropriate control of temperature to minimise the effect of localised
temperature changes or include a dark control in the same environment unless otherwise
justified. For both options 1 and 2, a pharmaceutical manufacturer/applicant may rely on the
spectral distribution specification of the light source manufacturer.
Option 1
Any light source that is designed to produce an output similar to the D65/ID65 emission
standard such as an artificial daylight fluorescent lamp combining visible and ultraviolet
(UV) outputs, xenon, or metal halide lamp. D65 is the internationally recognised standard
for outdoor daylight as defined in ISO 10977 (1993). ID65 is the equivalent indoor indirect
daylight standard. For a light source emitting significant radiation below 320 nm, an
appropriate filter(s) may be fitted to eliminate such radiation.
Option 2
For option 2 the same sample should be exposed to both the cool white fluorescent and near
ultraviolet lamp.
1.
A cool white fluorescent lamp designed to produce an output similar to that specified i n
ISO 10977(1993); and
2.
A near UV fluorescent lamp having a spectral distribution from 320 nm to 400 nm with
a maximum energy emission between 350 nm and 370 nm; a significant proportion of
UV should be in both bands of 320 to 360 nm and 360 to 400 nm.
1.3
Procedure
2.
ACTIVE SUBSTANCE
For active substances, photostability testing should consist of two parts: forced degradation
testing and confirmatory testing.
The purpose of forced degradation testing studies is to evaluate the overall photosensitivity of
the material for method development purposes and/or degradation pathway elucidation. This
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testing may involve the active substance alone and/or in simple solutions/suspensions to
validate the analytical procedures. In these studies, the samples should be in chemically
inert and transparent containers. In these forced degradation studies, a variety of exposure
conditions may be used, depending on the photosensitivity of the active substance involved
and the intensity of the light sources used. For development and validation purposes it is
appropriate to limit exposure and end the studies if extensive decomposition occurs. For
photostable materials, studies may be terminated after an appropriate exposure level has been
used. The design of these experiments is left to the applicant's discretion although the
exposure levels used should be justified.
Under forcing conditions, decomposition products may be observed that are unlikely to be
formed under the conditions used for confirmatory studies. This information may be useful
in developing and validating suitable analytical methods. If in practice it has been
demonstrated they are not formed in the confirmatory studies, these degradation products
need not be further examined.
Confirmatory studies should then be undertaken to provide the information necessary for
handling, packaging, and labelling (see section 1.3, Procedure, and 2.1, Presentation, for
information on the design of these studies).
Normally, only one batch of active substance is tested during the development phase, and
then the photostability characteristics should be confirmed on a single batch selected as
described in the Parent Guideline if the active substance is clearly photostable or photolabile.
If the results of the confirmatory study are equivocal, testing of up to two additional batches
should be conducted. Samples should be selected as described in the Parent Guideline.
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Where solid active substance samples are involved, sampling should ensure that a
representative portion is used in individual tests. Similar sampling considerations, such as
homogenisation of the entire sample, apply to other materials that may not be homogeneous
after exposure. The analysis of the exposed sample should be performed concomitantly with
that of any protected samples used as dark controls if these are used in the test.
2.3 E v a l u a t i o n of r e s u l t s
The forced degradation studies should be designed to provide suitable information to develop
and validate test methods for the confirmatory studies. These test methods should be capable
of resolving and detecting photolytic dgradants that appear during the confirmatory studies.
When evaluating the results of these studies, it is important to recognise that they form part
of the stress testing and are not therefore designed to establish qualitative or quantitative
limits for change.
The confirmatory studies should identify precautionary measures needed in manufacturing
or in formulation of the product, and if light resistant packaging is needed. When
evaluating the results of confirmatory studies to determine whether change due to exposure to
light is acceptable, it is important to consider the results from other formal stability studies
in order to assure that the active substance will be within justified limits at time of use (see
the relevant Stability and Impurity Guidelines).
3.
MEDICINAL PRODUCT
Normally, the studies on products should be carried out in a sequential manner starting with
testing the fully exposed product then progressing as necessary to the product in the
immediate pack and then in the marketing pack. Testing should progress until the results
demonstrate that the product is adequately protected from exposure to light. The product
should be exposed to the light conditions described under the procedure in section 1.3.
Normally, only one batch of product is tested during the development phase, and then the
photostability characteristics should be confirmed on a single batch selected as described i n
the Parent Guideline if the product is clearly photostable or photolabile. If the results of the
confirmatory study are equivocal, testing of up to two additional batches should be conducted.
For some products where it has been demonstrated that the immediate pack is completely
impenetrable to light, such as aluminium tubes or cans, testing should normally only be
conducted on directly exposed product.
It may be appropriate to test certain products such as infusion liquids, dermal creams, etc., to
support their photostability in-use. The extent of this testing should depend on and relate to the
directions for use, and is left to the applicant's discretion.
The analytical procedures used should be suitably validated.
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interactions between the samples and any material used for containers or for general
protection of the sample should also be considered and eliminated wherever not relevant to
the test being carried out.
Where practicable when testing samples of the product outside of the primary pack, these
should be presented in a way similar to the conditions mentioned for the active substance.
The samples should be positioned to provide maximum area of exposure to the light source.
For example, tablets, capsules, etc., should be spread in a single layer.
If direct exposure is not practical (e.g., due to oxidation of a product), the sample should be
placed in a suitable protective inert transparent container (e.g., quartz).
If testing of the product in the immediate container or as marketed is needed, the samples
should be placed horizontally or transversely with respect to the light source, whichever
provides for the most uniform exposure of the samples. Some adjustment of testing conditions
may have to be made when testing large volume containers (e.g., dispensing packs).
3.2 A n a l y s i s of s a m p l e s
At the end of the exposure period, the samples should be examined for any changes in
physical properties (e.g., appearance, clarity or colour of solution, dissolution/disintegration
for dosage forms such as capsules, etc.) and for assay and dgradants by a method suitably
validated for products likely to arise from photochemical degradation processes.
When powder samples are involved, sampling should ensure that a representative portion is
used in individual tests. For solid oral dosage form products, testing should be conducted on
an appropriately sized composite of, for example, 20 tablets or capsules. Similar sampling
considerations, such as homogenisation or solubilisation of the entire sample, apply to other
materials that may not be homogeneous after exposure (e.g., creams, ointments, suspensions,
etc.). The analysis of the exposed sample should be performed concomitantly with that of any
protected samples used as dark controls if these are used in the test.
4.
ANNEX
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Option 1
Put 10 millilitres (ml) of the solution into a 20 ml colourless ampoule seal it hermetically,
and use this as the sample. Separately, put 10 ml of the solution into a 20 ml colourless
ampoule (see note 1), seal it hermetically, wrap in aluminium foil to protect completely from
light, and use this as the control. Expose the sample and control to the light source for a n
appropriate number of hours. After exposure determine the absorbances of the sample (AT)
and the control (A) at 400 nm. Calculate the change in absorbance, A = Ap - A0. The length
of exposure should be sufficient to ensure a change in absorbance of at least 0.9.
Option 2
Fill a 1 cm quartz cell and use this as the sample. Separately fill a 1 cm quartz cell, wrap in
aluminium foil to protect completely from light, and use this as the control. Expose the
sample and control to the light source for an appropriate number of hours. After exposure
determine the absorbances of the sample (AT) and the control (A0) at 400 nm. Calculate the
change in absorbance, A = AT - AQ. The length of exposure should be sufficient to ensure a
change in absorbance of at least 0.5.
Alternative packaging configurations may be used if appropriately validated. Alternative
validated chemical actinometers may be used.
Note 1:
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5.
GLOSSARY
Immediate (primary) pack is that constituent of the packaging that is in direct contact with
the active substance or finished product, and includes any appropriate label.
Marketing pack is the combination of immediate packaging and other secondary packaging
such as a carton.
Forced degradation testing studies are those undertaken to degrade the sample deliberately.
These studies, which may be undertaken in the development phase normally on the active
substances, are used to evaluate the overall photosensitivity of the material for method
development purposes and/or degradation pathway elucidation.
Confirmatory studies are those undertaken to establish photostability characteristics under
standardised conditions. These studies are used to identify precautionary measures needed
in manufacturing or formulation and whether light resistant packaging and/or special
labelling is needed to mitigate exposure to light. For the confirmatory studies, the batch(es)
should be selected according to batch selection for long-term and accelerated testing which is
described in the Parent Guideline.
6.
REFERENCES
Quinine Actinometry as a method for calibrating ultraviolet radiation intensity in lightstability testing of pharmaceuticals.
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Guideline Title
Legislative basis
Date of first adoption
Date of entry into
force
Status
Previous titles/other
references
Additional Notes
CONTENTS
1.
INTRODUCTION
2.
DEFINITIONS
3.
DEVELOPMENT PHARMACEUTICS
4.
5.
CONTROL TESTS
6.
STABILITY
7.
CHANGES TO PRODUCTS
167
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INTRODUCTION
Pharmaceutical dosage forms may be developed in which the rate of release of active
substance(s) has in some way been modified compared with conventional formulations. Such
modification in release of active substances may have a number of objectives, but the
intention of this note for guidance is to cover those formulations in which the release of the
active substance is prolonged in some way in order to maintain therapeutic activity, to
reduce toxic effects or for some other therapeutic purpose.
The details required in the application for marketing authorisation will reflect:
-
and data must be provided in the various sections of the dossier in support of the application
taking into account these various requirements. The note for guidance will cover the various
parts 2.A to 2.F of the application for marketing authorisation and highlight areas which
need to be addressed. It is clear therefore that this note for guidance will cross-refer to other
quality guidelines, to the Notice to Applicants and in particular to the note for guidance
Clinical Testing of Prolonged Action Forms with Special Reference to Extended Release Forms.
The note for guidance concerns quality aspects, including in vitro testing, of oral solid
dosage forms in which release of active substance forms the rate-limiting step in absorption.
While the note for guidance is intended to be specific for prolonged release oral solid dosage
forms, many of the principles discussed will be relevant to other prolonged action dosage
forms intended for administration via other routes.
2.
DEFINITIONS
It is important to clearly define the terminology used to describe the different types of release
models to which the note for guidance relates. The definitions of various types of release
characteristics should be considered in relation to the pharmacokinetic properties and to the
therapeutic intention of the formulation (see note for guidance Clinical Testing of Prolonged
Action Forms with Special Reference to Extended Release Forms), and are correlated as
closely as possible with pharmacopoeial definitions. The more general terms "prolonged
release" can be applied to a range of different release models; terms such as "controlled
release" and "sustained release" are looser than those defined and should be avoided.
169
3AQ19a
rate or place at
wide range of
and "prolonged
pharmaceutical
release
A modified release product in which the release of active substance is delayed for a finite
"lag time", after which release is unhindered [e.g. enteric coated or "Gastro resistant" (Ph.
Eur.) oral tablets or capsules which remain intact in the stomach and only disintegrate in
the higher pH of the small intestine]. Delayed release results in a longer Tmax but with
Tmax and elimination half life unchanged.
2.1.2 Prolonged
release
A product in which the rate of release of active substance from the formulation after
administration has been reduced, in order to maintain therapeutic activity, to reduce toxic
effects, or for some other therapeutic purpose.
3.
DEVELOPMENT PHARMACEUTICS
3.1
Therapeutic objectives
The therapeutic objectives and rationale for developing the prolonged release product should
be provided. Pharmacokinetic and physico-chemical characteristics of the active substance
relevant to the development of the product should be given.
3.2
Principle of t h e p r o l o n g e d release s y s t e m
3.3
T e s t i n g of t h e prolonged-release s y s t e m
3.3.1 In vitro
testing
The release rate should be tested in vitro by a dissolution test method which has been shown
to discriminate between batches with acceptable and unacceptable in vivo performance. Test
conditions providing the most suitable discrimination should be chosen.
The dissolution apparatus should be one of those described in the European Pharmacopoeia.
The continuous flow-through method of the European Pharmacopoeia monograph may be of
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particular value in testing poorly soluble substances. The use of methods other than the
official methods in the European Pharmacopoeia should be justified.
The choice of rotation speed should be justified by carrying out the test at different speeds and
the speed giving appropriate discrimination between batches with acceptable and
unacceptable bioavailability should be chosen.
The test medium should preferably be aqueous-based; organic or aqueous-organic media
should be avoided. For poorly soluble substances, a minimal content of an appropriate
surfactant may be added. Buffer solutions at a number of pH values spanning the
physiological rate (pH 0.8-2, stomach; pH 5-6.5, jejunum; pH 6-7.5, ileum; Davis et al 1989)
should be used to determine the relationship between dissolution and pH. The data obtained
could usefully be represented using three-dimensional dissolution profiles (i.e. % dissolved
as a function of time and pH).
In order to achieve adequate discrimination, it may be necessary to limit the solubility of the
medicinal product and still achieve sink conditions in the dissolution medium. It may also
be necessary to consider the ionic strength and surface tension of the medium. The volume
of medium used should preferably ensure sink conditions which may be assumed if the
amount of substance in solution does not exceed 30% of the saturation concentration. The
solubility of the substance in the chosen dissolution medium should be stated.
Identical test conditions should be used for different strengths of the same product.
The robustness of the dissolution test should be determined by examining the effect on the
dissolution rate of variations in temperature, pH and speed of rotation.
Dissolution profiles should be determined for:
-
each strength of the prolonged release product if more than one strength is to be
marketed;
halved tablets where the release mechanism permits tablets to be broken in half for
dosage purposes;
At each time point individual dosage unit results (n _ 6), the mean value and a measure of
variability should be presented.
The definitive dissolution profile and the corresponding specification will be based on i n
vitro results of batches used in in vivo testing and will provide an assurance that batches
will routinely give the desired in vivo behaviour. It may be necessary to validate the
specification for any variations in the substance or excipients, e.g. the particle size or
polymorphic form of the active substance, the gelling properties or particle size of the
release-controlling excipients.
The content of any key excipient which has a determining effect on the release of the active
substance should not vary outside validated limits. These limits should be established on a
case by case basis during the development of each product.
3.3.2 In vivo
testing
A summary of the bioavailability testing should be given. The data should include batch
numbers, formulations (if different from the proposed marketing formulation) and
dissolution results of batches used in in vivo studies.
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comparison
To justify the specification limits of the in vitro dissolution test, an attempt should be made to
establish a meaningful correlation between in vitro release characteristics and In vivo
bioavailability parameters. In order to accomplish this, a number of techniques may be
employed. These include, In order of decreasing predictive power:
a)
comparison of the in vitro dissolution curve of the product with the in vivo dissolution
curves generated by deconvolution of plasma level data or by other appropriate
methods;
b)
comparison of the mean in vitro dissolution time of the product to either the mean i n
vivo residence time or the mean in vivo dissolution time of the product derived by
using the principles of statistical moment analysis;
c)
pharmacokinetic
Other approaches are acceptable especially if the above methods fail to demonstrate a
correlation. Examples of other approaches include demonstrating bioequivalence of the
proposed formulation to formulations with dissolution profiles at the upper and lower limits
of the specification, or alternatively, the specification limits may be derived from the spread
of in vitro dissolution results of batches used in bioavailability testing the choice of approach
should be justified by the applicant.
4.
Precise details of the manufacturing process should be given. Critical process parameters
which can significantly affect the release of the substance (e.g. tablet hardness, coating
procedure, moisture content) should be identified. If an in vitro-in vivo correlation has been
established, limits for the critical parameters should be validated by dissolution testing of the
product made under different processing conditions to demonstrate that allowable variations
in these parameters do not result in unacceptable changes to the dissolution profile.
If the manufacturing process has been validated using small-sized batches the effect of scale
up on the dissolution characteristics of the product should be established.
5.
CONTROL TESTS
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5.4 B a t c h r e s u l t s
Batch analytical results should be provided for at least three batches one of which should be
production scale for each product strength. Individual dosage unit dissolution results should
be included.
6.
STABILITY
It must be demonstrated that the dissolution profile of the active substance is maintained
within specification throughout the proposed shelf life of the product. The results of
dissolution testing should include mean values of individual dosage units together with
maximum and minimum values for all the batches undergoing the stability tests.
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7.
CHANGES TO PRODUCTS
Where the dissolution specification has been correlated with in vivo results minor changes
to the data may be acceptable on the basis of in vitro testing. Minor changes include changes
to the composition (e.g. nature and/or quantity of excipients which do not influence the
release characteristics) method or site of manufacture or manufacturing equipment. Other
changes may however necessitate further in vitro-in vivo correlation studies or in vivo
bioavailability studies.
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RADIOPHARMACEUTICALS
Guideline Title
Legislative basis
Date of first adoption
Date of entry into
force
Status
Previous titles/other
references
Additional Notes
Radiopharmaceuticals
Directives 65/65/EEC, 75/318/EEC as amended, Directive
89/343/EEC
December 1990
June 1991
Last revised December 1990
None/III/3936/89
This note for guidance concerns the application to
radiopharmaceuticals of Directive 65/65/EEC and of parts
2, 3 and 4 of the Annex to Directive 75/318/EEC as
amended, with a view to the granting of a marketing
authorisation for a radiopharmaceutical.
CONTENTS
1.
INTRODUCTION
2.
3.
4.
CLINICAL DOCUMENTATION
5.
RADIATION DOSIMETRY
6.
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RADIOPHARMACEUTICALS
INTRODUCTION
ready-for-use radiopharmaceuticals;
radionuclide generators;
other
substances
prior
to
administration
177
3AQ20a
2.
necessary
adequately
during
size)
final
after
in the Development
Generators
The recommendations for use of the generators should be discussed and documented.
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3AQ20a
c)
Precursors
The recommendations for use of the precursors should be discussed and documented.
2.3 Control of s t a r t i n g m a t e r i a l s
For the purposes of this section "starting materials" shall mean all the constituents of the
medicinal product, if necessary, all the constituents of its containers and closures and where
applicable, all constituents of the radionuclide source and any other materials used in the
final process prior to administration. A full description is required of the separation of
radionuclides and the control of radionuclide purity, as well as specific activity (with respect
to impurities and degradation products). Specifications of components of the container
(including the name of the approved producers) should be given. Specifications of' any
radiation shielding of the finished products should also be given.
For some radiopharmaceuticals, it is difficult to distinguish between control of starting
materials and control of the finished product. For such products, all the information should
preferably be placed in the section "Control tests on the finished product".
2.4 Control t e s t s o n t h e
finished
product
2.5 Stability t e s t s
For all radiopharmaceuticals, the shelf life of the product as supplied by the manufacturer
should be specified and justified, as should a shelf life after reconstitution where applicable,
taking into account radiochemical and radionuclide degradation products.
For radiopharmaceutical kits, the shelf life of the prepared product should be defined; in this
case, data should be submitted which detail the minimum and maximum levels of
radioactivity (and maximum and minimum volumes) and other relevant factors that are
recommended for use in the preparation of the product to be administered to the patient.
For radiopharmaceuticals prepared in multiple-dose vials, the stability following removal of
successive doses should be discussed.
For the purpose of this guideline, process parameter release is denned as "the decision to release a batch of
product for sale or supply based on an assessment of measured and recorded information relating to the
validation, maintenance, operation and control of a process."
179
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3.
It is appreciated that toxicity may be associated with a radiation dose. This toxicity is a
consequence of the use of radiopharmaceuticals in diagnosis and the wanted property of
radiopharmaceuticals used in therapy. The evaluation of safety and efficacy of
radiopharmaceuticals should, therefore, address both general parameters of the medicinal
product and radiation dosimetry aspects.
Toxicity studies should be designed to examine the chemical rather than the radiation
aspects of toxicity.
Knowledge of the toxicology of the ligand perse is of value. However, if the radiolabel is
likely to produce chemical changes in the ligand, it would be preferable to carry out the
toxicity studies on material in which radioactive decay has proceeded for long enough as to
expose the test animals to breakdown products as well as to the parent complex.
For other radiopharmaceuticals, consideration should be given to ascertaining the toxicity of
the parent molecule, either by reacting the ligand with a non-radioactive isotope of the
element in question, or, if appropriate, by allowing decay of the product to occur so that there
is no significant residual radioactivity.
Whatever the strategy and method chosen, it should be justified.
Distribution and elimination studies should be performed with the labelled compound.
For no-carrier-added radioactive elements and simple salts thereof, if the toxicity of the
element or simple salt is known and can be submitted in the application, no additional
toxicity studies would normally be required.
The contents in many final preparations (e.g. kit preparations) may be so small that is may
be justified to use a bulk preparation for toxicity testing but the stability of the bulk material
over the period of testing should be validated. The duration of animal toxicology testing will
be determined by the anticipated duration of clinical use. In cases where the
pharmacokinetic properties of the radiopharmaceutical (e.g. retention in certain organs)
may lead to long term exposure, the observation period of the toxicity study may have to be
extended.
Radiation dose should be evaluated with respect to target organs and physiological functions.
For radiopharmaceuticals, studies should be designed to assess:
a)
b)
c)
d)
of
tissues
resulting
from
administration
of
the
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3.2 E x a m i n a t i o n of r e p r o d u c t i v e f u n c t i o n a n d foetal t o x i c i t y
Although radiopharmaceuticals are not normally recommended for potentially pregnant
women, studies on reproduction may be required in certain cases, especially if the
radiopharmaceutical is intended for repeated use in women of child-bearing potential.
Otherwise the study on reproductive function may justifiably be limited to ascertaining the
effect on fertility.
3.3 M u t a g e n i c p o t e n t i a l
Mutagenicity testing may be limited to screening for gene and chromosome mutations and
should be performed to allow characterisation of the mutagenic potential of the nonradioactive equivalent of the product.
3.4 C a r c i n o g e n i c p o t e n t i a l
An evaluation of any carcinogenic potential of the substances involved must be presented. If
no carcinogenicity tests are performed, this must be clearly indicated in the "Summary of
product characteristics".
3.5
Pharmacodynamics
3.6
Pharmacokinetics
4.
CLINICAL DOCUMENTATION
Diagnostic radiopharmaceuticals differ in many ways from therapeutic radiopharmaceuticals. Consequently clinical documentation on diagnostic radiopharmaceuticals will be
different from that relating to therapeutic radiopharmaceuticals.
Radiopharmaceuticals for diagnostic use are part of a diagnostic system where other factors
such as instrumentation, time schedule, etc. also play an important role which should be
discussed. The same criteria as for non-radioactive therapeutic medicinal products apply to
therapeutic radiopharmaceuticals.
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4.1 Clinical p h a r m a c o l o g y
Whenever possible, initial pharmacodynamic and pharmacokinetic studies with
radiopharmaceuticals should be performed in suitable patients, rather than in healthy
volunteers.
Pharmacodynamics:
It is expected that many radiopharmaceuticals will not have any pharmacological action.
During early studies, the subjects should be monitored for a sufficient period to ascertain
any change in major organ function. Any adverse events should be reported, giving nature
and frequency.
Pharmacokinetics:
Pharmacokinetic studies should always provide the data necessary for the calculation of
radiation doses.
The results should be presented in a form which allows evaluation of the proposed radiation
dose and discussion of the in vivo stability of any radionuclide/carrier complex.
5.
RADIATION DOSIMETRY
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6.
6.1 Labelling
The label on the container should state:
-
for liquid preparations, the total radioactivity in the container, or the radioactive
concentration per millilitre, at a stated date and, if necessary, hour, and the volume of
liquid in container;
for capsules, the radioactivity of each capsule at a stated date and, if necessary, hour
and the number of capsules in the container;
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6.2 Packaging
The suitability of packaging material for the product and for the labelling procedure to be
carried out should be described. It may be necessary to describe special radiation shielding.
precautions to be taken by the user and the patient during the preparation and
administration of the product and special precautions for the disposal of the container
and its unused contents;
and, if it is appropriate for particular kits (i.e. those subject to variability beyond the
recommended limits) the leaflet should contain the methods and specifications needed
to check radiochemical purity.
184
and
the
prepared
and the
3AQ21a
RADIOPHARMACEUTICALS BASED ON
MONOCLONAL ANTIBODIES
Guideline Title
Legislative basis
Date of first adoption
Date of entry into
force
Status
Previous titles/other
references
Additional Notes
CONTENTS
1.
INTRODUCTION
2.
SOURCE MATERIALS
3.
PRODUCTION FACILHTES
4.
5.
6.
RADIOPHARMACEUTICAL ASPECTS
7.
8.
CLINICAL DOCUMENTATION
9.
RADIATION DOSIMETRY
10.
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RADIOPHARMACEUTICALS BASED ON
MONOCLONAL ANTIBODIES
INTRODUCTION
Monoclonal antibodies may form the basis of radiopharmaceuticals for in vivo diagnosis or
therapy. The antibody or antibody fragment is thus only one component of the medicinal
product and in the evaluation of quality and safety of this group of products, the
radiopharmaceutical and radiation protection aspects must be considered in addition to those
of the antibody component. The same principle would apply to monoclonal antibodies used in
conjunction with other agents e.g. toxins, though such products are not covered in this
document. The monoclonal antibodies used as the basis of such products may be of murine
origin, prepared in human cell lines or "humanised" using rDNA techniques. As regards
monoclonal antibodies and radiopharmaceuticals, different notes for guidance have already
been adopted by the CPMP: a note on "Production and quality control of monoclonal
antibodies", and a note on radiopharmaceuticals is published in this volume.
The notes for guidance are intended to be used by manufacturers submitting applications for
marketing authorisation. They are not intended for non-commercial producers. The notes
for guidance are advisory, not mandatory, and (as stated in the notes for guidance on
murine monoclonal antibodies) "a flexible approach" should be adopted.
A special consideration with this class of products is that chemical modification of the
antibody may be carried out. This may take the form of preparation of sub-fragments of
antibody (e.g. Fab or F(ab')2) and the antibody molecule (or a fragment of it) may also be
modified by addition of a conjugating agent for the radionuclide. These modified forms
require consideration with respect to quality, in addition to that for the monoclonal
antibodies from which they were derived.
Consideration of radiolabelling procedures encompasses the quality control of the
manufacturing steps and of the radiopharmaceutical aspects. In addition, the use of
radionuclides of short half-life will require specifications or instructions for the antibody
derivative/conjugate, the radionuclide (especially where specific purity requirements apply),
and the preparation and quality control of the final product intended for administration to
the patient, which typically, will be prepared by the user immediately prior to clinical use.
Frequently, the radionuclide and the monoclonal antibody components are marketed by
different manufacturers who are responsible independently for the marketing authorisation
and control of their product(s). The radionuclide (e.g. m i n ) may be authorised for use with a
number of monoclonal antibodies (or indeed with any antibody). In each specific case the
antibody manufacturer is responsible for providing the clinical and pharmaceutical data on
the radiolabelled antibody.
2.
SOURCE MATERIALS
The development and establishment of cell lines for the production of monoclonal antibodies
in this field has often taken place in non-commercial institutions. The initial development
may therefore not be as well documented as is generally required in the pharmaceutical
187
3AQ21a
industry. In cases where the history of the myeloma cell line and parental cell line are
limited by available data, more emphasis must be put on the characterisation at the seed lot
stage and of the final product to ensure quality (e.g. freedom from adventitious agents).
However, every effort should be made to provide evidence of the origin and acceptability of
the cell line. Non-commercial organisations collaborating with industry in the production of
monoclonal antibodies that will form part of a marketed product should be strongly
encouraged to improve their record-keeping so that full information on production of
antigen, immunisation, establishment of cell lines, testing of antibodies, etc. can be
provided.
3.
PRODUCTION FACILITIES
Even though production may be on a smaller scale than is usual in the pharmaceutical
industry, the appropriate good manufacturing practice should be followed for both the
radionuclide and the antibody components. A strategy for avoiding cross-contamination of
cell lines should be developed and specified if more than one antibody is produced within the
same facility.
4.
Although the points to consider in the manufacture of the antibodies are in essence those
outlined in the notes for guidance on murine and human monoclonal antibodies, special
considerations apply in the case of antibodies intended for use with radiopharmaceuticals:
-
for monoclonal antibodies prepared in only small amounts, it may not be essential to
have both a master cell bank and a manufacturer's working cell bank;
where these batches are subdivided for further processing, evidence of consistency of
this must be provided.
Purification of the antibody remains crucial and, in particular, virus contamination should
be carefully attended to. Steps should be included that will inactivate or remove
contaminating viruses that may be present. The purified bulk antibody should be tested for
extraneous proteins and DNA. Reference should be made to the note for guidance on Virus
Validation Studies: The Design, Contribution and Interpretation of Studies Validating the
Inactivation and Removal of Viruses.
5.
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3AQ21a
Each relevant step in the production of chemically modified monoclonal antibodies requires
validation and quality control covering source materials, limits for impurities arising from
the production process, evidence for consistency of the process, etc.
Initial immunological studies may be carried out on the unmodified antibody but for the
purposes of market authorisation the determination of definitive immunological properties
should be performed at an appropriate stage. For example, characteristics such as class,
subclass, and interaction with Fc receptors can best be determined with the monoclonal
antibody in the unmodified form. In contrast, for regulatory purposes, any tests of toxicity,
biological half-life, immunoreactivity and tissue cross-reactivity should be carried out on a
form that is as close as possible to the product to be administered to the patient, e.g. for a
chemically modified antibody on the derivatised form rather than the parent antibody. For
the radiolabelled form, appropriate studies should be undertaken on the product intended for
clinical use using "Cold" non-radioactive labelled material wherever possible. It should be
noted that in the case of 99Tcm, there is no equivalent non-radioactive isotope and often a
small percentage of the modified antibodies actually carries the label.
In the instances in which the final product is a chemically modified or derivatised
monoclonal antibody, criteria and specification limits for purity and potency should be
applied to the derivatised form and include a test for immunoreactivity.
Material from an early batch that has been clinically evaluated should be retained as the
manufacturer's reference batch for purity and potency of subsequent batches. A secondary
working standard may be established providing that equivalence with the primary reference
is demonstrated.
6.
RADIOPHARMACEUTICAL ASPECTS
6.1
Radionuclide
The radionuclide to be used for labelling the monoclonal antibody (whether derivatised or
not) needs to be an authorised medicinal product indicated for use for that purpose (see
Introduction). The radionuclide may be supplied as a component in the kit or separately.
Special purity measurements may apply and the radionuclide should have specifications for:
a)
b)
c)
d)
6.2 R a d i o l a b e l l i n g
chemical
method
Where this is carried out by the manufacturer: The process should be validated.
includes quantitative relationships between the (derivatised) antibody
radionuclide, purification of the labelled product and removal of excess reagents,
for radiochemical purity, quantity of radioactive material in the container,
stability data.
This
and
tests
and
189
3AQ21a
b)
Where this is carried out by the user: This is likely to be in the form of a
radiopharmaceutical kit consisting of a (derivatised) monoclonal antibody, reagents
and materials necessary for the radiolabelling procedure including any necessary
purification of the product plus a package insert giving clear, precise instructions for
the use of the kit, quality control, and potential hazards. Radiolabelling methods and
quality requirements for the necessary reagents should form part of the product
marketing authorisation application.
The radiolabelling procedure for kit preparations should be validated under relevant
circumstances and the detailed specifications for the radiolabelling medium (e.g.
99Tcm or i n l n ) should be discussed. Quantitative relationships between the antibody (in
particular the immunoreactivity), the conjugating agent and the radionuclide should
be presented.
c)
Specifications to be fulfilled should be part of the application and could include the following:
-
If it is considered necessary for the user to carry out appropriate quality control tests on the
final radiolabelled product, the methods should be fully described and validated.
Samples of reference materials, antigens and special reagents should be made available
upon request.
7.
P R E C L I N I C A L SAFETY TESTS
7.1 General
Due account should be taken of the note for guidance on Pre-clinical Biological Safety
Testing on Medicinal Products derived from Biotechnology.
Radiolabelled monoclonal antibodies may be used for diagnosis and therapy. While the
diagnostic use may cover many different types of diseases, the therapeutic use is currently
limited to treatment of cancer as a means of getting a high radiation dose to the target organ.
Testing requirements may therefore be different for the two uses. It is characteristic for the
diagnostic use that smaller amounts of antibodies are needed.
It is appreciated that toxicity may be associated
consequence of the use of radiopharmaceuticals
radiopharmaceuticals used in therapy. The
radiopharmaceuticals should therefore address
radiation dosimetry aspects.
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7.3 E x a m i n a t i o n of r e p r o d u c t i v e f u n c t i o n ; f o e t a l t o x i c i t y ; m u t a g e n i c
potential; carcinogenic potential
Due account should be taken of the note for guidance on
7.4
Radiopharmaceuticals.
Pharmacodynamics
7.5
Pharmacokinetics
8.
CLINICAL DOCUMENTATION
8.1 General
There are two quite different types of radiopharmaceuticals; first radiopharmaceuticals
which are used to effect a medical diagnosis and which are part of a diagnostic system
where other factors such as instrumentation, time schedule etc., also play an important role
which should be discussed; second, radiopharmaceuticals which are used for the treatment of
diseases. Diagnostic radiopharmaceuticals differ in many ways from therapeutic
radiopharmaceuticals and consequently clinical documentation has to be different. The
same criteria as for non-radiolabelled therapeutic substances apply to therapeutic
radiopharmaceuticals.
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9.
RADIATION DOSIMETRY
192
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for liquid preparations, the total radioactivity in the container, or the radioactive
concentration per millilitre, at a stated date and, if necessary, hour (and state the time
zone used), and the volume of liquid in the container;
for capsules, the radioactivity of each capsule at a stated date and, if necessary, hour
(and state the time zone used), and the number of capsules in the container;
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10.2 P a c k a g i n g m a t e r i a l
The suitability of packaging material for the product and for the radiolabelling procedure to
be carried out should be described. It may be necessary to describe special radiation
shielding.
10.3 P a c k a g e leaflets
Package leaflets play a particularly important role for semi-manufactured products such as
preparation kits for radiolabelled monoclonal antibodies. This is the responsibility of the
antibody manufacturer and should at least show:
-
precautions to be taken by the user and the patient during the preparation and
administration of the product and special precautions for the disposal of the container
and its unused contents;
if it is appropriate for particular kits (i.e. those subject to variability beyond the
recommended limits) the leaflet should contain the methods and specification needed
to check radiochemical purity.
194
and
the
prepared
and the
3AQ22a
Guideline Title
Legislative basis
Date of first adoption
Date of entry into
force
Status
Previous titles/other
references
Additional Notes
CONTENTS
A
DESCRIPTI
O N O F THE METHO D O F PREPARATIO N
O
C NTR
O L O F STARTING MATERIALS
O
C NTR
O L O F TESTS CARRHD O UT AT AN INTERMEDIATE STAGE O F THE
MANUFACTURING PRO CESS O F THE FINISHED PRO DUCT
O
C NTR
O L TESTS O N FINISHED PRO DUCT
STABILITY TESTS
ANNEX
195
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Note for guidance concerning the application of Part 2 of the Annex to Directive 75/318/EEC
as amended. The special problems of herbal remedies and the differences between medicinal
products containing chemically defined active substances are described in this note for
guidance.
Consistent quality for products of vegetable origin can only be assured if the starting
materials are defined in a rigorous and detailed manner including especially the specific
botanical identification of the plant material used. It is also important to know, the
geographical source and the conditions under which the vegetable substance is obtained to
ensure material of consistent quality.
Reference substances used in the control of all stages of the manufacturing process should be
clearly defined.
either
a)
or
b)
EXAMPLE
a)
Active substance
Name
Quantity
Sennae folium
900 mg
or
b)
Active substance
Name
Quantity
Sennae folium
of
as
Other substance
Name
Quantity
0-170 mg, corresponding to the quantity of Sennae
folium
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2.
either
a)
the equivalent quantity, or the ratio e.g. 8:1 of the vegetable substance to the
vegetable substance preparation must be stated (this does not apply to fatty or
essentials oils).
or
b)
The composition of any solvent or solvent mixture and the physical state of the extract must
be indicated.
If any other substance is added during the manufacture of the vegetable substance
preparation to adjust the vegetable substance preparation to a certain level of constituents
with known therapeutic activity, or for any other purpose, the added substance must be
mentioned as an "other substance" and the genuine extract as the "active substance".
EXAMPLE
a)
Active substance
Name
Sennae folium
Quantity
125 mg
or
Sennae folium
Active substance
Name
Sennae folium
dry 60% ethanolic extract (8:1)
Quantity
100-130 mg,
corresponding
to 25 mg
hydroxyanthracene
glycosides,
calculated
Sennoside
Other substances
Name
Dextrin
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Quantity
20-50 mg
of
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The manufacturing process within the meaning of this section is the preparation of the
finished product from the starting materials. The description should include details of any
comminution or size reduction step, and details of any process such as fumigation etc. used
to reduce the levels of microbial contamination together with the controls exercised over the
process. If vegetable substance preparations are the starting material, the manufacture of the
vegetable substance preparations and their controls do not belong under this section but
under section C.
A complete monograph for each vegetable substance must be submitted, even if the starting
material is a vegetable substance preparation. This also applies if the applicant is not the
manufacturer of the preparation. In the case of fatty or essential oils a complete monograph
for the vegetable substance is not required, only the scientific name of the parent plant and
its part(s) have to be stated.
If no monograph for the vegetable substance is given in a Pharmacopoeia referred to in
Directive 75/318/EEC as amended, Annex 1, a monograph on the vegetable substance must be
supplied and should be set out in the same way where practicable, as the monographs on
vegetable substances in the European Pharmacopoeia. This should include the botanical
name and authority and the common name if used for labelling purposes. Information on
the site of collection, the time of harvesting and stage of growth, treatment during growth
with pesticides etc., and drying and storage conditions should be included if possible. The
monograph should be established on the basis of recent scientific data. In the case of
vegetable substances with constituents of known therapeutic activity, assays of their content
(with test procedure) are required. The content must be included as a range, so as to ensure
reproducibility of the quality of the finished product.
As a general rule, vegetable substances must be tested for microbiological quality and for
residues of pesticides and fumigation agents, radioactivity, toxic metals, likely
contaminants and adulterants, etc., unless otherwise justified. Specifications and
descriptions of the analytical procedures must be submitted, together with the limits applied.
Reference samples of the vegetable substances must be available for use in comparative tests
e.g. macro and microscopic examination, chromatography etc.
2.
If the herbal remedy contains not the vegetable substance itself but a preparation, the
monograph on the substance must be followed by a description and validation of the
manufacturing process for the vegetable substance preparation.
For each vegetable substance preparation, a monograph must be submitted. This must be
established on the basis of recent scientific data and must give particulars of the
characteristics, identification tests and purity tests. This has to be done e.g. by different
appropriate chromatographic methods. If deemed necessary by the results of the analysis of
the starting material, tests on microbiological quality, residues of pesticides, fumigation
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agents, radioactivity, solvents and toxic metals have to be carried out. Quantitative
determination (assay) of characteristic constituents is required. The content must be
indicated with the lowest possible tolerance. The test methods must be described in detail.
If preparations from vegetable substances with constituents with known therapeutic activity
are standardised (i.e. adjusted to a certain level of constituents with known therapeutic
activity) it must be stated how such standardisation is achieved. If another substance is used
for this purpose, it is necessary to specify as a range the quantity that can be added.
Details of all control tests with details of test procedures and limits applied at any
intermediate stages of the manufacturing processes are required, especially if these tests
cannot be done in the finished product.
The control tests on the finished product must be such as to allow the qualitative and
quantitative determination of the composition of the active substances and a specification has
to be given which may be done by using markers if constituents with known therapeutic
activity are unknown. In the case of vegetable substances or vegetable substance preparations
with constituents of known therapeutic activity, these constituents must also be specified and
quantitatively determined.
If a herbal remedy contains several vegetable substances or preparations of several vegetable
substances and it is not possible to perform a quantitative determination of each active
substance, the determination may be carried out jointly for several active substances. The
need for this procedure must be justified.
STABILITY TESTS
Since the vegetable substance or vegetable substance preparation in its entirety is regarded
as the active substance, a mere determination of the stability of the constituents with known
therapeutic activity will not suffice. It must also be shown, as far as possible e.g. by means of
appropriate fingerprint chromatograms, that other substances present in the vegetable
substance or in the vegetable substance preparation are likewise stable and that their
proportional content remains constant.
If a herbal remedy contains several vegetable substances or preparations of several vegetable
substances and if it is not possible to determine the stability of each active substance, the
stability of the medicinal product should be determined by appropriate
fingerprint
chromatograms, appropriate overall methods of assay and physical and sensory tests or other
appropriate tests.
If the only evidence that can be submitted concerning the stability of the finished product
consists of results of trials in which each active substance was separately tested in a
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formulation corresponding to that of the finished product, the reasons why it is not possible to
carry out stability tests on the finished product must be stated in full. It must furthermore be
shown that interactions between the active substances and the excipients in the finished
product are unlikely to occur.
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ANNEX
GlossaryHerbal remedies (herbal medicines) are medicinal products containing as active substances
exclusively plant material and/or vegetable substance preparations.
Vegetable substances are plant material used for a medicinal purpose. A vegetable substance
or a preparation thereof is regarded as one active substance in its entirety whether or not the
constituents with therapeutic activity are known.
Vegetable substance preparations are comminuted or powdered vegetable substances,
extracts, tinctures, fatty or essential oils, expressed juices etc. prepared from vegetable
substances, and preparations whose production involves a fractionation, purification or
concentration process. However, chemically defined isolated constituents or their mixtures
are not vegetable substance preparations. Other substances such as solvents, diluents,
preservatives may form part of vegetable substance preparations. These substances must be
indicated.
Constituents with known therapeutic activity are chemically defined substances or groups of
substances which are known to contribute to the therapeutic activity of a vegetable substance
or of a preparation.
Markers are chemically defined constituents of a vegetable substance which are of interest
for control purposes. Markers may serve to calculate the quantity of vegetable substance or
preparation in the finished product if that marker has been quantitatively determined in the
vegetable substance or preparation when the starting materials were tested.
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BIOTECHNOLOGY GUIDELINES
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CONTENTS
1.
INTRODUCTION
2.
3.
DEVELOPMENT GENETICS
4.
5.
6.
7.
ACTrVE SUBSTANCE
8.
9.
10.
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INTRODUCTION
Developments in molecular genetics and nucleic acid chemistry enable the genes coding for
natural, biologically active proteins to be identified, analysed in fine detail, transferred
between organisms, and expressed under controlled conditions so as to obtain synthesis of
the polypeptide for which they code.
Sufficient quantities of medicinal products which were previously difficult to prepare from
natural sources can now be produced using such recombinant DNA (rDNA) technology. In
addition, the ability to synthesise and manipulate nucleic acids allows the construction of
genes coding for modified products possessing different properties from their natural
counterpart, or even entirely novel products.
A common strategy in the development of rDNA derived products is the insertion of
naturally occurring or intentionally modified natural sequences or novel nucleotide
sequences into a vector which is introduced into a suitable host organism so as to ensure the
efficient expression of the desired gene product. Both prokaryotic and eukaryotic vector/host
cell expression systems have been developed and are in use for production. The factors
affecting the expression of foreign genes introduced into a new host using a suitable vector
are complex and the efficient, controlled expression of stable, cloned DNA sequences is a n
important aspect of product development.
A flexible approach to the control of these products should be adopted so that recommendations
can be modified in the light of experience of production and use, and with the further
development of new technologies. Implementation of these recommendations for an
individual product should reflect its intended clinical use.
This note for guidance is intended to facilitate the collection and submission of data to
support applications for marketing authorisation within the European Union for polypeptide
based products derived by rDNA technology and intended for medicinal use in man. It
should be read in conjunction with the European Directives and other specialised guidelines
where appropriate.
2.
Thus, appropriate attention needs to be given to the quality of all reagents used in production,
including components of fermentation media; specifications for these are to be included in
documentation and they must comply with any relevant European recommendations (e.g.
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Spongiform
Tests for potency, abnormal toxicity, pyrogenicity and sterility etc., which apply to products
made by conventional methods, will also apply to products made by rDNA technology. It is
undesirable to use in production agents which are known to provoke sensitivity in certain
individuals, such as, for example, penicillin or other -lactam antibiotics.
Although comprehensive characterisation of the final product is essential, considerable
emphasis must also be placed on "in-process" control, a concept which has been highly
effective in the quality control of bacterial and viral vaccines prepared by conventional
methods.
Certain factors may compromise the consistency, safety and efficacy
products; these should be given special attention and are outlined below:
of rDNA-derived
a)
All biological systems are inherently subject to genetic alteration through mutation
and selection and foreign genes inserted into new host cells may exhibit increased
genetic instability. The purpose of molecular genetic studies is to establish that the
correct sequence has been made and incorporated in the host cell and that both the
structure and the number of copies of the inserted sequence are maintained within the
cell during culture to the end of production. Such studies can provide valuable
information which should be considered in conjunction with tests performed at the
protein level for assuring the quality and consistency of the product.
b)
c)
The choice of manufacturing procedure will influence the nature, range and amount
of potential impurities in the final product and which the purification processes must be
shown to be capable of removing. Examples of these are endotoxins in products
expressed in bacterial cells, and adventitious agents and DNA in products expressed
in mammalian cells.
d)
Unintended variability in the culture during production may lead to changes which
favour the expression of other genes in the host/vector system or which cause alteration
in the product. Such variation might result in differing yield, in change to the product
itself (e.g. in the nature and degree of glycosylation) and/or in quantitative and
qualitative differences in the impurities present. Consequently, procedures to ensure
consistency of production conditions as well as the final product are imperative.
e)
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Whilst the recommendations set out below should be considered to be generally applicable,
individual products may present particular quality control issues. Thus, the production and
control of each product must be given careful individual consideration taking fully into
account any special features.
3.
DEVELOPMENT GENETICS
3.4
Expression
The strategy by which the expression of the relevant gene is promoted and controlled during
production should be described in detail.
3.5 Stability of t h e e x p r e s s i o n s y s t e m
The stability of host/vector genetic and phenotypic characteristics should be investigated up
to and beyond the population doubling level or generation number used for routine
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production (End of Production Cells). The expression construct should be analysed in the
End of Production Cells, as described above, at least once for each MCB.
Stability studies should also provide detailed information on:
i)
ii)
iii)
For this purpose, analysis should be performed in such a way that the results can confirm
that the number of variants is below an acceptable limit to be established on a case by case
basis depending on the nature and proposed use of the product. Analysis at the protein and/or
at the DNA level can be envisaged. Whichever method is used, it should be validated and the
detection limit given.
4.
It is essential that production is based on a well defined master and working cell bank
system. During the establishment of the banks no other cell lines should be handled
simultaneously in the same laboratory suite or by the same persons. The origin, form,
storage, use, and expected duration at the anticipated rate of use must be described in full for
all cell banks. New working cell banks should be fully characterised.
A critical part of quality control will involve the full characterisation of cells. Where
eukaryotic cells are used for production, distinguishing genetic, phenotypic and
immunological markers of the cell will be useful in establishing the identity of the cells.
Likewise, where microbial cultures are used, specific phenotypic features which form a basis
for identification should be described.
The cell banks should be examined for adventitious agents (viral, bacterial, fungal and
mycoplasmal). Special attention should be given to viruses which can commonly
contaminate the animal species from which the cell line has been derived. Certain cell lines
contain endogenous viruses, e.g. retroviruses, which may not readily be eliminated. The
possibility of mutations of endogenous viruses during prolonged culture should be
considered. Furthermore, the purification process should be shown to be capable of removing
and/or inactivating any such virus which may inevitably be present in the cells as an
endogenous agent.
Cell banks should be periodically tested for cell viability, genetic and phenotypic stability
and any other relevant parameters.
5.
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information to confirm the adequate sensitivity of the methods used to detect contamination
should be provided and acceptable limits of contamination set.
Ideally not more than one cell line should be cultivated simultaneously in the same
production area. If other cell lines are cultivated in parallel, records must be kept of the cell
lines handled and validation data presented for the absence of cross-contamination between
them.
5.1
The maximum permitted generation number or population doubling level for production
should be defined and should be based on information concerning the stability of the host
cell/vector system up to and beyond the level of production. Data on consistency of growth of
the culture and on the maintenance of yield within specified limits should be presented.
Appropriate monitoring of host cell/vector characteristics at the end of the production cycles
should also be undertaken. Evidence should be provided that the yield does not vary beyond
defined limits and that the nature and quality of the product does not change with respect to
specific parameters.
5.2 M u l t i p l e h a r v e s t p r o d u c t i o n
The period of continuous cultivation should be specified and this should be based on
information concerning the stability of the system and consistency of the product up to and
beyond this limit. Monitoring of the production system is necessary throughout the duration
of the culture. The required frequency and type of monitoring will depend upon several
factors including the nature of the expression system and product, as well as the total length
of the period of continuous cultivation undertaken. The acceptance of harvests for further
processing should be clearly linked to the schedule of monitoring applied. Evidence should
be provided that the yield does not vary beyond defined limits and that the nature and
quality of the product does not change with respect to specific parameters.
6.
6.1 Methods
Methods used to purify the product and their in-process controls including their specification
limits should be described in detail, justified and validated. Procedures which make use of
affinity chromatography, for example employing monoclonal antibodies, should be
accompanied by appropriate measures to ensure that these substances, or any additional
potential contaminants arising from their use, do not compromise the quality and safety of
the final product. Attention is drawn to the notes for guidance "Production and Quality
Control of Monoclonal Antibodies" and "Virus validation studies: The design, contribution
and interpretation of studies validating the inactivation and removal of viruses".
The criteria for reprocessing of any intermediate or final bulk should be carefully defined,
validated and justified.
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7.
ACTDTE S U B S T A N C E
7.1 C h a r a c t e r i s a t i o n of t h e active s u b s t a n c e
7.1.1 Physico-chemical characterisation, relative molecular mass, pi value
Rigorous characterisation of the active substance by chemical and biological methods will be
essential. Particular attention should be given to using a wide range of analytical
techniques exploiting different physico-chemical properties of the molecule; for instance,
size, charge, isoelectric point and hydrophobicity. A list of the analytical possibilities is
beyond the scope of this guideline. In the following there are only examples of the type of
analysis.
with
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7.1.3 Post-translational
modifications
data for
macromolecules
It is desirable to include suitable tests to establish that the product has the desired
conformational structure and state of aggregation. Examples of techniques suitable for such
purposes are: Polyacrylamide gel electrophoresis; isoelectric focusing; size exclusion,
reversed phase ion exchange, hydrophobic interaction or affinity chromatography; peptide
mapping and subsequent amino acid sequencing; light scattering; UV spectroscopy; circular
dichroism and mass spectrometry. Additional characterisation of the product using for
example NMR spectra, X-ray crystallography or relevant immunochemical techniques may
provide valuable information.
7.1.5 Biological,
immunological
characterisation,
expression
of
strength
7.2 Purity
Data should be provided on contaminants whose presence is anticipated in the final
processed product. The level of contamination considered as acceptable should be justified,
and criteria for acceptance or rejection of a production batch should be given. It is important
that the techniques used to demonstrate purity be assessed using as wide a range of methods
as possible, including physico-chemical and immunological techniques. Unwanted
materials of host origin, as well as materials which may have been added during the
production or purification processes, and where appropriate, viral and nucleic acid
contamination should be tested.
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8.
8.1. Consistency
An acceptable number, for example 5 (smaller numbers could be acceptable where justified),
of successive batches of the bulk processed product should be characterised as fully as
possible to determine consistency of composition. In the case of a production where multiple
harvests are applied, batches from different fermentation runs should normally be studied.
The studies should include biological, chemical and immunological methods to characterise
and assay the active substance (including methods showing the consistency of the
glycosylation pattern for glycoproteins) and methods to detect and identify impurities. Any
differences which occur between batches should be noted.
82.2. Purity
The degree of purity desirable and attainable will depend on several factors; these include
the nature and intended use of the product, the method of its production and purification and
also the degree of consistency of the production process. In general, a very high degree of
purity can be achieved for most products by modern manufacturing procedures.
The purity of each batch should be established and be within specified limits. The analysis
should include sensitive and reliable assays for DNA of host cell origin and/or of the vector
applied to each batch of product prepared from cell lines of mammalian origin, in which
case upper limits should be set. It is recommended that DNA analyses are also performed on
each batch of bulk product obtained from other eukaryotic cell systems and limits set for
DNA content. DNA of prokaryotic expression systems should be tested for wherever
appropriate to consideration of the quality of the product. The residual cellular proteins
should also be determined by an assay with appropriate sensitivity (e.g. ppm) and strict
upper limits set. In some instances, potential impurities such as DNA can only be
determined on intermediates of purification, at an earlier step.
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9.
The studies described in section 7 will contribute to a definitive specification for the product
when justified by the information obtained from the examination of successive batches and
results of batch analysis, as indicated in section 8.
A suitable batch of the product, preferably one which has been clinically evaluated, should be
fully characterised in terms of its chemical composition, purity, potency and biological
activity, including where possible full amino acid sequencing, and retained for use as a
chemical and biological reference material.
Criteria for expiration and possible re-testing and re-qualification of reference standards
should be established.
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GLOSSARY
1.
Cell Banks
a)
A homogeneous suspension of cells derived from the master cell bank(s) by a finite passage
level, aliquoted into individual containers for storage (e.g. in a liquid nitrogen
refrigerator).
In both cell banks, all containers are treated identically during storage, and once removed
from storage, the containers are not returned to the cell bank stock.
2.
Production method
a)
The number of population doublings (or duration of culture for certain production systems)
are specified based on information concerning the stability of the system and the
consistency of the product. Criteria for the termination has to be defined by the
manufacturer.
3.
Bulk harvest
This is the final product, after completion of the production process, obtained from a bulk
harvest. It is maintained in a single container or multiple identical containers where
necessary and used in the preparation of the final dosage form. The generation of this final
batch has to be clearly defined and unambiguously recorded by the producer.
5.
Finished product
The active substance is formulated and filled into final, sealed containers which hold the
product in its final dosage form, i.e. the finished product. The containers of a filling lot are
processed together and uniform in their contents and biological potency.
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CONTENTS
I.
INTRODUCTION
II.
CONCLUSION
V.
GLOSSARY
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I.
INTRODUCTION
This document presents guidance regarding the characterisation of the expression construct
for the production of recombinant DNA (rDNA) protein products in eukaryotic and
prokaryotic cells. This document is intended to describe the types of information that are
considered valuable in assessing the structure of the expression construct used to produce
rDNA derived proteins. This document is not intended to cover the whole quality aspect of
rDNA derived medicinal products.
The expression construct is defined as the expression vector containing the coding sequence
of the recombinant protein. Segments of the expression construct should be analysed using
nucleic acid techniques in conjunction with other tests performed on the purified
recombinant protein for assuring the quality and consistency of the final product. Analysis
of the expression construct at the nucleic acid level should be considered as part of the
overall evaluation of quality, taking into account that this testing only evaluates the coding
sequence of a recombinant gene and not the translational fidelity nor other characteristics
of the recombinant protein, such as secondary structure, tertiary structure, and posttranslational modifications.
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analysis of genomic DNA. Analytical approaches that examine a bulk population of nucleic
acids, such as those performed on pooled clones or material amplified by the polymerase
chain reaction, may be considered as an alternative to approaches that depend on selection of
individual DNA clones. Other techniques could be considered that allow for rapid and
sensitive confirmation of the sequence coding for the recombinant protein in the expression
construct.
The following sections describe information that should be supplied regarding the
characterisation of the expression construct during the development and validation of the
production system. Analytical methodologies should be validated for the intended purpose of
confirmation of sequence. The validation documentation should at a minimum include
estimates of the limits of detection for variant sequences. This should be performed for either
nucleic acid or protein sequencing methods. The philosophy and recommendations for
analysis expressed in this document should be periodically reviewed to take advantage of
new advances in technology and scientific information.
IH. C H A R A C T E R I S A T I O N O F T H E E X P R E S S I O N SYSTEM
A.
The manufacturer should describe the origin of the nucleotide sequence coding for the
protein. This should include identification and source of the cell from which the nucleotide
sequence was originally obtained. Methods used to prepare the DNA coding for the protein
should be described.
The steps in the assembly of the expression construct should be described in detail. This
description should include the source and function of the component parts of the expression
construct, e.g. origins of replication, antibiotic resistance genes, promoters, enhancers,
whether or not the protein is being synthesised as a fusion protein. A detailed component
map and a complete annotated sequence of the plasmid should be given, indicating those
regions that have been sequenced during the construction and those taken from the
literature. Other expressed proteins encoded by the plasmid should be indicated. The
nucleotide sequence of the coding region of the gene of interest and associated flanking
regions that are inserted into the vector, up to and including the junctions of insertion,
should be determined by DNA sequencing of the construct.
A description of the method of transfer of the expression construct into the host cell should be
provided. In addition, methods used to amplify the expression construct and criteria used to
select the cell clone for production should be described in detail.
B.
Cell B a n k System
Production of the recombinant protein should be based on well-defined Master and Working
Cell Banks. A cell bank is a collection of ampoules of uniform composition stored under
defined conditions each containing an aliquot of a single pool of cells. The Master Cell
Bank (MCB) is generally derived from the selected cell clone containing the expression
construct. The Working Cell Bank (WCB) is derived by expansion of one or more ampoules
of the MCB. The cell line history and production of the cell banks should be described i n
detail, including methods and reagents used during culture, in vitro cell age, and storage
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conditions. All cell banks should be characterised for relevant phenotypic and genotypic
markers which could include the expression of the recombinant protein or presence of the
expression construct.
The expression construct in the MCB should be analysed as described below. If the testing
cannot be carried out on the MCB, it should be carried out on each WCB.
Restriction endonuclease mapping or other suitable techniques should be used to analyse the
expression construct for copy number, for insertions or deletions, and for the number of
integration sites. For extrachromosomal expression systems, the percent of host cells
retaining the expression construct should be determined.
The protein coding sequence for the recombinant protein product of the expression construct
should be verified. For extrachromosomal expression systems, the expression construct
should be isolated and the nucleotide sequence encoding the product should be verified
without further cloning. For cells with chromosomal copies of the expression construct, the
nucleotide sequence encoding the product could be verified by recloning and sequencing of
chromosomal copies. Alternatively, the nucleic acid sequence encoding the product could be
verified by techniques such as sequencing of pooled cDNA clones or material amplified by
the polymerase chain reaction. The nucleic acid sequence should be identical, within the
limits of detection of the methodology, to that determined for the expression construct as
described in Section III.A. and should correspond to that expected for the protein sequence.
C.
The limit for in vitro cell age for production should be based on data derived from
production cells expanded under pilot or full scale conditions to the proposed in vitro cell age
or beyond. Generally, the production cells are obtained by expansion of the Working Cell
Bank; the Master Cell Bank could be used to prepare the production cells with appropriate
justification.
The expression construct of the production cells should be analysed once for the MCB as
described in Section III.B, except that the protein coding sequence of the expression construct
in the production cells could be verified by either nucleic acid testing or analysis of the final
protein product. Increases in the defined limit for in vitro cell age for production should be
supported by data from cells which have been expanded to an in vitro cell age which is equal
to or greater than the new limit for in vitro cell age.
IV. CONCLUSION
The characterisation of the expression construct and the final purified protein are both
important to ensure the consistent production of a rDNA derived product. As described above,
it is considered that analytical data derived from both nucleic acid analysis and evaluation
of the final purified protein should be evaluated to ensure the quality of a recombinant
protein product.
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GLOSSARY
Expression Construct
The expression vector which contains the coding sequence of the recombinant protein and the
elements necessary for its expression.
Flanking Control Regions
Non-coding nucleotide sequences that are adjacent to the 5' and 3' end
sequence of the product which contain important elements that affect the
translation, or stability of the coding sequence. These regions include,
enhancer, and splicing sequences and do not include origins of replication
resistance genes.
of the coding
transcription,
e.g. promoter,
and antibiotic
Integration Site
The site where one or more copies of the expression construct is integrated into the host cell
genome.
In vitro Cell Age
Measure of time between thaw of the MCB vial(s) to harvest of the production vessel
measured by elapsed chronological time in culture, by population doubling level of the cells,
or by passage level of the cells when subcultivated by a defined procedure for dilution of the
culture.
Master Cell Bank (MCB)
An aliquot of a single pool of cells which generally has been prepared from the selected cell
clone under defined conditions, dispensed into multiple containers and stored under defined
conditions. The MCB is used to derive all working cell banks. The testing performed on a
new MCB (from a previous initial cell clone, MCB or WCB) should be the same as for the
MCB unless justified.
Pilot Plant Scale
The production of a recombinant protein by a procedure fully representative of and
simulating that to be applied on a full commercial manufacturing scale. The methods of cell
expansion, harvest, and product purification should be identical except for the scale of
production.
Relevant Genotypic and Phenotypic Markers
Those markers permitting the identification of the strain of the cell line which should
include the expression of the recombinant protein or presence of the expression construct.
Working Cell Bank (WCB)
The Working Cell Bank is prepared from aliquots of a homogeneous suspension of cells
obtained from culturing the MCB under defined culture conditions.
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CONTENTS
1.
INTRODUCTION
2.
3.
MANUFACTURING REQUIREMENTS
4.
FINISHED PRODUCT
5.
GLOSSARY
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INTRODUCTION
2.
This note for guidance is intended to facilitate the collection and submission of data to
support applications for marketing authorisation within the European Union for cytokine
products derived from biotechnological processes. Cytokines may be produced in quantity by
the large scale cultivation either of (i) transformed cell lines producing particular cytokines
or (ii) prokaryotic or eukaryotic, including mammalian, cells in which the relevant
cytokine genes have been inserted by rDNA techniques. The large scale production of
cytokines by these procedures may affect the quality of particular cytokine products, and thus
have implications for control testing. Although comprehensive characterisation of the final,
purified cytokine product will be essential, particular emphasis must also be placed on "inprocess" control and the consistency of the manufacturing process, a concept which has been
very effective in the control of other biological products.
Requirements relating to establishments in which biological products are manufactured
(e.g. Revised Requirements for Biological Substances No 1; WHO TRS 323) will apply to the
manufacturers of cytokine products as will several of the general requirements for the
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quality control of biological products. Thus, appropriate attention needs to be given to the
quality of all reagents used in production, including components of fermentation or
cultivation media; specifications for these are to be included in documentation and they
must comply with any relevant European requirements. Tests for potency, abnormal
toxicity, pyrogenicity and sterility, etc., which apply to products made by conventional
methods, will also apply to products of biotechnological processes. It is undesirable to use in
production agents which are known to provoke sensitivity reactions in certain individuals,
such as, for example, penicillin or other beta-lactam antibiotics.
Certain factors may compromise the safety and efficacy of cytokine products; these should be
given special attention and are outlined below:
a)
The use of a transformed cell line, particularly one of neoplastic origin, as the
substrate for the manufacture of particular cytokines raises questions of safety. These
questions, pertaining mainly to the potential oncogenicity of contaminating
heterogeneous DNA derived from the substrate, have been debated for a number of
years at many national and international scientific meetings. There is a growing
international consensus that transformed cell lines can be used for the manufacture of
biologicals, although their use as yet has only been approved in certain cases (e.g. the
manufacture of interferon derived from the Namalwa human lymphoblastoid cell
line) where the product may be subjected to rigorous purification.
b)
c)
The choice of manufacturing procedure may influence the nature and range of
potential contaminants. Examples of these are endotoxins in products expressed in
bacterial cells and DNA or oncogenic potential in products expressed in transformed
mammalian cells. Thus cytokine products may contain potentially hazardous
contaminants which the purification processes must be shown to be capable of
removing.
d)
Unintended variability in the culture during production may lead to changes which
favour the expression of other genes in the host/vector system or which cause
alterations in the polypeptide product. Such variation might result in decreased yield of
the products and/or quantitative and qualitative differences in the impurities present
in the product. Consequently, procedures to ensure the consistency of production
conditions as well as the consistency of the final product are imperative.
Whilst the requirements set out below should be applied wherever they are appropriate for
safety and efficacy, individual cytokine products may present particular quality control
problems. Thus, the production and control of each cytokine product must be given careful
individual consideration taking fully into account any special features.
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3.
MANUFACTURING
3.1
Definitions
3.1.1 International
REQUIREMENTS
name
A cytokine shall be named in accordance with international consensus. Proper names shall
be the equivalent of the international name in the language of the country of origin. The use
of the international name should be limited to suitably purified preparations.
3.12, Descriptive
definition
standards
and international
unit
A number of international standards for particular cytokines have been or are being
developed. These have assigned potencies in terms of biological activity which are quoted i n
International Units (IU).
3.2
Strategy for p r o d u c t i o n
by transformed
cell
lines
A full description of the biological characteristics of the transformed cell line and any
additions, e.g. the inducer and any enhancer or other substances (e.g. antibiotics) used in
production, shall be given. The information includes:
a)
documentation concerning the origin of the cell line, and the nature of any known
relevant condition of the donor, e.g. Burkitt's lymphoma, active infectious
mononucleosis, infection with viruses or mycoplasma in general;
b)
data which can be used to establish the identity of the cell line;
c)
d)
data which document the stability of the cell line under the cultivation conditions used,
especially if cells are to be sub cultured for an extended or indefinite period;
e)
if serum is included in the medium of production cell cultures, evidence that it is free
from bacteria, fungi, mycoplasma and viruses.
engineered
organisms
- strategy
for
Expression vector and host cell: A description of the host cell and expression vector
used in production should be given. This should include details of the origin and
identification of the gene which is being cloned and the construction, genetics and
structure of the expression vector. The method by which the vector is introduced into the
host cell and the state of the vector within the host should be described. The association
of the vector and host cell may be permanent, allowing continuous expression of the
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Sequence of the cloned gene: Full details of the nucleotide sequence of the gene insert
and of flanking control regions of the expression vector should be provided. All
relevant expressed sequences should be clearly identified. The DNA sequence of the
cloned gene should normally be confirmed at the seed lot stage and at least once after
a full scale fermentation. In certain systems, for example where multiple copies of the
gene are inserted into the genome of a continuous cell line, it may be inappropriate to
sequence the cloned gene at the production level. Under these circumstances, Southern
blot analysis of total cellular DNA or sequence analysis of the mRNA may be helpful
and particular attention should be paid to the characterisation of the final product.
c)
Expression: The strategy by which the expression of the relevant gene is promoted and
controlled during production should be described in detail.
3.3.2 Production
Ideally not more than one cell line should be cultivated simultaneously in the same
production area or the cultivation should be carried out in closed vessels with effective
barriers which would prevent contamination with other adventitious agents or other cell
lines. If other cell lines are cultivated in parallel, records must be kept of the cell lines
handled and evidence presented for the absence of cross-contamination between them.
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a)
b)
Continuous culture production: This approach should only be undertaken when special
consideration has been given to the control of production based on continuous culture.
Where it is undertaken, monitoring of the production system is necessary throughout
the life of the culture. The required frequency and type of monitoring will depend upon
several factors including the nature of the expression system and product. Information
should be obtained on the molecular integrity of the gene being expressed and of the
phenotypic and genotypic characteristics of the host cell after long term cultivation.
Evidence should be provided that the yield does not vary beyond defined limits and that
the nature and quality of the product does not change with respect to specific
parameters. The acceptance of harvests for further processing should be clearly linked
to the schedule of monitoring applied. The period of continuous cultivation should be
specified and this should be based on information concerning the stability of the
system and consistency of the product up to and beyond this limit. In cases on long
term continuous cultivation, the cell line and product should be completely re-evaluated
at intervals based on information concerning the stability of the system and the
characteristics of the product.
A clear definition of a "batch" of product for further processing should be provided.
Regular tests for microbial contamination should be performed in relation to the
strategy for harvesting. Criteria for rejection of harvests and premature termination of
the culture should be defined.
3.4 P u r i f i c a t i o n of t h e p r o d u c t
3.4.1
Methods
Methods used to purify the product should be described in detail. Procedures which make use
of affinity chromatography, for example employing monoclonal antibodies, should be
accompanied by appropriate measures to ensure that these substances, or any additional
potential contaminants arising from their use, do not compromise the quality and safety of
the final product. Attention is drawn to the note for guidance Production and Quality Control
of Monoclonal Antibodies.
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3.4.2 Validation
of the purification
procedure
The capacity of the purification procedure to remove unwanted host cell derived proteins,
nucleic acids, viruses and other impurities should be investigated thoroughly, as should the
reproducibility of the purification process as regards its ability to remove specific
contaminants and the consistent composition of the purified product with respect to any
impurities which may be present. Laboratory scale pilot studies which mimic the production
process using, for example, a carefully selected group of viruses which exhibit a range of
physico-chemical features relevant to their behaviour during the process of purification, or
radioactively labelled DNA intentionally mixed with the crude preparation (spiking) should
be undertaken. A reduction factor for such contaminants at each stage of purification should
be established by using, if necessary, concentrations of DNA and viruses in excess of that
expected during normal production.
of the purified
cytokine
Purity
Data should be provided on contaminants which might be present in the final processed
product. The level of contamination considered as acceptable and criteria for acceptance or
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rejection of a production batch should be given. It is important that the techniques used to
demonstrate purity be assessed using as wide a range of methods as possible, including
physico-chemical and immunological techniques. Particular emphasis should be placed on
tests for viral and nucleic acid contamination and for other unwanted materials of host
origin, as well as on materials which may have been added during the production or
purification processes.
3.6.1 Consistency
An acceptable number of successive batches of the purified cytokine bulk solution should be
characterised as fully as possible to determine consistency of composition. The studies
should include biological, chemical and immunological methods to characterise and assay
the cytokine and methods to detect and identify impurities. Any differences which occur
between batches should be noted. The data obtained from these consistency studies should be
used as the basis for product specification.
3.6.2
Identity
A selection of the tests used to characterise the purified cytokine (see section 3.4) should be
used to confirm the product identity for each batch. The methods employed should include
tests for the anticipated biological activity as well as physico-chemical and immunological
methods. Depending on the extent of other identification tests, sequence verification of a
number of amino acids at the N- and C- terminus or other methods such as peptide mapping
should be performed.
3.6.3
Purity
The purity of each batch should be established and be within specified limits. The analysis
should include sensitive and reliable assays for DNA of host cell origin applied to each
batch of product prepared from continuous lines of transformed mammalian cells. Strict
upper limits should be set for DNA in the product. It is recommended that DNA analyses are
also performed on each batch of product obtained from other eukaryotic cells, and limits set
for DNA content, until further information on safety is obtained. DNA of prokaryotic
expression systems (e.g. of vector or plasmid origin) should be tested for when considering
the quality and safety of the product. For products to be administered chronically, or in high
doses, the residual cellular proteins should also be determined by an assay with appropriate
sensitivity (e.g. ppm) and limits be established for these.
3.6.4
Potency
The potency of each batch of the cytokine product should be established (e.g. units of
biological activity per ml) using, wherever possible, an appropriate national or international
reference preparation calibrated in units of biological activity (see section 3.7).
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In addition, information on specific activity (units of biological activity per unit weight of
product) is of considerable value and should be reported. A highly purified reference
preparation is required to standardise measurements of specific activity (see section 3.7).
It is recommended that correlations between potency measurements, involving biological
tests, and the results of physico-chemical methods of assay are made and the information
reported. If possible, batches should be calibrated using accurate physico-chemical tests, and
the biological assays used to confirm - within stated limits - that the product is biologically
potent.
4.
FINISHED PRODUCT
Where appropriate, the product in final containers should be shown to comply with the
requirements of the relevant European directives. In circumstances where this is not
possible, the omission of tests should be justified by the manufacturer.
The manufacturer shall take samples from each final lot for the following tests.
4.3 Potency
The test for potency should utilise a bioassay unless otherwise justified. An appropriate
reference preparation should be tested in parallel. These tests shall be performed on samples
representative of the final filling lots. Statistical analysis of the data should show that the
mean potency value obtained has the confidence limits accepted by the competent authority.
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GLOSSARY
1.
A quantity of cells able to produce the intended cytokine stored at -70C or below in aliquots of
uniform composition, one or more of which is used for the production of a Manufacturer's
Working Cell Bank.
2.
A single uniform suspension of cells which have been dispensed in a single working
session into a number of containers which are stored at -70C or below. Cells revived from
one or more of these containers are used as a source of Production Cell Cultures.
3.
The cell cultures derived from one or more containers of MWCB which in the production
process are able to form or are induced to form the intended cytokine.
4.
Inducer
A substance added to a cell culture which leads to or stimulates the production of the intended
cytokine.
5.
Enhancers
Any substances added to an induced cell culture which improve the final yield of the
intended cytokine.
6.
Single Harvest
The resultant cytokine solution from a single harvest, which has been taken through a
designated purification process.
8.
Either the purified cytokine solution of the resultant of blending two or more batches of
purified cytokine solution.
9.
The purified cytokine bulk solution to which excipients, e.g. stabilisers, have been added.
10.
Final Bulk
The formulated cytokine bulk solution (or the finished biological material) present in the
container from which the final containers are filled.
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11.
Final Lot
A collection of sealed final containers that are homogeneous with respect to the risk of
contamination during filling or preparation of the finished product. A final lot must
therefore consist of finished material obtained during one working session from a single
final bulk.
12.
Numerous samples from one or more final lots of material which has been shown to be
active in clinical use, or are directly related to such material e.g. purified cytokine bulk
solution, and which has been fully characterised in ways to be specified by the competent
authority, appropriately stored by the manufacturer to serve as a reference material for
several years. For certain critical tests, such reference material shall be included i n
parallel with each lot of production material, which must match the specification of the
reference batch within limits to be agreed by the competent authority.
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Guideline Title
CONTENTS
1.
INTRODUCTION
2.
3.
SOURCE CELLS
4.
5.
6.
7.
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8.
PROD
U CTION
9.
U
P RIFICATION OF THE ANTD30DY
10.
11.
12.
13.
14.
15.
ANNEX I
ANNEX II
ANNEX III (GLOSSARY)
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INTRODUCTION
In this document the requirements for murine, human and engineered monoclonal
antibodies for therapeutic (including ex vivo application) and in vivo diagnostic use i n
humans are outlined. Monoclonal antibodies intended for use in the purification of other
products should be shown to be pure and free from adventitious agents by the methods
described. Monoclonal antibodies to be used for diagnostic purposes in vitro are not the
concern of this note for guidance.
Monoclonal antibodies are antibodies with a defined specificity derived from cloned cells or
organisms. They can be obtained from immortalised lymphocytes that are cloned and
expanded as continuous cell lines (murine and human monoclonal antibodies) or from
rDNA-engineered mammalian or bacterial cell lines (engineered monoclonal antibodies).
Important considerations for the clinical use of monoclonal antibodies include the possible
unintentional immunological cross-reactivity of the antibody with human tissue antigens
other than those desired, and the possible presence of viruses in the products.
11
M o n o c l o n a l a n t i b o d i e s of m u r i n e o r i g i n
Murine monoclonal antibodies are obtained from murine hybridomas produced by fusion of
B-lymphocytes from immunised mice or rats with murine myeloma cells.
A general problem with the therapeutic use of murine monoclonal antibodies in man is the
possible induction of antibodies in the recipient against murine immunoglobulin (human
anti murine antibody or HAMA response). This may result in adverse reactions and limit
the duration of effective antibody therapy. In addition the in vivo half life of murine
monoclonal antibodies is relatively short. It may be prudent to minimise the murine protein
load administered to the patient by the use of a parental myeloma cell lines which does not
itself synthesise immunoglobulin chains.
1.2
Human monoclonal
antibodies
The advantages of human monoclonal antibodies over murine monoclonal antibodies are
that human recipients are less likely to develop antibodies against them (although antiidiotypic and possibly anti-allotypic antibodies may still be produced) and that human
antibodies are likely to have the full range of biological functions, such as those of the Fc
region which may be species specific. There may be other advantages such as selection of a
subclass of antibody with particular properties.
Murine monoclonal antibodies are almost always prepared using cell lines (hybridomas)
made by fusion of lymphocytes from an immunised donor with myeloma cells. This is not
the case for human monoclonal antibodies as, despite encouraging early reports, there is
still no really satisfactory human myeloma fusion partner. As a result, a major difficulty
with the production of human monoclonal antibodies has been the generation of hybridoma
lines of acceptable stability. It is also difficult in many cases to obtain antigen-primed
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lymphocytes suitable for fusion. In view of this, a number of alternative strategies have been
devised for production of human monoclonal antibodies. These are:
a)
b)
c)
d)
Other methods for generating stable lines secreting human antibodies may be developed or
exploited in future.
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2.
3.
SOURCE CELLS
Whenever possible, murine tissue and animals used as source materials should be shown to
be free of viruses as indicated in Annex I (a), table 2.
Monoclonal antibodies obtained from human cells present particular concerns regarding
safety. Human monoclonal antibodies for use in humans are currently unique in that they
are often derived from cells which are likely immortalised by the deliberate introduction of
EBV, a potential human pathogen. They are likely to be obtained from a transformed human
cell line which is potentially oncogenic. Evidence of contamination with viruses originating
from the donor is cause for concern, as they will by definition be viruses capable of infecting
humans. Cells from human origin should be shown free of viruses indicated in Annex I (a)
table 3.
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3.1
C h a r a c t e r i s a t i o n of n o n - s p e c i f i c c e l l s
3.1.1 Feeder
cells
Whenever appropriate, the origin of feeder cells used should be defined. Feeder cells should
be derived from SPF (specific pathogen free) animals or cell seed stocks shown to be free of
microbial contamination such as mycoplasma, bacteria and fungi and special consideration
should be given to possible exogenous viral contamination.
3.1.2 Fusion
partner(s)
The fusion partner used (e.g. myeloma, human lymphoblastoid B-cell line) should be fully
described and documented. The source, name and characteristics of the parental cell line
should be given. It should be shown that the cell line is a pure culture and is not
contaminated with cells of other types. If possible the cell line used as fusion partner should
be selected as one which does not synthesise any immunoglobulin chains. Cryopreserved
samples of the cell line used as fusion partner should be retained in case retrospective
investigations become necessary.
3.1.3 Host cell for the expression
of the recombinant
monoclonal
antibody
A description of the starting host strain or cell line should be provided including the history
of the strain or cell line, its identification characteristics and potential viral contaminants.
Special attention should be given to the possibility of unintended cross-contamination with
other cell lines or viruses not endogenous to a particular cell line.
The cell line used should not synthesise any endogenous immunoglobulin chains before and
after transfection.
Cryopreserved samples of the host cell line should be retained in case retrospective
investigations become necessary.
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4.
5.
5.1 Cloning and characterisation of the DNA coding for the nonspecific part of the recombinant mAb
For both chimeric and humanised monoclonal antibody a description of the origin, isolation
and cloning strategy of the heavy and the light chain genes should be provided. In addition
the following information is required:
i)
the introduced framework residue substitutions in humanised monoclonal antibodies
to improve the CDR conformation (where applicable).
ii)
iii)
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cell lines and vectors used in the generation of the monoclonal antibody, and a
description of the expression vectors used for the transfection of rDNA antibody genes
into the mammalian host cell line, including the origin, structure and selection
markers.
ii)
for both the heavy and light chain expression vectors the nucleotide sequences of the
genes of interest and the flanking control regions. A detailed map indicating the
regions which have been sequenced during construction and those deduced from the
literature should be given. All the junction regions created by ligation during
construction should be confirmed by sequencing.
iii)
iv)
the methods used for introducing the vector into the host cell, the selection and cloning
of the transformants.
ii)
iii)
a detailed study using various restriction enzymes and Southern blot analysis
providing convincing data on the integrity in the host cell. Useful information is
provided on the expression system by Northern blot analysis.
iv)
ii)
characterisation of the monoclonal antibody. Analysis at the protein level and/or DNA
level can be envisaged. Whichever method is used, it should be validated and the
detection limit should be given.
iii)
the level and consistency of expression of both the heavy and light chain.
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6.
6.1
It is essential that production is based on a well defined cell bank system. This will
normally involve a Master Cell Bank (MCB) and a Manufacturer's Working Cell Bank
(MWCB). During the establishment of the cell bank no other cell lines should be handled
simultaneously in the same laboratory suite or by the same persons. The origin, form,
storage, use, and details of life expectancy at the anticipated rate of use must be described i n
full for all cell banks. New working cell banks should be fully characterised.
Samples of the working cell bank should be retained until at least after the expiry date of the
resulting final lot.
6.2 C o n t r o l of v i r o l o g i c a l a n d m i c r o b i a l c o n t a m i n a t i o n
The various cell levels, including MCB, MWCB and PPCB (Post Production Cell Bank; see
6.5) should be examined for adventitious agents (viral, bacterial, fungal or mycoplasmal).
Special attention should be given to viruses which can commonly contaminate the species
from which the cell line has been derived. Appropriate tests to demonstrate the absence of
virus contamination as described in Annex I should be performed.
Certain cell lines contain endogenous viruses, e.g. retroviruses, which may not readily be
eliminated. Furthermore, potential viral contamination may take the form of complete viral
genomes or subgenomic fragments resulting in the expression of infectious viral particles.
Therefore the possibility of mutations of endogenous viruses during prolonged culture should
be considered. The presence of sequences from viral genomes may not disqualify use of the
cells, but any exogenous viral nucleic acid found should be identified. If the
heterohybridoma approach is used for construction of the antibody secreting cell line the cell
bank should be examined for the presence of murine and human viruses.
A cell line which produces any infectious virus capable of infecting human cells would be
acceptable only in exceptional circumstances. All products derived from such lines would
have to be considered on a case by case basis. If the cell line secretes infectious virus,
appropriate precautions should be taken to protect personnel involved in production from
infection.
There is special concern about the use of cell lines transformed by the deliberate introduc
tion of EBV for the production of human monoclonal antibodies. Despite the fact that EBV
transformed human cells in general do not secrete viral particles these cells contain
complex copies of the viral genome and EBV sequences should be sought by PCR or by cocultivation with suitable indicator cell lines.
6.3
Characterisation
A critical part of quality control will involve the full characterisation of cells. The identity
of the cells should be established by distinguishing markers of the cell, such as specific
isoenzyme and immunological features, and phenotypic characterisation.
If the EBV transformation procedure is used alone for the generation of a cell line for the
production of human monoclonal antibodies difficulties can arise in the cloning procedure.
It is therefore essential that manufacturers show convincing evidence that the cell line i s
monoclonal.
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7.
8.
PRODUCTION
In vitro production is the preferred method of production. During the last few years the
technology for in vitro production of monoclonal antibodies has been considerably improved.
In vitro production of monoclonal antibodies offers a high degree of control and
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standardisation and has important advantages over in vivo production with respect to viral
safety, consistency of production and the absence of contaminant immunoglobulins in the
crude harvest. In vitro production in serum free medium is also now feasible. Another
advantage of this method of production is the considerable reduction of animal usage.
Manufacturers should be aware of Directive 86/609/EEC concerning the protection of
animals used for experimental and other scientific purposes.
If in vivo production is chosen it must be justified by the manufacturer.
A clear definition of a "batch" of product for further processing should be provided. A
production batch should normally originate from a fresh ampoule of the MWCB. Details of
the culture with the in-process controls should be provided. Criteria for rejection of the
harvests and premature termination of the culture should be defined.
8.1
In vitro production
For each production run, the presence, extent and nature of any microbial contamination i n
the culture vessels immediately prior to all harvesting must be thoroughly examined.
Detailed information to confirm the adequate sensitivity of the methods used to detect
contamination should be provided and acceptable limits of contamination set. The bulk
culture fluid should be shown to be free from mycoplasmal, mycotic and bacterial
contamination and should be tested for the presence of viruses using a general test
involving inoculation into suitable cell substrates (see Annex I, b).
The composition and source of the cell culture medium used for production should be
recorded. If animal serum-derived additives are used, they should be shown to be free from
adventitious agents (See 2.2.).
Ideally not more than one cell line should be cultivated simultaneously in the same
production area. If other cell lines are cultivated in parallel, records must be kept of the cell
lines handled and evidence presented for the absence of cross contamination between them.
production
The maximum permitted generation number for production should be defined and should be
based on information concerning the stability of the cell line or the up to and beyond the
level of production. Data on consistency of growth of the culture and on the maintenance of
yield within specified limits should be presented. Appropriate monitoring of the cell line
characteristics at the end of the production cycles should also be undertaken. Evidence
should be provided that the yield does not vary beyond defined limits and that the nature and
quality of the product does not change with respect to specific parameters.
production
The period of continuous cultivation should be specified and this should be based on
information concerning the stability of the system and consistency of the product up to and
beyond this limit. Monitoring of the production system is necessary throughout the life of the
culture. The required frequency and type of monitoring will depend upon several factors
including the nature of the expression system and monoclonal antibody, as well as the total
length of the period of continuous cultivation undertaken. The acceptance of harvests for
further processing should be clearly linked to the schedule of monitoring applied. Evidence
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should be provided that the yield does not vary beyond defined limits and that the nature and
quality of the monoclonal antibody does not change with respect to specific parameters.
of the animals
used
The strain and origin of the animals used for production should be specified, together with
their genotype and age. They should be from a closed, specific pathogen-free (SPF) colony
which is routinely monitored for those viruses listed in Annex I Table 2. The long term
records of the breeding colony in respect of freedom from viral contamination should be
considered in relation to the reliability of maintenance of the colony. Evidence should also
be presented that animals are maintained under SPF conditions at all times during transfer
and use.
8.2.2 Harvest
and handling
of ascitic
fluid
Each production batch should originate from a fresh ampoule of the MWCB. The maximum
permissible number of serial passages in vivo during normal production should be defined
and restricted: justification of this limit should include information concerning the yield of
monoclonal antibody and the stability of the hybridoma characteristics on in vivo passage up
to beyond that used in production. Indefinite passage in animals is not acceptable. A scheme
of priming, inoculation and harvesting should be provided.
The number of animals and the procedure used to prepare the bulk ascitic harvest should be
given in detail. Full details should be provided on any substances used to pre-treat mice or
rats to facilitate growth of hybridomas. Description, volume and concentration of cell
inoculum should be given. Data concerning the titre of the antibody in and storage
conditions of the bulk ascitic fluid should be provided (e.g. temperature, duration, details of
any proteolytic enzyme inhibitors added). Particular attention should be paid to the degree
and nature of any microbial contamination (bacterial, mycotic and mycoplasmal) in the
bulk ascitic fluid. Testing procedures capable of detecting all of the murine viruses listed i n
Annex I Table 2 should be performed, as indicated in Annex 1(a) and (b), on at least the first
five bulk harvests of the product. However, it may be expected that general testing methods
for viruses may be sufficient as experience of production is gained. Consequently, after the
first five production runs, general testing for viruses, limited to those described in Annex I
(b), may be considered adequate. Any infectious agent should be identified and tests for
viruses in Group I Table 2 should be negative. If the source of mice is changed to a different
colony or supplier, tests described in Annex 1(a) should be performed on at least the first five
bulk harvests to re-establish consistency of freedom from contaminant agents.
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9.
9.1 Methods
Methods used to purify the product and their in-process controls including their specification
limits should be described in detail, justified and validated. It is important to ensure that
purification procedures do not impair relevant immunobiological features of the
immunoglobulin. Procedures which make use of affinity chromatography, for example
employing monoclonal antibodies, should be accompanied by appropriate measures to ensure
that these substances, or any additional potential contaminants arising from their use, do
not compromise the quality and safety of the final product. Cross reference is made to the
note for guidance Virus Validation Studies: The Design, Contribution and Interpretation of
Studies Validating the Inactivation and Removal of Viruses.
The criteria for reprocessing of any intermediate or final bulk should be carefully defined,
validated and justified.
Consideration should be given to incorporating procedures which inactivate/eliminate
potential viral contaminants where such methods will not compromise the biological activity
of the product.
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10. T h e b u l k final p r o c e s s e d p r o d u c t
10.1 T h e monoclonal a n t i b o d y
Rigorous characterisation of the purified monoclonal antibody by chemical and biological
methods will be essential. At least the following parameters should be determined: class,
subclass and light-chain composition, glycosylation pattern, integrity of the molecule by
analysis of the ratio heavy/light chain, microheterogeneity, molecular weight, N- and Cterminal amino acid sequence, and secondary and tertiary structure of the antibody. With
increasing experience, th tests for subclass, light chain composition, N- and C- terminal
amino acid sequence and secondary and tertiary structure could be omitted. The total protein
content, the degree of aggregation and molecular fragmentation of the immunoglobulin
should be determined. Appropriate specifications for these parameters, with acceptance
limits, should be set. Especially for engineered and humanised antibodies sufficient
sequence information to characterise the gene product adequately should be obtained by
peptide mapping or amino acid sequencing.
Particular attention should be given to use a wide range of analytical techniques exploiting
different physico-chemical properties of the molecule. Examples of suitable techniques are:
SDS-polyacrylamide gel electrophoresis under reducing and non reducing conditions,
isoelectric focusing, column chromatography (including HPLC), peptide mapping, amino
acid analysis, circular dichroism and carbohydrate mapping. The manufacturer should
provide clear photographs of the gels, etc..
The immuno-reactivity of the antibody should be assessed. The specific activity of the
purified monoclonal antibody should be determined (units of activity/weight of product).
A clear difference should be made between the analytical tests performed during
development, in order to fully characterise the monoclonal antibody and tests performed
routinely on each batch of purified bulk product. Quality control tests should be carried out
routinely on each batch of purified bulk product according to the Guide to GMP.
10.2 P u r i t y
Data should be provided on contaminants whose presence is anticipated in the final
processed product. The level of contamination considered as acceptable should be justified,
and criteria of acceptance or rejection of a production batch should be given. It is important
that the techniques used to demonstrate purity be assessed using as wide a range of methods
as possible, including physico-chemical and immunological techniques. These should
include tests for viral and cellular nucleic acid and protein contamination of parental,
hybridoma, or host cell origin, as well as on materials derived from tissue culture medium
or materials which have been added during the production or purification processes.
Measurements of total protein and cellular DNA concentrations, specific activity,
microbiological and chemical purity should be reported for the final product. Assays of
endotoxin level should also be carried out.
10.3 A d v e n t i t i o u s a g e n t s
The final bulk product should be shown to be free from bacterial, fungal and mycoplasmal
contamination. Evidence should be presented to show that any viral contaminant known to
be possibly present in the bulk harvest has been eliminated or inactivated (see Annex I).
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111 Consistency
Evidence should be provided on the consistency of production, for example on at least five
consecutive full scale production batches. This should include information on the bulk
harvest and final dosage form as well as on in-process controls. In the case of a production
where multiple harvests are applied, batches from different fermentation runs are needed.
The studies should include biological, chemical and immunological methods to characterise
and assay the monoclonal antibodies and methods to detect and identify impurities. Any
differences which occur between batches should be noted.
Purity
The degree of purity desirable and attainable will depend on several factors; these include
the nature and intended use of the product, method of its production and purification and also
the degree of consistency of the production process. The purity of each batch should be
established and be within specified limits. For engineered monoclonal antibodies the
analysis should include sensitive and reliable assays for DNA of host cell origin and the
vectors applied to each batch of product. Strict upper limits should be set for DNA in the
product.
The product should be shown to be free from microbial contamination. Evidence should be
presented to show that any viral contaminant known to be present in the bulk harvest has
been eliminated or inactivated (see Annex I). Pyrogenicity should be tested for.
Particular attention should be given to assessment of the degree of aggregation or molecular
fragmentation of the immunoglobulin. All possible steps should be taken to prevent
aggregation. Limits for the presence of oligomeric immunoglobulin molecules should be
justified.
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European directives and pharmacopoeias. In circumstances where this is not possible the
omission of tests should be justified by the manufacturer.
15.1 I n v i t r o s t u d i e s o n p r o d u c t e q u i v a l e n c e
The physico-chemical characteristics of the monoclonal antibody, like isotype, subclass,
microheterogeneity, molecular weight, primary structure, secondary structure, glycosylation
pattern, structural integrity, should be determined. The biological characterisation should
include immunoreactivity and crossreactivity, the determination of relevant functional
characteristics and binding studies to determine affinity.
When there are changes in the cell culture procedure/conditions without changes in the
MCB, relevant parameters such as morphology, cell growth, viability, isoenzymes, and
stability of production should be analysed.
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ANNEX I
Testing for viruses should be performed in laboratories with experience in routine virus
testing and should be performed in accordance with good laboratory practice.
Table 1 lists the tests for viruses to be performed at the different stages of production.
Table 2 lists viruses which should be considered as potential contaminants
manufacture of monoclonal antibodies produced by cell lines of murine origin.
in the
in the
(ii)
254
Inoculation of cell cultures capable of detecting a wide range of murine, human, and,
if relevant, bovine viruses. Examples of useful cell types (substrates) are: murine
fibroblast cultures, e.g. mouse embryo cultures; human fibroblast cultures, e.g. human
diploid cells such as MRC5; continuous cell lines of human, murine and bovine
origin. The indicator cell lines should additionally be tested for haemadsorbing
viruses (with erythrocytes from human blood group O, guinea pig, chicken) at the end
of the observation time. Tests for retroviruses should be included.
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c)
2 litters of suckling mice, comprising at least 10 animals less than 24 hours old
10 adult mice
5 guinea-pigs
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TABLE 1
TESTING SCHEME FOR VIRAL CONTAMINANTS
Annex I sections which are applicable
MCB or MWCB
Mouse breeding Colony
Ascitic fluid harvest
In vitro bulk harvest
(a)
(a)
(a)*
256
(b)
(c)
(b)
(b)
Specified tests of (b) if virus contamination was detected i n
the bulk harvest
It is proposed that these tests should be carried out on at least the first five production
runs.
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TABLE 2
MURINE VIRUSES
Group
Virus
Species Affected
M, R
M
R
M, R
M, R
II
Ectromelia virus*
K virus
Kilham rat virus (KRV)
Lactic dehydrogenase virus (LDH)
Minute virus of mice (MVM)
Mouse adenovirus (MAV)
Mouse cytomegalovirus (MCMV)
Mouse encephalomyelitis virus (MEV, Theiler's or GDVII)
Mouse hepatitis virus (mhv)
Mouse rotavirus (EDIM)
Pneumonia virus of mice (PVM)
Polyoma virus
Rat cornavirus (RCV)
Retrovirus*
Sialodacryoadenitis virus (SDA)
Thymic virus
Toolan virus (HI)
M
M
R
M
M, R
M
M
M
M
M
M, R
M
R
M, R
R
M
R
M - mouse
R-rat
Viruses for which evidence exists of a capacity to infect man or primates are to be found i n
Group I. Those viruses for which there is no evidence of infection in man but which could
nevertheless pose a potential danger, for example in immunocompromised individuals, are
listed in Group II. Viruses which are known to be capable of replicating in vitro in cells of
human and monkey origin are indicated by * in Table 2.
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TABLE 3
HUMAN VIRUSES
Virus
Human Immunodeficiency Virus (Type 1, Type 2)
Human cell Leukaemia Virus (Type I, Type II)
Cytomegalo virus
HHV6
Epstein - Barr virus
Hepatitis virus
Hepatitis C virus
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ANNEX II
Suggested list of human tissues to be used for immunohistochemical or cytochemical
investigations of cross reactivity of monoclonal antibodies. This list should reflect the
specificity of the antibody and its particular use.
Tonsil, thymus, lymph node
Bone marrow, blood cells
Lung, liver, kidney, bladder, spleen, stomach, intestine
Pancreas, paratid, thyroid, para-thyroid, adrenal, pituitary
Brain, peripheral nerve
Heart, striated muscle
Ovary, testis
Skin
Blood vessels
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ANNEX III
Glossary
1.
Murine
"Murine" means derived from an animal belonging to the M uridae family which includes
mice and rats.
2.
a)
Cell Banks
M aster cell bank (M CB)
A homogeneous suspension of the original cells on which production is based, aliquoted into
individual containers for storage (e.g. in a liquid nitrogen refrigerator). The original cell
line may not necessarily have been produced by the manufacturer.
For engineered products the cells in the master cell bank are already transformed by the
expression vector containing the desired gene. In some cases it may be necessary to esta
blish separate master cell banks for the expression vector and the host cells.
b)
A homogeneous suspension of cells derived from the master cell bank(s) by a finite passage
level, aliquoted into individual containers for storage (e.g. in a liquid nitrogen
refrigerator).
In both cell banks, all containers are treated identically during storage, and once removed
from storage, the containers are not returned to the cell bank stock.
c)
Post production cells are the cells cultured up to 10 or more population doublings beyond the
maximum population doubling level used for routine production (single harvest production)
or cells cultured for a period of time which exceeds the total length of the cultivation period
by one third (multiple harvest production).
3.
Production Method
a)
The number of population doublings are specified based on information concerning the
stability of the system and the consistency of the product criteria for the termination has to be
defined by the manufacturer.
4.
Bulk Harvest
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5.
This is the final product, after completion of the production process, obtained from a bulk
harvest. It is maintained in one or more containers and used in the preparation of the final
dosage form. The generation of this final batch has to be clearly defined and unambiguously
recorded by the producer.
6.
Finished Product
The active substance is formulated and filled into final, sealed containers which hold the
product in its final dosage form, i.e. the finished product. The containers of a filling lot are
processed together and uniform in their contents and biological potency.
7.
A human monoclonal antibody in which critical amino acid residues are replaced by
molecular technology.
8.
Fusion Partner
A cell line fused with the antibody producing cell with the intention to immortalise this cell.
9.
Feeder Cells
Cells or cell line co-cultivated with the antibody producing cell line to provide optimal
growth conditions.
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CONTENTS
1.
PREAMBLE
2.
3.
TERMINOLOGY
4.
SELECTION OF BATCHES
5.
6.
STORAGE CONDITIONS
7.
TESTING FREQUENCY
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8.
SPECIFICATIONS
9.
LABELLING
GLOSSARY
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PREAMBLE
The guidance stated in the ICH Harmonised Tripartite Guideline Stability Testing of New
Drug Substances and Products, hereafter called Tripartite Guideline on Stability, (published
in this volume under the title Stability Testing of New Active Substances and Medicinal
Products)
applies in general
to biotechnological/biological
products. However,
biotechnological/biological products do have distinguishing characteristics to which
consideration should be given in any well-defined testing program designed to confirm
their stability during the intended storage period. For such products, in which the active
components are typically proteins and/or polypeptides, maintenance of molecular
conformation and, hence of biological activity, is dependent on noncovalent as well as
covalent forces. The products are particularly sensitive to environmental factors such as
temperature changes, oxidation, light, ionic content, and shear. In order to ensure
maintenance of biological activity and to avoid degradation, stringent conditions for their
storage are usually necessary.
The evaluation of stability may necessitate complex analytical methodologies. Assays for
biological activity, where applicable, should be part of the pivotal stability studies.
Appropriate physico-chemical, biochemical and immunochemical methods for the analysis
of the molecular entity and the quantitative detection of degradation products should also be
part of the stability program whenever purity and molecular characteristics of the product
permit use of these methodologies.
With the above concerns in mind, the applicant should develop the proper supporting stability
data for a biotechnological/biological product and consider many external conditions which
can affect the product's potency, purity and quality. Primary data to support a requested
storage period for either an active substance or medicinal product should always be based on
long-term, real-time, real-condition stability studies. Thus, the development of a proper longterm stability program becomes critical to the successful development of a commercial
product. The purpose of this document is to give guidance to applicants regarding the type of
stability studies that should be provided in support of marketing applications. It is understood
that during the review and evaluation process, continuing updates of initial stability data
may occur.
2.
The guidance stated in this annex applies to well-characterised proteins and polypeptides,
their derivatives and products of which they are components, and which are isolated from
tissues, body fluids, cell cultures, or produced using rDNA technology. Thus, the document
covers the generation and submission of stability data for products such as cytokines
(interferons, interleukins, colony-stimulating
factors, tumour necrosis
factors),
erythropoietins, plasminogen activators, blood plasma factors, growth hormones and growth
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3.
TERMINOLOGY
For the basic terms used in this annex the reader is referred to the "Glossary" in the
Tripartite
Guideline
on
Stability.
However,
since
manufacturers
of
biotechnological/biological products sometimes use traditional terminology, traditional
terms are specified in parentheses to assist the reader. A supplemental glossary is also
included that explains certain terms used in the production of biotechnological/biological
products.
4.
SELECTION OF BATCHES
4.2 Intermediates
During manufacture of biotechnological/biological products, the quality and control of
certain intermediates may be critical to the production of the final product. In general, the
manufacturer should identify intermediates and generate in-house data and process limits
that assure their stability within the bounds of the developed process. While the use of pilot-
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plant-scale data is permissible, the manufacturer should establish the suitability of such data
using the manufacturing-scale process.
4.4
Where one product is distributed in batches differing in fill volume (e.g. 1 millilitre (ml),
2 ml, or 10 ml), unitage (e.g. 10 units, 20 units, or 50 units), or mass (e.g. 1 milligram (mg),
2 mg, or 5 mg) samples to be entered into the stability program may be selected on the basis
of a matrix system and/or by bracketing.
Matrixing, i.e. the statistical design of a stability study in which different fractions of
samples are tested at different sampling . points, should only be applied when appropriate
documentation is provided that confirms that the stability of the samples tested represents the
stability of all samples. The differences in the samples for the same medicinal product
should be identified as, for example, covering different batches, different strengths, different
sizes of the same closure and possibly, in some cases, different container/closure systems.
Matrixing should not be applied to samples with differences that may affect stability, such as
different strengths and different containers/closures, where it cannot be confirmed that the
products respond similarly under storage conditions.
Where the same strength and exact container/closure system is used for three or more fill
contents, the manufacturer may elect to place only the smallest and largest container size
into the stability program, i.e., bracketing. The design of a protocol that incorporates
bracketing assumes that the stability of the intermediate condition samples are represented
by those at the extremes. In certain cases, data may be needed to demonstrate that all
samples are properly represented by data collected for the extremes.
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5.
STABILITY-INDICATING PROFILE
On the whole, there is no single stability-indicating assay or parameter that profiles the
stability characteristics of a biotechnological/biological product. Consequently, the
manufacturer should propose a stability-indicating profile that provides assurance that
changes in the identity, purity and potency of the product will be detected.
At the time of submission, applicants should have validated the methods that comprise the
stability-indicating profile and the data should be available for review. The determination of
which tests should be included will be product-specific. The items emphasised in the
following subsections are not intended to be all-inclusive, but represent product
characteristics that should typically be documented to adequately demonstrate product
stability.
5.1
Protocol
The dossier accompanying the application for marketing authorisation should include a
detailed protocol for the assessment of the stability of both active substance and medicinal
product in support of the proposed storage conditions and expiration dating periods. The
protocol should include all necessary information which demonstrates the stability of the
biotechnological/biological product throughout the proposed expiration dating period
including, for example, well-defined specifications and test intervals. The statistical
methods that should be used are described in the Tripartite Guideline on Stability.
5.2
Potency
When the intended use of a product is linked to a definable and measurable biological
activity, testing for potency should be part of the stability studies. For the purpose of stability
testing of the products described in this guideline, potency is the specific ability or capacity of
a product to achieve its intended effect. It is based on the measurement of some attribute of
the product and is determined by a suitable quantitative method. In general, potencies of
biotechnological/biological products tested by different laboratories can be compared in a
meaningful way only if expressed in relation to that of an appropriate reference material.
For that purpose, a reference material calibrated directly or indirectly against the
corresponding national or international reference material should be included in the assay.
Potency studies should be performed at appropriate intervals as defined in the stability
protocol and the results should be reported in units of biological activity calibrated, whenever
possible, against nationally or internationally recognised standard. Where no national or
international standards exists, the assay results may be reported in in-house derived units
using a characterised reference material.
In some biotechnological/biological products, potency is dependent upon the conjugation of
the active substance(s) to a second moiety or binding to an adjuvant. Dissociation of the
active substance(s) from the carrier used in conjugates or adjuvants should be examined i n
real-time/real-temperature studies (including conditions encountered during shipment). The
assessment of the stability of such products may be difficult since, in some cases, in vitro
tests for biological activity and physico-chemical characterisation are impractical or provide
inaccurate results. Appropriate strategies (e.g. testing the product prior to
conjugation/binding, assessing the release of the active compound from the second moiety,
in vivo assays) or the use of an appropriate surrogate test should be considered to overcome
the inadequacies of in vitro testing.
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the medicinal product, these items may need to be monitored during the stability
program.
The container/closure has the potential to adversely affect the product and should be
carefully evaluated (see below).
6.
STORAGE CONDITIONS
6.1 Temperature
Since most finished biotechnological/biological products need precisely defined storage
temperatures, the storage conditions for the real-time/real-temperature stability studies may
be confined to the proposed storage temperature.
6.2 Humidity
Biotechnological/biological products are generally distributed in containers protecting them
against humidity. Therefore, where it can be demonstrated that the proposed containers (and
conditions of storage) afford sufficient protection against high and low humidity, stability
tests at different relative humidities can usually be omitted. Where humidity-protecting
containers are not used, appropriate stability data should be provided.
6.4 Light
Applicants should consult the appropriate regulatory authorities on a case by case basis to
determine guidance for testing.
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6.5
Container/Closure
Changes in the quality of the product may occur due to the interactions between the
formulated biotechnological/biological product and container/closure. Where the lack of
interactions cannot be excluded in liquid products (other than sealed ampoules), stability
studies should include samples maintained in the inverted or horizontal position (i.e., in
contact with the closure), as well as in the upright position, to determine the effects of the
closure on product quality. Data should be supplied for all different container/closure
combinations that will be marketed.
In addition to the standard data necessary for a conventional single-use vial, the applicant
should demonstrate that the closure used with a multiple-dose vial is capable of withstanding
the conditions of repeated insertions and withdrawals so that the product retains its full
potency, purity, and quality for the maximum period specified in the instructions-for-use on
containers, packages, and/or package inserts. Such labelling should be in accordance with
relevant national/regional requirements.
6.6
Stability after R e c o n s t i t u t i o n of F r e e z e - D r i e d P r o d u c t
The stability of freeze-dried products after their reconstitution should be demonstrated for the
conditions and the maximum storage period specified on containers, packages, and/or
package inserts. Such labelling should be in accordance with relevant national/regional
requirements.
7.
TESTING FREQUENCY
The shelf-lives of biotechnological/biological products may vary from days to several years.
Thus, it is difficult to draft uniform guidelines regarding the stability study duration and
testing frequency that would be applicable to all types of biotechnological/biological products.
With only a few exceptions, however, the shelf-lives for existing products and potential
future products will be within the range of 0.5 to five years. Therefore, the guidance is based
upon expected shelf-lives in that range. This takes into account the fact that degradation of
biotechnological/biological products may not be governed by the same factors during
different intervals of a long storage period.
When shelf-lives of one year or less are proposed, the real-time stability studies should be
conducted monthly for the first three months and at three-month intervals thereafter.
For products with proposed shelf-lives of greater than one year, the studies should be
conducted every three months during the first year of storage, every six months during the
second year, and annually thereafter.
While the testing intervals listed above may be appropriate in the pre-approval or pre-license
stage, reduced testing may be appropriate after approval or licensure where data are
available that demonstrate adequate stability. Where data exist that indicate the stability of a
product is not compromised, the applicant is encouraged to submit a protocol which supports
elimination of specific test intervals (e.g. nine-month testing) for post-approval/postlicensure, long-term studies.
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8.
SPECIFICATIONS
9.
LABELLING
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GLOSSARY
Conjugated Product
A conjugated product is made up of an active substance (for example, peptide, carbohydrate)
bound covalently or noncovalently to a carrier (for example, protein, peptide, inorganic
mineral) with the objective of improving the efficacy or stability of the product.
Degradation Product
A molecule resulting from a change in the active substance (bulk material) brought about
over time. For the purpose of stability testing of the products described in this guideline, such
changes could occur as a result of processing or storage (e.g. by deamidation, oxidation,
aggregation, proteolysis). For biotechnological/biological products some degradation
products may be active.
Impurity
Any component of the active substance (bulk material) or medicinal product (final container
product) which is not the chemical entity defined as the active substance, an excipient, or
other additives to the medicinal product.
Intermediate
For biotechnological/biological products, a material produced during a manufacturing
process which is not the active substance or the medicinal product but whose manufacture is
critical to the successful production of the active substance or the medicinal product.
Generally, an intermediate will be quantifiable and specifications will be established to
determine the successful completion of the manufacturing step prior to continuation of the
manufacturing process. This includes material which may undergo further molecular
modification or be held for an extended period of time prior to further processing.
Manufacturing-Scale Production
Manufacture at the scale typically encountered in a facility intended for product production
for marketing.
Pilot-Plant Scale
The production of the active substance or medicinal product by a procedure fully
representative of and simulating that to be applied at manufacturing scale. The methods of
cell expansion, harvest, and product purification should be identical except for the scale of
production.
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Guideline Title
Legislative basis
Date of first adoption
Date of entry into
force
Status
Previous titles/other
references
Additional Notes
CONTENTS
1.
INTRODUCTION
2.
3.
DEVELOPMENT GENETICS
4.
PRODUCTION
5.
PURIFICATION
6.
PRODUCT CHARACTERISATION
7.
8.
SAFETY REGULATIONS
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INTRODUCTION
Somatic gene therapy encompasses medical interventions which involve the deliberate
modification of the genetic material of somatic cells. Scientific progress over the past decade
has led to the development of novel methods for the transfer of new genetic material into
patients' cells. The aims of these methods include the efficient transfer and functional
expression or manifestation of the transferred genetic material in a target somatic cell
population for therapeutic, prophylactic or diagnostic purposes.
Although in the majority of cases the intention is the addition and expression of a gene to
yield a protein product, the transfer of nucleic acids with the aim of modifying the function
or expression of an endogenous gene, e.g. by homologous recombination, is also included i n
the definition of gene therapy. This will also include transfer of genetic material that
specify nucleic acid products, e.g. ribozymes, anti-sense nucleotides, designed to modify
endogenous gene expression at either transcriptional or translational levels. The transfer of
genetic material for the purposes of (i) marking or following the migration of particular
somatic cell populations, and (ii) protective vaccination against foreign antigens should be
included, since the products used to achieve these ends will have the same or similar
characteristics to those used in gene therapy.
There are several approaches to the introduction of genetic material into a somatic cell.
These include the transfer of naked nucleic acid, nucleic acid complexed with a carrier and
the use of replication deficient viruses. Defective viruses and nucleic acid complexes used
for nucleic acid transfer into cells are called gene transfer vectors and the nucleic acid
transferred is called the expression construct. Modification of somatic cells can be achieved
by in vivo administration of nucleic acids, with or without a carrier or transfer vector, or
performed ex vivo, after which the genetically modified autologous, allogenic or xenogenic
somatic cells are administered (Table 1).
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TABLE 1
POTENTIAL PRODUCTS FOR GENE THERAPY
(a)
(b)
(i)
(ii)
Replication-deficient
(d)
Genetically-modified
fibroblasts,
myoblasts or other cell which could somatic
cells be introduced/engrafted into appropriate patient's
tissues/organs.
This document covers quality aspects in the production of the gene transfer vectors and
genetically modified somatic cells included in Table 1. However, it is not intended to apply
to chemically synthesised, short polynucleotides, e.g. anti-sense nucleotides, where quality
control in manufacture will be different.
This note for guidance is intended to facilitate the collection and submission of data to
support applications for marketing authorisation within the EC for gene therapy products
derived by biotechnology/high technology and intended for medicinal use in man. It should
be read in conjunction with the European Directives and other specialised guidelines where
appropriate. Any commercially manufactured gene therapy products will require marketing
authorisation by the European Medicines Evaluation Agency through the centralised
procedure. A flexible approach to the control of these products has been adopted so that
recommendations can be modified in the light of experience of production and use, and of
further developments. Implementation of these recommendations for an individual product
should reflect its intended clinical use.
2.
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Appropriate attention needs to be given to the quality of all reagents used in production:
specifications for these are to be included in documentation and they should comply with any
relevant EU recommendations (e.g. note for guidance on Minimising the Risk of Transmitting
Agents causing Spongiform Encephalopathy via Medicinal Products.
It is undesirable to use in production agents which are known to provoke sensitivity i n
certain individuals, such as, for example, penicillin or other -lactam antibiotics.
Although comprehensive characterisation of the final product is essential, considerable
emphasis must also be placed on 'in-process' control, a concept which has been highly
effective in the quality control of bacterial and viral vaccines prepared by conventional
methods and, more recently, of rDNA-derived products.
Certain factors may compromise the quality, and thus the safety and efficacy, of gene
therapy products and should be given special attention:
a)
The genetic material involved, a defined nucleic acid will require amplification
within a replicating organism or by an in vitro technique, e.g. polymerase chain
reaction (PCR). Uncertainties over the fidelity of the replication systems raise
concerns about the homogeneity of the amplified
product. For example, a gene
containing errors in base sequences may specify an abnormal protein which may
have undesirable biological and immunological activities. Transference procedures
are intended to introduce copies of the genetic material involved into large numbers of
target cells. Therefore, it is essential to purify and characterise the genetic material
involved as thoroughly as possible before use. Where possible, evidence should be
obtained that the correct nucleotide sequence, or that at least the correct coding capacity,
has been made and that this has been stably maintained during the amplification steps
before transfer and that the sequence/coding capacity remains unmodified following
transfer.
b)
In most instances, the genetic material (nucleic acid) involved will be ligated into
appropriate plasmids or cassettes having promoters which regulate its expression. The
resulting expression constructs may be complexed with salts, proteins (e.g.
transferrin) or polymers (e.g. polylysine), or linked to carriers (e.g. liposomes or
gold), or adsorbed to replication-deficient viruses (e.g. adenovirus), to increase the
specificity or efficiency of transfer of genetic material (Table 1). This may mean that
some products are manufactured as components of the final vector, which is constituted
just prior to use (cf. monoclonal antibodies which are radiolabelled just before
application). In these cases, all components of the final transfer vector should be
thoroughly characterised.
c)
Virus vectors raise particular issues regarding manufacture and safety. For example,
viruses proposed as vectors are themselves likely to produce pathological effects under
certain circumstances. It is however expected that viral vectors will have been
'engineered' to lack viral genes (encoding structural and enzymatic proteins) that are
required for replication and viral particle formation. Viral nucleic acid sequences
known to be associated with pathological effects should also be deleted. Replicationdeficient viruses are propagated in special "packaging" cell lines genetically
modified to express the viral proteins necessary for the recombinant genomes to be
replicated and packaged. The aim should be the construction of packaging cell lines
which make the production of replication-competent (infectious) virus(es) by
recombination with the viral genome of the gene transfer vector used impossible. One
way to do this (e.g. for retroviruses) is to separate the genes encoding the viral
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structural and enzymatic proteins and to express them from separate constructs which
are inserted into separate chromosomal integration sites. To further minimise the risk
of recombination within the packaging cell line, packaging cell lines containing any
endogenous viral sequences that could complement the recombinant viral genome
should be avoided. Precautions must also be taken to prevent infection of the packaging
cell line by wild-type viruses that might also lead to the formation of replicationcompetent recombinant viruses. In addition, the recombinant genome may be subject to
mutation during replication in the packaging cell line. Complete characterisation and
safety-testing of such vectors may be difficult, especially because purification to
homogeneity, e.g. for retroviral vectors, may not be readily attainable.
d)
e)
f)
Unintended variability in culture during production may lead to changes which cause
alteration of the product, reduce the yield of product and/or result in quantitative and
qualitative differences in the impurities present. Procedures to ensure consistency of
production conditions as well as of the final product are imperative.
g)
of
be
in
in
Whilst the recommendations set out below should be considered to be generally applicable,
individual products may present particular quality control problems. The production and
control of each product will be considered on a case by case basis.
mutagenesis
Most existing vectors can only transfer genetic material into target cells leading to either
random integration with chromosomal DNA or to localisation in extra-chromosomal sites,
suggesting a number of undesirable possibilities. Random integration of vector nucleic acid
could result in:
(i)
(ii)
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(iii) integration at sites which affect cellular responsiveness to exogenous agents, such as
growth factors, cytokines or hormones.
In all three examples cited, the affected cells may acquire tumourigenic potential. However,
oncogenesis (transformation of a cell to the tumorigenic phenotype) is generally regarded as
a multi-step process involving the disruption of many genes, and thus occurrence of singlesite insertional mutagenesis may only carry a very low risk of the development of tumour
cells. Nevertheless, a high vector nucleic acid copy number per cell, randomly integrated
into chromosomal DNA, may be cause for concern. Vector nucleic acid copy number could
increase in cases where repeated vector application is necessary.
2.2.2 Induced
cellular
changes
Following transfer of vector nucleic acid to target cells, certain unintended and unexpected
observable changes to target cell appearance, functions and behaviour, e.g. migratory
characteristics, may occur compared with the original unmodified target cell population.
These should be well-documented. In addition, certain non-observable changes may occur.
For example, several members of the herpesvirus family which are latent in human cells
are also reactivatable under certain conditions leading to the production of infectious virus.
Therefore, where possible, transduced target cells should be screened for the presence of
likely re-activatable viruses such as herpes zoster, Epstein-Barr virus and cytomegalovirus.
There may also be the possibility that transfer of vector nucleic acid increases the
immunogenicity of the target cells. For example, this could be the case where the vector
nucleic acid encodes viral or other non-human proteins, or proteins that were previously not
expressed within the patient treated due to the specific genetic defect.
2.2.3 Vector DNA
mobilisation
Vector nucleic acid mobilisation is not likely to occur for those vectors which have no
replication potential. Replication-deficient viral vectors however while not normally
expected to replicate may infrequently be rescued either by co-infection with wild-type,
replication-competent viruses or by recombination with endogenous viral nucleic acid
sequences. There may also be a low risk that the recombinant viral genome itself
recombines with the genomes of co-infecting viruses to produce novel recombinant viruses.
Vector nucleic acid mobilisation may lead to non-target cells receiving this (e.g. germline
cells being transduced with vector nucleic acid) and a risk of its horizontal spread to
clinical staff and members of the public.
3.
DEVELOPMENT GENETICS
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Vector
3.3.1 Complexed
construction
nucleic
acid as
vector
viruses
as
vector
Full documentation on the origin, history and other characteristics of the parental virus,
current virus stocks and methods of propagation should be provided. A full description and
characterisation of the genetic material, the part(s) of the viral genome to which the genetic
material is inserted or ligated, modifications of remaining viral nucleic acid sequences
and any other nucleic acid sequences (e.g. promoters) to be included in the recombinant
viral genome should be provided.
Details of the construction of the packaging cell line should be given, including the nature
and, where possible, the location of the helper viral nucleic acid and its encoded
proteins/functions. The origin, identity and biological characteristics of the cell line
together with details of the presence or absence of endogenous viral particles and sequences
should be described. A well-defined master and working cell bank should be established.
Evidence of freedom from contamination with adventitious microorganisms, including
viruses, bacteria, mycoplasma, yeasts, moulds (fungi), is essential.
A complete description of the procedures used to transfect/transfer the recombinant viral
genome containing the genetic material into the packaging cells should be provided. Where
the packaging cells do not contain integrated helper viral nucleic acid sequences, but
packaging of the recombinant viral genome is reliant on transfecting an additional nucleic
acid construct, full details of this construct should be given.
The processes resulting in recombinant viral genome replication and its packaging into
virus particles should be described. Where possible, the stability of the recombinant viral
genome in the packaging cells should be assessed.
Where selection techniques are required to isolate cells producing replication-deficient viral
vector, details of the methods used should be provided.
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3.4 G e n e t i c a l l y - m o d i f i e d s o m a t i c c e l l s a s p r o d u c t s
Full documentation of the origin, history, construction and characteristics of the geneticallymodified somatic cells should be provided. The homogeneity and genetic stability of the cells
should be demonstrated or thoroughly characterised. Any observable changes in cell
morphology, functions and behaviour, e.g. migration characteristics, of the geneticallymodified somatic cells compared with the original unmodified cells should be well
documented. A well-defined master and working cell bank should be established, where
appropriate. Evidence of freedom from contamination with adventitious microorganisms,
including viruses, bacteria, mycoplasma, yeasts, moulds (fungi), is essential.
4.
PRODUCTION
Details of the production process including volumes, times, harvest and storage should be
given. A clear definition of a "batch" of product, which may be subjected to further
processing, should be provided. Acceptable limits for the purity, consistency and yield of
product should be specified and justified.
5.
PURIFICATION
6.
PRODUCT CHARACTERISATION
Rigorous characterisation of the product and of its stability by a range of molecular and
biological methods is essential. It is desirable to include suitable tests to establish that
complexed nucleic acid has the desired biochemical and biological characteristics required
for its intended use. Immunological and immunochemical tests may provide valuable
information. In the case of replication-deficient viral vectors tests should, where possible, be
included to show integrity and homogeneity of the recombinant viral genome. Tests to
establish the cellular tropism and, if expected, tissue-specific transcription of gene transfer
vectors and, where appropriate, the inducibility of the desired gene, should also be
undertaken.
When appropriate, the purity of the final processed product should be determined, and the
level of contamination considered as acceptable should be justified. The criteria for
acceptance or rejection of a production batch must be given. In the case of replicationdeficient viral vectors, tests to show they are free from replication-competent viruses are
essential. For example, replication-competent retroviruses, even of xenogenic origin, are
able to promote oncogenesis, probably because they can randomly integrate many copies of
their genome through multiple-infection cycles in target cells. It is essential therefore that
all measures/steps be taken to exclude the possibility that replication-deficient retroviral
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7.
7.1 Consistency
An acceptable number, e.g. five, of successive batches of the bulk product should be
characterised as fully as possible to determine consistency of composition. The studies
should include molecular, biological, and immunological methods to characterise and assay
the product as well as methods to detect and identify impurities. Any differences which occur
among batches should be noted.
7.2
7.2.1
A selection of tests used to characterise the purified product (see section 6) should be used to
confirm product identity for each batch. The methods employed should include tests for the
genetic composition and physico-chemical and immunological characteristics, together with
tests for the expected biological activity (see section 7.2.3).
7.2.2
Purity
The degree of purity desirable and attainable will depend on several factors; these include
the nature and intended use of the product, the method of its production and purification and
also the degree of consistency of the production process. The purity of each batch should be
established and be within specified limits. Tests should be applied to determine levels of
contaminants of cellular origin, e.g. from the packaging cell line, as well as materials
which may have been added during the production processes. A strict upper limit for each
identifiable contaminant should be set.
72.3 Efficacy potency
tests
For estimating the efficacy/potency of vectors, biological tests should be applied that permit
the efficiency of transfer and the level and stability of expression of genetic material, or its
effects, to be determined. Wherever possible, a reference batch of vector of assigned potency
should be established and used to calibrate tests.
The efficiency with which vectors transfer the genetic material to target/test cells together
with information on the resulting level of gene expression will provide the basis for
assessing their potency. When tests are conducted in vitro, the target cell population should
be carefully characterised. The variability of the biological system as a whole should be
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monitored, particularly where target cells may be derived from different sources/donors and
long term expression or manifestation of the transfected genetic material is being followed.
Where appropriate and for vectors intended for direct in vivo application, biological potency
tests in animal tissues maintained ex vivo or in whole animals should be carried out.
Transgenic animals or animals with transplanted human tissues or systems may be
suitable for this purpose.
Where possible, suitable ways for expressing potency of vectors should be established and
results reported in a reference unitage. It is recommended that the reference unitage be
correlated if possible with a physico-chemical parameter of the vector, e.g. weight of DNA, to
provide information on the 'specific activity" of the vector. Stated limits for the potency and
specific activity of batches of vector should be provided.
Where possible, the particle :infectivity ratio of replication-deficient viruses should be
determined and when this is excessively high rejection of the batch should be considered.
72.4 Safety
tests
8.
SAFETY REGULATIONS
Currently, gene therapy products with viruses as vectors fall under the scope of
90/219/EEC on contained use of genetically modified microorganisms and
90/220/EEC on the deliberate release of genetically modified organisms. The
competent authorities for the implementation of these Directives have adopted the
approach:
a)
Directive
Directive
group of
following
genetic modification of somatic cells, as well as the culture, storage and use of
the genetically modified somatic cells carried out in laboratory or hospital
facilities.
(ii)
Since 1 January 1995, the deliberate release of medicinal products containing or consisting
of GMOs for the purpose of placing them on the market falls within the scope of Council
Regulation (EEC) 2309/93, which provides for a specific environmental risk assessment
similar to that laid down in Directive 90/220/EEC. Thus, in its opinion on applications for
marketing authorisation of such medicinal products, the CPMP shall ensure that all
appropriate measures are taken to avoid adverse effects on human health and the
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environment which might arise from the deliberate release or placing on the market of
genetically modified organisms.
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Guideline Title
Legislative basis
Date of first adoption
Date of entry into
force
Status
Previous titles/other
references
Additional Notes
CONTENTS
1.
INTRODUCTION
2.
DEFINITIONS
3.
4.
5.
PRODUCTION
6.
CONCLUSION
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INTRODUCTION
Transgenic organisms contain a foreign gene which has been experimentally inserted into
the normal genetic component, and currently include many plants and a number of a n i m a l
species. They have been used experimentally to investigate gene function, development and
disease. Transgenic animals have also been proposed as a means of testing agents for
oncogenicity or virulence.
This document is concerned with the use of transgenic animals to produce biological
pharmaceutical materials for use in human recipients. Transgenic animals may produce
higher quantities of material in more concentrated form than existing culture methods, and
therefore have considerable advantages in both the cost of producing the starting material
and in its downstream processing. In some instances where very large amounts of material
are required for therapy the use of transgenic animals may be one of the few viable
production strategies. However in some respects the products resemble classical biologicais
in that they derive from a whole animal rather than from definable culture systems. The
considerations which apply are therefore a blend of those relevant to recombinant DNA
(rDNA) derived materials and materials from less defined sources.
2.
DEFINITIONS
Forebears: the animals from which the egg and sperm used to create the genetic founder
were derived
Host:
the recipient mother in whom the embryonic genetic founder was implanted
Genetic founder: the transgenic animal resulting from the introduction of the foreign DNA
into the embryo or fertilised egg
Production founder:
animal herds
Production animals:
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4.
4.1 Origin
Animals which have been proposed as hosts for production include among others sheep, cows,
pigs, rabbits and mice, and much interest currently centres on the use of transgenes
expressed in milk or colostrum. The choice of animal will be determined by a variety of
factors. For example pigs breed rapidly and produce large litters, so that establishing a
suitable transgenic line of animals may be technically simpler than if the same process is
attempted in cows. On the other hand pigs are difficult to milk, while milk production in
cows is well understood.
Each species will raise its own microbiological and virological concerns which should be
addressed. Many of the potential host animals are not conventional laboratory animals, but
infectious agents of agricultural significance are likely to be well known. The
microbiological status of the production animal, its forebears and host animals involved i n
derivation of the transgenic line should be documented as far as is possible. Consideration
should be given to the use of breeds of animals resistant to specific agents such as scrapie
resistant breeds of sheep. The founder animals and their offspring should be shown to
comply with the existing guidelines Minimising the Risk of Transmitting Agents causing
Spongiform Encephalopathy via Medicinal Products.
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appropriate tissue should be clearly described. The complete sequence of the final construct
should be determined.
4.3 C r e a t i o n of t h e t r a n s g e n i c a n i m a l
A number of methods are currently in use for the creation of transgenic animals. One
favoured method involves the inoculation of the DNA into the pronucleus of a fertilised
ovum, followed by implantation into pseudo pregnant females. This results in a proportion of
animals carrying the transgene in the germ line which may be high in some species (e.g.
mouse 5-30%) or low in others (e.g. cows and sheep, 1-5%). Depending on the time when the
transgene is incorporated into the cellular DNA, mosaic animals may develop in which
certain cell lineages carry the transgene while others do not. Other methods of creating
transgenic animals involving retroviral infection of the embryo at an early cleavage stage
in the blastocyst result in only a proportion of the cells carrying the transgene, and therefore
a high proportion of mosaic animals some of which may not have the transgene incorporated
in the germ line at all. Methods for predictable site specific integration of sequences into the
host genome would have advantages for both controlled expression and safety.
The method used to create the transgenic animal should be described in detail, including the
isolation of ova, in vitro fertilisation, insertion of the transgene, reimplantation and
delivery. The use of retroviral vectors raises additional quality considerations related to
preparation of the vector, its virological purity and its persistence. Consideration of
guidelines related to regulatory aspects of gene therapy is advisable.
The genealogy of the production animals must be documented. A transgenic line will derive
from a single genetic founder animal, and materials from different transgenic lines
should not be mixed. The founder animal and the production animals should be defined as
diploid or haploid with respect to the inserted sequence.
The level of expression of the incorporated gene should be assessed and the tissue
distribution of expression should wherever possible be shown to be consistent with the chosen
strategy of expression. Estimates of the copy number should be made and evidence as t the
accuracy of the incorporated gene squence should be presented. It is believed that while
multiple copies of the transgene are usually incorporated, there is usually only a single site
of integration. Thus, even where multiple copies are introduced it will be possible to identify
the expressed sequence or sequences with confidence at the level of the genomic DNA.
It is of doubtful value to determine multiple sequences of the insert but evidence that the
correct sequence is present should be obtained. Some sequence data for example of cDNA
clones will be valuable as will restriction endonuclease maps, which will serve to
demonstrate that the site of integration has not changed in offspring of the founder a n i m a l
where these are used. It should be clearly stated whether the animals used for production are
haploid or diploid for the transgene. The animals used in production should be characterised
to ensure an acceptable level of consistency.
The virological status of the donors and host animals should be shown to be acceptable; for
example calves born to mothers infected with BVDV are likely to be persistently infected,
and vertical transmission of BSE has not been eliminated as a possibility. Similarly bovine
immunodeficiency virus (BIV) may be transmissible through semen. These are examples
only.
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4.4 S t a b i l i t y of t h e g e n e
Transgenic animals produced by microinjection of DNA have the highest probability of
incorporating the transgene into the germ line and therefore expressing it in the appropriate
intended tissue. However this method often results in the insertion of multiple head to tail
copies of the transgene, and rearrangements and eliminations may occur on breeding. The
stability of the gene on breeding will be an issue where numbers of animals derived from a
founder animal are to be used. Greater consistency of production will be achievable if a
uniform production herd can be bred in a reproducible manner. The strategy used to
generate a herd of animals of similar productivity should be clearly delineated. Evidence
should be presented that the animals are similar, in the yield of product and genetically i n
terms of numbers of copies of the gene incorporated and the site of integration in the genome.
Restriction length polymorphisms may be of value in providing evidence for a constant
integration site.
5.
PRODUCTION
5.1
There are major veterinary and ethical difficulties in raising and maintaining
agricultural animals under specific pathogen free conditions although this is desirable if it
can be achieved. Otherwise good husbandry and agricultural practice may contribute to
virological and microbiological safety.
However the general conditions suitable for
satisfactory agricultural production are likely to be less stringent than those applicable to the
manufacture of pharmaceutical materials, so that good husbandry and agricultural practice
are unlikely to be sufficient alone to ensure adequate safety of a pharmaceutical product.
The conditions under which the animals are bred and maintained should be described and
precautions taken to ensure that the site is free of disease likely to affect the production
animal species prior to use. Potential sources of infection may include foodstuff, a n i m a l
handlers and veterinary surgeons, and the environment especially if the animals are kept
outside. The health and virological status of the animals should be documented and animals
subjected to regular veterinary examination. If the source material is milk the health of the
udder should be subject to special examination. Administration of antibiotics and hormones
for prophylactic or therapeutic reasons at any time when they may contaminate the product is
not permitted. Cows should be shown to be free of bovine tuberculosis.
Many cow herds are known to be infected with bovine viral diarrhoea virus, and other
infections include bovine polyoma and infectious rhinotracheitis virus which may or may
not be apparent. Sheep are susceptible to many agents including orf virus and Louping 111
virus, and pigs to swine vesicular disease and porcine parvovirus. These examples do not
constitute an exhaustive list. Many infectious agents of agricultural animals may establish
persistent infections, and some are also able to infect humans. In general animals which
are known to be infected with an agent should not be used for production.
5.2 T h e s o u r c e m a t e r i a l
Different litter mates have been reported to express the transgene to different levels for
unknown reasons unrelated to copy number or accuracy of the incorporated sequence.
During the period of lactation the expression of the gene may vary, and it may also vary
between different lactations. The source material may therefore be variable, m a k i n g
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purification procedures potentially less consistent. The nature of the source material (for
example milk or colostrum) should be clearly stated and justified. There is wide variation
in the composition of milk and the purification process must be shown to be satisfactory i n
dealing with the range of materials expected. Acceptable limits for the level of active
substance in the source material should be set. Where the source material is milk,
specifications could be set in terms of product activity per unit of non fat dry solid. A single
batch of source material may involve pooling separate harvests and should be clearly
defined. While milk is a source material with a long history in which the safety issues are
generally well understood, pharmaceutical proteins may be given parenterally, not orally,
and it may not be possible to pasteurise or sterilise them in the ways which have been applied
to milk.
Limits for the microbiological status of the source material should be set. Milk is likely to be
contaminated with bacteria, although such contamination may be minimised by good
husbandry. Contamination by certain agents, such as zoonotic mycobacteria, would make
the material unacceptable. While bacteria may be removed by sterile filtration of the product,
mycoplasma may not and efforts should be made to exclude them from the source material.
5.3 P u r i t y of t h e a c t i v e s u b s t a n c e a n d v a l i d a t i o n of d o w n s t r e a m
processing
The purity of the active substance should be in accordance with criteria accepted for products
of rDNA technology. Most such products are currently manufactured by in vitro culture
methods involving either the fermentation of microorganisms or the large scale culture of
cells from higher organisms. A transgenic animal is unlikely to be free of pathogens to the
same degree as a well characterised cell bank. Validation of the purification process is
therefore important in ensuring the safety of the product. Guidelines on Virus Validation
Studies: The Design, Contribution and Interpretation of Studies Validating the Inactivation and
Removal of Viruses have been prepared. Where the source material is milk or colostrum,
contamination with mycoplasma is possible, and the process should be validated for their
removal, as well as limits set for their levels in the starting material.
The source material, whether blood, milk, colostrum or other tissue will contain large
numbers of host derived proteins other than the desired product, some of which may be
present in large amounts which must be removed. Milk is known to contain proteases, and
the possible effect of these on the product should be addressed; if degradation occurs,
acceptable limits should be set for the products in the final material. Care should be taken to
document and if necessary eliminate host proteins homologous to the required product.
Limits should be set for contaminants which may copurify with the desired material.
Hypersensitivity to milk is common, and materials must therefore be of high purity.
Data on the carbohydrate components of the product should be presented. The non enzymic
glycosylation or glycation of proteins in the presence of free carbohydrate such as lactose
should be considered. This process is likely to be inevitable to some degree for a product
derived from milk but attempts should be made to reduce it to a minimum. Glycated proteins
can cause the activation of end stage macrophages to produce cytokines, and long term
exposure to a glycated product is likely to be harmful.
The attractions of transgenic animals as a means of production include the ability to
produce materials required on a scale which may otherwise be prohibitive because of the
large amounts required in therapy. This increases the concerns associated with the
immunogenicity of the proteins because of trace impurities or imperfect post translational
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modifications, and close attention should be given to the purity, quality and consistency of
the product.
6.
CONCLUSION
Transgenic animals may have advantages over existing production methods with respect to
the quantity and quality of the source material, which may reduce production costs and
simplify downstream processing. Other than veterinary and environmental concerns,
which are outside the scope of this document, the issues they raise are principally those of
using a whole organism in production rather than a potentially more predictable cell culture
or fermentation system based on a seed lot. These include microbiological and virological
concerns, possible difficulties in purification and the consistency of the production and
purification process.
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Guideline Title
Legislative basis
Date of first adoption
Date of entry into
force
Status
Previous titles/other
references
Additional Notes
Procedures
CONTENTS
1.
INTRODUCTION
2.
3.
4.
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5.
6.
INTERPRETATI
O N O F DATA
7.
LIMITATI
O NS O F VALIDATIO N STUDHS
8.
RE-EVALUATI
O N STUDIES
APPENDE* I:
APPENDED II:
CALCULATI
O N O F REDUCTIO N FACTO RS
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INTRODUCTION
1.1
This guideline discusses the need for and the contribution of viral validation studies
towards the viral safety of biological products. The principal aims of the guideline are to
provide guidance on the design of a validation study including the choice of viruses to be
used and on the interpretation of the ensuing data especially with respect to defining a
process step which can be considered to be effective in the inactivation and/or removal of
viruses.
1.2
The guideline concerns the validation of virus inactivation and/or removal
procedures for all categories of medicinal biological products for human use with the
exception of live viral vaccines including genetically engineered live vectors. The type of
products covered include:
products derived from in vitro culture of cell lines of human or animal origin,
products derived from in vivo culture of cell lines, or from organs or tissues of human
or animal origin,
products derived from blood or urine or other biological fluids of human or a n i m a l
origin.
1.3
The risk of viral contamination is a feature common to all biologicals whose
production involves the use of material of animal or human origin. Viral contamination of
a biological may arise from the source material, e.g. cell banks of animal origin, human
blood, human or animal tissues, or as adventitious agents introduced by the production
process, e.g. the use of animal sera in cell culture.
1.4
In the past, a number of biologicals administered to humans have been contaminated
with viruses. In several instances, the virus was only identified many years after the
product had been introduced into the market since contamination occurred prior to adequate
knowledge concerning the presence of the infectious agents. The primary cause of these
viral transmissions has been contamination of the starting or source materials. Examples
include Yellow Fever vaccine which was contaminated by avian leukosis virus by virtue of
its production in naturally infected hens eggs, whilst SV40 was a contaminant of poliovirus
and adenovirus vaccines prepared in the 1950's on primary cultures of kidney cells obtained
from Rhesus monkeys naturally harbouring a clinically inapparent infection with SV40. In
addition, viruses present in human plasma, e.g. and HCV, have contaminated blood
products whilst human growth hormone extracted from the pituitaries of cadavers has been
implicated in the transmission of the aetiological agent responsible for Creutzfeldt-Jakob
disease. Contamination of a biological can also arise from the use of infected material
during production or as an excipient. Perhaps the most notable was Yellow Fever vaccine
contaminated with HBV present in human serum used as a stabiliser in the 1940's.
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1.5
Three principal complementary approaches can be adopted to control potential viral
contamination of biologicals:
(i)
selecting and testing source material for the absence of detectable viruses,
(ii)
(iii) testing the product at appropriate stages of production for freedom from detectable
viruses.
No approach provides a sufficient level of assurance alone and this will only be achieved
using a combination of the above.
1.6
Testing of starting materials is essential to minimise viral contamination. While
tests may be able to detect one or more virus species, no single test will be able to
demonstrate the presence of all known viruses. Moreover all test systems require a
minimum level of viral contamination to record a positive and tests are also limited by
statistical considerations in sampling. Some tests, e.g. the test for antibody to HCV in
human plasma, may measure markers of infection which only become positive sometime
after infection. Similar considerations apply to testing of the final product.
1.7
Therefore establishing the freedom of a biological from infectious virus will in
many instances not derive solely from direct testing for their presence, but also from a
demonstration that the manufacturing process is capable of removing or inactivating them.
Validation of the process for viral inactivation/removal can play an essential and
important role in establishing the safety of biological products especially when there is a
high potential for the source material to be contaminated with a virus known to be pathogenic
for man, e.g. plasma derived products. Also, since many instances of contamination in the
past have occurred with agents whose presence was not known or even suspected at the time
of manufacture, an evaluation of the process can provide a measure of confidence that a
wide range of viruses including unknown, harmful viruses, may be eliminated.
1.8
The intention of this note for guidance is to provide a general framework for
validation studies and the virological approach which should be used in the design of virus
validation studies. Manufacturers should apply the recommendations presented here to their
specific product taking into consideration the nature of the source material, the procedures
used for production and purification and any other factors which can have consequences on
this safety issue. The approach used by manufacturers in studies for evaluating virus
elimination should be explained and justified.
2.
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22
Cells may have a latent or persistent infection, for example, a herpesvirus or a
retrovirus, which may be transmitted vertically from one cell generation to the next as a
viral genome and which may be expressed intermittently as infectious virus.
2.3
The process of construction of a production cell line may introduce a contaminant
virus indigenous to another species, e.g. an EBV transformed human lymphoblastoid cell
line secreting a monoclonal antibody can be infected with a murine retrovirus after fusion
with a murine myeloma.
2.4
Adventitious viruses may be introduced by the use of contaminated animal products
in the production process e.g. cell cultures may be contaminated with bovine viruses through
the use of bovine sera or a murine monoclonal antibody used in affinity chromatography
may contaminate a product with a murine virus.
2.5
Other sources of contamination, e.g. operating personnel or raw materials of nonbiological origin, are possible.
3.
3.1
(i)
(ii)
to provide indirect evidence that the production process might inactivate/remove novel
or unpredictable virus contamination.
This is achieved by deliberately adding ('spiking') a virus
production steps and measuring its removal or inactivation
individual step or steps. This will identify production steps
reducing the level of infectious virus and provide an estimate
the process to eliminate contaminating viral infectivity.
to material at various
during the subsequent
which are effective i n
of the overall ability of
3.2
Virus validation studies, as with direct testing of materials at appropriate steps,
contribute to confidence in the virological safety of the product. However, all virus
validation studies must be regarded as an approximation to the true capacity of the process
since it may be difficult or impossible to conduct a perfect validation study of a process
because of the large numbers of complex variables involved. Results have shown that even
small modifications in procedure or the particular laboratory strain of virus used can have
a large effect on virus removal or inactivation.
3.3
Where the starting or source material is less well characterised, such as blood,
tissues and organs of human or animal origin, or when cells have been cultured by in vivo
techniques, there is a higher possibility of viral contamination and the manufacturing
process will normally incorporate one or more effective virus inactivation/removal steps.
Products derived from human plasma raise particular viral safety concerns and specific
guidance is given in the guideline on Medicinal Products Derived From Human Plasma.
3.4
In the past, where the starting material posed a lower virological risk, such as a fully
characterised cell bank, the purification process often did not contain a specific virus
inactivation/removal step and a validated purification process was considered to give
sufficient levels of viral inactivation/removal. Clinical experience has not revealed any
problems with this approach. However, some manufacturers of monoclonal antibodies
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(mAbs) are introducing specific viral inactivation/removal steps into their production
process since mAb producing cell lines of murine origin inevitably secrete variable
quantities of retroviruses which may be infectious.
3.5
It should be borne in mind that cell culture systems inherently support virus
replication. Therefore, a distinct low level of risk of viral contamination of the culture
persists despite a high level of cell bank characterisation and occasional cases of
adventitious virus contamination have been reported.
3.6
The justification for, and the extent of, the required validation studies will vary
depending on the manufacturing process and type of product (e.g. species of origin of
starting material, whether source material is variable or defined, stability of the active
material, etc.). The appropriateness of the studies will be reviewed on a case by case basis.
4.
4.1
Viruses for validation should be chosen firstly to resemble viruses which may
contaminate the product as closely as possible and secondly to represent as wide a range of
physico-chemical properties as possible in order to test the ability of the system to eliminate
viruses in general.
4.2
Most validation studies employ laboratory strains of virus which can be produced
and assayed conveniently. However, experience has shown, and manufacturers should be
aware, that different laboratory strains of virus may have different properties from each
other and from naturally occurring viruses. Consequently, any virus used in a validation
study is actually a model virus. The manufacturer should justify the choice of viruses in
accordance with the aims of the validation study and the principles laid down in this
guideline.
Unless otherwise justified, where two similar viruses could be used for
validation studies either because of their equal resemblance to possible contaminants or
similarities in their properties, the virus considered to be the more resistant should be used.
4.3
(i)
(ii)
Cell lines derived from rodents usually contain endogenous retroviral particles which
may be infectious (C-type particles) or non-infectious (A-type particles). Where the
source material is obtained from rodent cell lines, the production process should be
evaluated for its ability to inactivate/remove one of the closely related laboratory
murine retroviruses.
b)
c)
Examples of viruses which have been used in the past in validation studies are given i n
Table 1. However, since these and the viruses mentioned above are merely examples, the use
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3AB8a
of any of them is not mandatory and manufacturers are invited to consider other viruses
especially those which may be more appropriate for their individual processes. Further
guidance on the choice of viruses for the validation of manufacturing processes of plasma
derivatives is provided in the guideline Medicinal Products Derived From Human Plasma.
4.4
There should be an efficient, sensitive and reliable infectivity assay for the viruses
used. Viruses which can be grown to high titre will be desirable, although this may not
always be possible.
4.5
Products derived from ovine, caprine or bovine tissues raise the problem of
contamination by agents of transmissible spongiform encephalopathy, such as scrapie,
which accumulate in the central nervous system and lymphoid tissue. These agents are the
subject of a separate note for guidance on Minimising the Risk of Transmitting Agents causing
Spongiform Encephalopathy via Medicinal Products.
5.
5.1
Validation studies involve the deliberate addition of a virus at various production
steps and measuring the extent of its removal/inactivation during the subsequent individual
step or steps. It is not necessary to validate every individual step of a manufacturing process.
Only those steps which are likely to contribute to inactivation/removal of a virus need to be
subject to a validation study.
5.2
GMP restraints prevent the deliberate introduction of any virus into the production
facilities. Therefore, the validation should be conducted in a separate laboratory equipped for
virological work on a scaled-down version of the production process and performed by staff
with virological expertise in conjunction with the production bioengineers. Studies should be
carried out in accordance with the principles of GLP.
5.3
The comparability of the model and full scale procedures is the premise on which the
results obtained with the scaled-down system can be accepted in evaluating the virus safety
of the product. Therefore, the validity of the scaling down should be demonstrated, by
comparison of process parameters such as pH, temperature, concentration of protein and
other components, reaction time, column bed height, linear flow rate, flow rate to bed height
ratio, elution profile and step efficiency (e.g. yield, balance, specific activity, composition).
Deviations which cannot be avoided should be discussed with regard to any potential
influence on the results.
5.4
Whenever possible, it should be shown whether the reduction in virus infectivity is
accomplished by inactivation of virus or by removal of virus particles. This may be
achieved by establishing the kinetics of loss and/or a balance of infectivity, as appropriate.
Processes which reduce virus infectivity by inactivation are potentially more easily
modelled than those which physically remove particles. For a viral inactivation step, the
kinetics of inactivation should be studied and included in both tabular and graphical form
in reports. Where the inactivation is too rapid to plot the kinetics using process conditions,
further studies should be performed in order to prove that infectivity is indeed lost by
inactivation. Thus appropriate controls should be introduced to detect possible interference
with the assay from the sample or the matrix into which it is introduced and the limits of
detection should be established.
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5.5
Production parameters which influence the effectiveness of a process step to
inactivate/remove viruses should be explored and the results used in setting appropriate inprocess limits. Critical parameters include:
mechanical parameters such as flow rates, mixing rates, column dimensions, column
reuse, etc.
physico-chemical parameters such as protein content, pH, temperature, moisture
content, etc.
5.6
Antibodies present in the starting material may affect the behaviour of a virus i n
partition or inactivation steps. Validation studies should take this into account.
5.7
The validity of the log reduction achieved will be established from investigation of
the effects of variation in critical process parameters used to set in-process limits.
5.8
Published work concerning the ability of related or generic processes to
inactivate/remove viruses may provide an indication of which steps are likely to be
effective. However, the variability intrinsic to validation studies arising from the need to
model the process, choose viruses to be used and explore full scale production parameters on
a laboratory scale, means that validation data must be based on experimental studies
provided by the
5.9
The amount of virus added to the starting material for the production step which is to
be studied should be as high as possible in order to determine the capacity of the production
step to inactivate/remove viruses adequately. However, the virus spike should be added such
that the composition of the production material is not significantly altered (typically the
volume of the virus spike will be less than 10%). Whenever possible, calculated reduction
factors should be based on the virus which can be detected in the spiked starting material
and not on the amount of virus added.
5.10
If possible, virus in samples from model experiments should be titrated without
further manipulations such as ultra-centrifugation, dialysis or storage. Where further
treatments are unavoidable, e.g. to remove inhibitors or toxic substances, or storage for a
period to ensure that all samples are titrated together, appropriate controls should be included
to determine what effect the procedures have on the result of the study. Effects of the sample
on the detection system, including toxic effects, should be recorded as they influence the
limits of detection.
5.11
Quantitative infectivity assays should be performed according to the principles of
GLP and may involve plaque formation, detection of other cytopathic effects such as syncytia
or foci formation, end point titrations (e.g. TCID50 assays), detection of virus antigen
synthesis or other methods. The method should have adequate sensitivity and reproducibility
and should be performed with sufficient replicates and controls to ensure adequate statistical
accuracy of the result (see Appendix I).
5.12
Nucleic acid amplification methods, e.g. PCR, are a promising approach capable of
great sensitivity in detecting viral genomes and also can detect viruses such as hepatitis
and C for which culture systems are not available. However, an important limitation of the
technology is that inactivated virus may still score positive in a genome amplification assay
and thus may underestimate the degree of virus inactivation obtained by a potentially
effective step. On the other hand, PCR may be of value in studies of processes which depend
on virus removal. The use of this technology poses major problems in terms of
quantification, standardisation, quality control and interpretation of results. Validation and
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6.
INTERPRETATION OF DATA
6.1
A combination of factors must be considered when judging the effectiveness of a
virus inactivation/removal step. Assessment of a step based solely on the quantity of virus
inactivated/removed can lead to the conclusion that a process meeting specified levels of
virus reduction will produce a safe product. This is not necessarily the case. The following
factors all contribute in defining the effectiveness of a step and the data must be carefully
evaluated in each case:
(i)
(ii)
(iii) The lgn, reduction achieved. Log reductions of the order of 4 logs or more are
indicative of a clear effect with the particular test virus under investigation. However,
it is emphasised that log number reduction cannot be used as the single, absolute
measure of the effectiveness of a step.
(iv) The kinetics of inactivation. This will indicate whether or not the measured log
reduction is a conservative estimate. Virus inactivation is usually not a simple first
order reaction and often has a fast initial phase followed by a slower phase. However,
a dramatic reduction in the rate of inactivation with time may suggest a loss of
effectiveness of the inactivating agent or that a residual virus fraction is resistant to
the inactivating agent and implies that the step is neither highly effective nor robust.
(v)
The nature of inactivation/removal and whether it is selective for only certain classes
of virus. A process step may be highly effective for some viruses but ineffective against
others, e.g. S/D treatment is effective against lipid-containing but not lipid-free
viruses.
variations
in-process
Where a process step is challenged with 6 logs of virus and 4 logs are recovered, the
step cannot be claimed to be effective, although it may contribute to overall removal.
(ii)
Where a process step is challenged with 6 logs of virus, but because of the cytotoxicity of
the product the limit of assay sensitivity in the product is 4 logs, only 2 logs of removal
have been demonstrated, and the step cannot be claimed to be effective. The process step
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may in fact be able to remove far greater quantities of virus, which might be
demonstrated by a different experimental design.
(iii) Where a process step is challenged with 6 logs of
substantial amounts of virus have been removed.
sterile. However, if this reduction is reproducible
variables, the step is of some efficacy. It contributes
and may be counted as such.
(iv) Where a process step is challenged with 6 logs of virus and no virus is detected in the
product with a limit of sensitivity of the order of 2 logs, approximately 4 logs of
removal have been demonstrated. This is substantial and the step may in fact remove
far greater quantities than can be quantified or claimed.
(v) Where virus is inactivated, the kinetics of loss of infectivity are important. If a
process step involves prolonged incubation, e.g. heating for ten hours, and infectivity
reaches the limits of detection rapidly, the process is likely to have a greater virucidal
effect than can often be demonstrated. On the other hand, if infectivity is lost slowly
and the limits of detection are reached towards the end of the treatment period, the step
provides less assurance of viral safety.
6.3
In general, partition processes are not considered to be effective viral removal steps
although it is recognised that they can contribute to virus removal. Partition processes
usually have a number of variables that are difficult to control and are often difficult to
scale down for validation purposes. Furthermore, partitioning is dependent on the extremely
specific physico-chemical properties of a virus which influence its interaction with gel
matrices and precipitation properties. Thus a model virus may be partitioned in a completely
different manner to a target virus because of relatively minor differences in surface
properties such as glycosylation. Even a relevant virus propagated in the laboratory may act
differently from the wild-type virus in this respect. However, if a partition process gives
reproducible reduction of virus load and if manufacturing parameters influencing the
partition can be properly defined and controlled and if the desired fraction can be reliably
separated from the putative virus-containing fraction, then it could fit the criteria of an
effective step.
6.4
The objective of the validation is to identify
steps effective in the
inactivation/removal of viruses and to obtain an estimate of the overall capacity of the
manufacturing process to inactivate/remove them. An overall reduction factor is generally
expressed as the sum of individual factors (see Appendix II). However, a simple summing of
low individual reduction factors may be misleading. Reductions in virus titre of the order of
1 log or less are considered to be unreliable because of the limitations of virus validation
studies and should be ignored. Manufacturers should differentiate effective steps from
process steps which may contribute to removal but upon which less reliance can be placed.
Consideration should also be given to whether virus surviving one step would be resistant to
a subsequent step or alternatively have increased susceptibility. In general, a single step
having a large effect gives more assurance of viral safety than several steps having the
same overall effect.
6.5
If little reduction of infectivity is achieved by the production process, and the removal
of virus is considered to be a major factor in the safety of the product, a specific, additional
inactivation/removal step or steps should be introduced.
6.6
304
For all viruses, manufacturers will be expected to justify the acceptability of the
reduction factors obtained. Results will be considered on a case by case basis.
3AB8a
6.7
The GMP principle that material subjected to an effective virus inactivation/removal
step should be segregated from untreated material should be rigorously applied.
7.
Validation studies are useful in contributing to the assurance that an acceptable level of
safety in the final product is established and do not by themselves establish safety. A number
of factors in the design and execution of virus validation experiments may lead to a n
incorrect estimate of the ability of the process to remove naturally occurring virus
infectivity. These factors include the following points.
7.1
Experience has shown that different laboratory strains of virus may differ in their
sensitivity to the same treatment. The particular virus chosen may therefore not resemble
the virus for which it has been chosen as a model. Native viruses may have unpredicted
properties, for example association with lipid, which may affect their properties. Virus
preparations used to validate a production process are likely to be produced in tissue culture.
The behaviour of tissue culture virus in a production step may be different from that of the
native virus for example if native and cultured viruses differ in purity or degree of
aggregation. The strains of virus, their cultivation and assay, and details of sampling and
storage should all be documented.
7.2
There are some situations in which it may not be valid to add logarithmic
reductions. For example, if a matrix is able to adsorb 104 infectious units of a virus and then
cannot adsorb further material with comparable affinity then it will remove all virus when
challenged with 104 infectious units, but only 1% when challenged with 106. The clearance
measured may therefore differ with the challenge titre.
7.3
Inactivation of virus infectivity frequently follows a biphasic curve in which a rapid
initial phase is followed by a slower phase. It is possible that virus escaping a first
inactivation step may be more resistant to subsequent steps. As a consequence, the overall
reduction factor is not necessarily the sum of reduction factors calculated from each
individual step in which a fresh virus spike suspension is used. For example if the resistant
fraction takes the form of virus aggregates, infectivity may be resistant to a range of
different chemical treatments and to heating.
7.4
Model scale processing is likely to differ from full scale processing despite care
taken to design the scaled down process.
7.5
The presence of antibodies to a native virus may affect partition of the virus or its
susceptibility to chemical inactivation; but it may also complicate the design of the study by
neutralising infectivity. The appropriateness of the study design may be difficult to judge.
The level of antibody present may be considered a significant process variable.
7.6
Small differences in production parameters such as protein content or temperature
can produce large differences in the reduction of virus infectivity by whatever mechanism.
8.
8.1
RE-EVALUATION STUDIES
Changes to the production process may necessitate a new validation study.
305
3AB8a
8.2
As scientific experience accumulates, processes will require re-examination
ensure that they remain of an acceptable standard.
306
to
3AB8a
APPENDE* 1
STATISTICAL EVALUATION OF VIRUS TITRES AND REDUCTION FACTORS AND
ASSESSMENT OF THEIR VALIDITY
Virus titrations suffer the problems of variation in common to all biological assay systems.
Assessment of the accuracy of the virus titrations and reduction factors derived from them
and the validity of the assays are therefore necessary to define the reliability of a study. The
objective of statistical evaluation is to establish that the study has been carried out to an
acceptable level of virological competence.
1.
2.
Variation can arise within an assay as a result of dilution errors, statistical effects
and differences within the assay system which are either unknown or difficult to
control. These effects are likely to be greater when different assay runs are compared
(between assay variation) than when results within a single assay run are compared
(within assay variation).
3.
The 95% confidence limits for within assay variation and for between assay variation
normally should be of the order 0.5 log10 or better. Between assay variation can be
monitored by the inclusion of a in-house reference preparation, the estimate of whose
potency should be within approximately 0.5 log10 of the mean estimate established in the
laboratory for the assay to be acceptable. Within assay variation can be assessed by
standard textbook methods. In any particular experiment, if the precision of the
titration is less than these target figures, the study may still be acceptable if justified.
4.
The reduction in virus load should be calculated from the experimentally determined
virus titres. The 95% confidence limits of the reduction factors should be obtained
wherever possible. They can be approximated by +^](s +a ), where s is the 95%
confidence limits for the viral assays of the starting material, and for the viral
assays of the material after the step.
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APPENDLX II
CALCULATION OF REDUCTION FACTORS
The virus reduction factor, R, for an individual inactivation or removal step is given by the
expression:
VI T\
R = log
V2 Tl
where, R = the reduction factor,
VI = volume of starting material,
T l = concentration of virus in starting material,
V2 = volume of material after the step, and
T2 = concentration of virus after the step.
This formula takes into account both the titre and the volume of the material before and after
the step.
Reduction factors are normally expressed on a logarithmic scale which implies that, while
residual virus infectivity may be greatly reduced, it will never be reduced to zero. The
European Pharmacopoeial convention* with respect to methods of sterilisation is that
processes which deliver a sterility assurance level (SAL) of 10'6 or better for bacteria, moulds
and yeasts are considered adequate. A SAL of 10"6 denotes a probability of not more than one
viable micro-organism in 1 IO"6 sterilised items of the final product.
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TABLE 1
EXAMPLES OF VIRUSES WHICH HAVE BEEN USED IN VIRUS VALIDATION STUDIES
Virus
Family
Genus
Natural
Host
Genome
Env
Size
Shape
Resistance to
Physicochemical
Treatment*
Vesicular
stomatitis virus
Rhabdo
Vesiculovirus
Equine
Bovine
RNA
Yes
70x175 nm
Bullet shaped
Low
Parainfluenza
virus
Paramy
zo
Paramyxovirus
Various
RNA
Yes
100-200nm
Pleo/Spher
Low
Retro
Lentivirus
Man
RNA
Yes
8)-100nm
Spherical
Low
Murine leukaemia
virus (MuLV)
Retro
TypeC
oncovirus
Mouse
RNA
Yes
8)-110nm
Spherical
Low
Sindbis virus
Toga
Alpha virus
Man?
RNA
Yes
60-70nm
Spherical
Low
Bovine viral
diarrhoeal virus
(BVDV)
Toga
Pesti virus
Bovine
RNA
Yes
50-70nm
Pleo-Spher
Low
Pseudorabies virus
Herpes
Varicellovirin
Swine
DNA
Yes
120-200nm
Spherical
Med
Poliovirus, Sabin
typel
Picorna
Enterovirus
Man
RNA
No
25-30nm
Icosahedral
Med
Encephalomyocard
itis virus (EMC)
Picorna
Cardiovirus
Mouse
RNA
No
25-30nm
Icosahedral
Med
Reovirus 3
Reo
Orthoreovirus
Various
RNA
No
60-80nm
Spherical
Med
Hepatitis A
Picorna
Hepato virus
Man
RNA
No
25-30nm
Icosahedral
High
SV40
Papova
Polyomavirus
Monkey
DNA
No
40-50nm
Icosahedral
V.High
Parvoviruses
(canine, porcine)
Parvo
Parvovirus
Canine
Porcine
DNA
No
18-24nm
Icosahedral
V.High
This Table gives an Incomplete list of viruses which have been used in validation studies. Consequently, the use of any of the viruses in the Table is not
mandatory and manufacturers are invited to consider other viruses especially those which may be more appropriate for their individual production processes.
This general classification is based on validation studies of production processes
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Guideline Title
Legislative basis
Date of first adoption
Date of entry into
force
Status
Previous titles/other
references
Additional Notes
311
3AB9a
Data on the validation of processes for the removal or inactivation of viruses are rapidly
accumulating. Validation studies should therefore be reviewed and updated if necessary at
intervals to ensure that they are consistent with current scientific knowledge.
Where possible studies should be performed with species of viruses which may be present i n
plasma, such as HIV. In some cases this will not be possible. For example hepatitis C virus
(HepCV) cannot be grown or assayed readily, so that model viruses must be used. The model
virus chosen should be as close in its relevant properties to HepCV as possible. In the past,
togaviruses such as Sindbis virus, flaviviruses such as yellow fever virus and pestiviruses
such as bovine viral diarrhoea virus have been used as models for HepCV. All have
properties in common with HepCV and the results have generally been consistent with the
safety of the product in clinical use. Further data on the behaviour of the viruses are needed
to identify the most appropriate model.
Hepatitis A virus is a non-enveloped virus of the Picornavirus family which is believed to
have been transmitted by clotting factors. Hepatitis A virus should be used whenever possible
as it is thought to be significantly more hardy than other picornaviruses. However plasma
pools can contain neutralising antibodies which may make validation studies difficult. In
addition, hepatitis A virus may be technically demanding to grow and assay. Alternative
viruses such as EMC or Theiler's virus or the use of pools screened for the absence of
antibodies to HAV in validation studies may be considered where relevant. The choice of
model viruses for hepatitis A, if any, will become clear with further studies.
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3 AB 10a
Guideline Title
Legislative basis
Date of first adoption
Date of entry into
force
Status
Previous titles/other
references
Additional Notes
CONTENTS
1.
GENERAL REMARKS
2.
3.
4.
5.
CONCLUDING REMARK
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3 AB 10a
GENERAL REMARKS
Bovine spongiform encephalopathy (BSE) was first recognised in the United Kingdom i n
1986. Since then a large number of cattle and individual herds have been affected. This note
for guidance considers the implication of the disease for medicinal products and methods for
minimising the risk of transmission by their use.
The naturally occurring spongiform encephalopathies include scrapie (in sheep and goats),
chronic wasting disease (in mule deer and elk), bovine spongiform encephalopathy (BSE; i n
cattle) as well as Creutzfeldt-Jakob Disease (CJD) and Kuru (in humans). Agents causing
these diseases replicate in infected individuals without being detectable by diagnostic tests
applicable to the living organism. After incubation periods of up to several years the agents
cause disease and, finally, lead to a fatal outcome. No means of therapy are known.
Diagnosis is based on clinical signs with post mortem confirmation of characteristic brain
lesions by histopathology or immunological detection of the fibrillary proteins specific for
the spongiform encephalopathies. The demonstration of infectivity by the inoculation of
suspect tissue into target species or laboratory animals may also be used for confirmation
but with an incubation period of months or years. Iatrogenic transmission of spongiform
encephalopathies has been reported. In sheep scrapie has been accidentally transmitted via
the application of Louping III vaccine prepared from pooled, formaldehyde treated ovine
brain and spleen in which material from scrapie infected sheep had been inadvertently
incorporated. In humans cases of transmission of CJD have been reported which have been
attributed to the repeated parenteral administration of growth hormone and gonadotropin
derived from human cadaveric pituitary glands. Cases of CJD have also been attributed to
the use of contaminated instruments in brain surgery and with the transplantation of
human meninges and cornea.
There is no evidence that spongiform encephalopathies have been transmitted from animals
to humans. However, the possibility of such transmissions, although remote, cannot be
dismissed. Therefore due prudence is warranted if biological materials are used for the
manufacture of medicinal products from species affected via non-experimental routes by
those diseases, primarily ruminants and among these especially cattle, sheep and goats.
Information on the characteristics of the agents is limited. They are extremely resistant to
the chemical and physical procedures that inactivate conventional viruses. They do not
induce a detectable immune response. There are natural barriers which limit the
interspecies spread of infection, but they can be crossed under appropriate circumstances
usually involving efficient routes of administration and high doses of agent. Studies on
laboratory animals have shown that intracerebral inoculation is much more efficient than
any other route and is followed in decreasing order of efficiency, by intravenous,
intraperitoneal and subcutaneous administration. The oral route is less efficient than the
parenteral routes. In some cases species barriers can be crossed only after passage of the
agent through intermediary species.
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3 AB 10a
Human beings must have been naturally exposed to the scrapie agent for at least 200 years,
but despite extensive epidemiological studies no sign of transmission of scrapie to humans
has been detected. Insofar as BSE is different from scrapie, it is conceivable that also the
species barriers may be different. Therefore the recommendations below should be followed.
The acceptability of a particular medicinal product containing or derived from bovine
materials will be influenced by a number of factors including the selection and processing
of source materials, the age and geographical origin of the individual source animal, the
intended use of the product, its stipulated dose and route of administration, production
process and quality control. The state of science and technology must be taken into
consideration. All products will be considered on a case by case basis.
2.
This note for guidance covers all medicinal products which contain active substances
and/or excipients derived from bovines, as well as medicinal products for which the
production process involves bovine materials.
The note also covers the use of such materials in procedures which are indirectly associated
with the manufacturing process, for example, in test media used in the validation of plant
and equipment to avoid cross-contamination.
3.
The safety of medicinal products can be further secured, and the risk of transmission
infectious agents greatly reduced by combinations of the measures specified in this note
guidance or other appropriate measures. The pharmaceutical manufacturers and
producers of medicinal products of animal origin are responsible for the selection
adequate measures.
of
for
the
of
the feeding to ruminants of ruminant protein derived from the specified offal (brain,
spinal cord, spleen, thymus, tonsil and intestine from duodenum to rectum, placenta),
either produced in the country or imported from other countries;
b)
318
3AB10a
c)
d)
scrapie-associated factors:
-
the relative geographical distribution of sheep and goats to cattle, where this
might have led to the use of sheep material in cattle feed in the past;
importation of cattle above the age of 6 months from countries where a high incidence
of BSE has occurred and/or importation of progeny of affected females.
3.1.2 Materials may also be sourced from countries where a low number of cases have
occurred, if in addition to the factors in paragraph 3.1.1:
-
3.1.3 Satisfactory source materials may be obtained from established and monitored herds,
where their feeding and breeding history is documented. This is possible even in countries
with a high incidence of BSE.
the potential risks will be influenced by the circumstances in which tissues were
removed, especially by contact of material of a low-risk group with that of a high-risk
group. Thus the contamination of some tissues may be increased if infected a n i m a l s
319
3AB10a
are slaughtered by penetrative brain stunning, or if the brain and/or spinal cord is
sawed;
-
dura mater, hypophysis and pineal gland from animals older than six months should
be regarded as belonging to group 2 only if contamination with brain tissue can be
avoided;
body fluids should be collected with minimal damage to tissue, and cellular
components should be removed; e.g. foetal blood should be collected without
contamination from placenta and amniotic fluids.
The information currently available suggests that, given assurances of adequate collection
and processing, certain materials and their derivatives are unlikely to present any risk of
contamination. These include: milk and its derivatives, for example, lactose and casein;
skin and its derivatives, for example, gelatine; hair and wool and their derivatives, for
example, wool alcohols and lanolin.
In addition, materials derived from rendered carcasses and subjected to rigorous processes
of extraction and purification (for example, triglycerides, glycerol, sorbitan esters, etc.
manufactured from tallow) may be considered unlikely to be contaminated.
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CATEGORY I
High infectivity
CATEGORY II
Medium infectivity
CATEGORY III
Low infectivity
CATEGORY IV
No detectable infectivity
4.
Removal and inactivation procedures contribute to the reduction of the risk of infection.
Their effectiveness in removing infectivity during a given production process must be tested
and validated using appropriate model systems (presently: animal infection experiments).
Tissues in brackets were not titrated in the original studies1""', but their relative infectivity is indicated by
other data on spongiform encephalopathies. Materials not listed may be classified by analogy to those
mentioned on the basis of their composition.
No infectivity was transmitted in bioassays involving inoculation of up to 5 mg tissue into rodent trains.
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Whereas none of the following procedures may guarantee complete inactivation of the
infectious agents, the efficiency of the first three methods on this list is considered greatly
superior to that of the remaining ones:
-
autoclaving at shorter times and/or lower temperatures than those given above;
5.
CONCLUDING REMARK
at least 2%
Although this note for guidance relates particularly to BSE and materials of bovine origin,
similar considerations are also applicable to material from sheep, goats and other species
affected via non-experimental routes by agents causing spongiform encephalopathies.
Finally, while this note for guidance has general applicability, it may not be necessary to
fulfil all of the listed measures for all products. The potential risks associated with a given
medicinal product will have to be considered individually in the light of specific
circumstances and current knowledge.
1)
2)
3)
4)
5)
Diringer H, Hilmert H, Simon D, Werner E, Ehlers (1983) Eur J Biochem 134, 555560
6)
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Guideline Title
Legislative basis
Previous titles/other
references
Additional Notes
None
This document provides basic guidance on the
presentation of data validating test procedures carried out
for toxicological and pharmacological studies as well as
for clinical trials provided for by Directive 75/318/EEC as
amended with a view to the granting of a marketing
authorisation in respect of a medicinal product.
CONTENTS
1.
INTRODUCTION
2.
3.
RECOMMENDATIONS
ANNEX
323
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INTRODUCTION
The objective of analytical validation on samples of biological origin (plasma, urine, faeces
etc.) is to demonstrate the reliability of results for active substances and metabolites obtained
from pharmacokinetic, metabolic and bioavailability studies.
2.
The validation criteria are those currently used in analytical chemistry (Good Laboratory
Practice) and consist of:
Specificity
Repeatability
Precision
Reproducibility
Accuracy
Linearity/Range/Sensitivity
Limit of detection
Limit of quantification
Each test procedure should be validated for each type of biological sample and each species
(animal, human).
If the same test procedure has been used during the development of the medicinal product (in
vitro) and during routine tests (in vivo), a revalidation is necessary.
The degree of validation depends, to a large extent, on the problem posed.
3.
RECOMMENDATIONS
3.1
For assays on samples of biological origin, the following specific problems can arise,
which may influence both the validation and the interpretation of the results.
3.1.1 The test procedures (assays) carried out are not necessarily done in a single
laboratory, but in many and sometimes even outside of those of the manufacturer. Therefore,
it is very important - for the same test - to be able to compare the results between the two.
There are two cases to consider:
a)
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b)
3.1.2 A significant time-lapse can pass between the moment of sampling and the moment
of analysis. For this reason, amongst others, it is necessary to know:
-
the stability of the substances being examined in the biological fluid in the precise
storage conditions;
the sorption of the substance by the sampling container and the stopper.
3.1.3 The test procedure may change over time according to the evolution of the problem
posed (clinical trials). In that case, a revalidation will be necessary.
A short description of the main principle of the test procedure should be indicated.
must be described
This includes:
-
the exact description of the test conditions including precautions, methods of extraction,
reagents, reference substances and preparations;
the verification of the test procedure under the defined operating conditions, for
example: verification of the separating power of a chromatographic system (system
suitability test);
3.2.3 The reference substances and preparations (in house standards) used for tests - if
pharmacopoeial or other official standards are not used- must be precisely described. Their
identity, purity and content must be fully established.
3.2.4
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In all cases, the complete data which demonstrate validity should be indicated.
3AB11a
ANNEX
Glossary
The annex is a glossary which should give the manufacturer a better understanding of the
different validation requirements and definitions. It should again be remembered here, that
the criteria which must be satisfied depend very much on the objective of the analysis.
1.
TEST PROCEDURE
The test procedure is the total operation necessary to perform the analysis of an analyte:
preparation of the sample, of the reference substances or preparations, of the reagents, use of
the apparatus, calibration curve, formulae for the calculation, number of replicates and
operating procedure for the replicates etc.
2.
SPECIFICITY
TESTS:
(Impurity content)
ASSAY:
(Content or Potency)
to ensure that the signal measured with the test procedure comes
only from the substance being analysed i.e. no interferences from
excipients and/or degradation product and/or impurities.
A routine assay may not necessarily comply with the criterion of specificity. This can be
compensated by using one or more adequate related substances test(s) (applies mainly to
bulk material, see pharmacopoeia).
Specificity is assessed either by a single determination or by the total results of the test
procedures.
a)
b)
c)
Under the heading, the means of satisfying the criteria of specificity may be different:
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3.
ACCURACY
The accuracy expresses the closeness of agreement between the value which is accepted
either as a conventional true value (in house standard) or an accepted reference
value(international standard, e.g. pharmacopoeial standard) and the value found (mean
value)obtained by applying the test procedure a number of times.
The accuracy provides an indication of systematic errors.
Several methods of determining accuracy are available of which the following are two
examples:
a)
Comparing the proposed test procedure with a second test procedure, the accuracy of
which is stated and/or defined (for instance: pharmacopoeial method), (applies
normally to starting material).
b)
4.
PRECISION
The precision of a test procedure expresses the closeness of agreement (degree of scatter)
between a series of measurements obtained from multiple sampling of the same
homogeneous sample under prescribed conditions.
Precision provides an indication of random errors.
4.1
Repeatability
same analyst, . .
same apparatus,
identical reagents.
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4.2
Reproducibility
laboratories,
analysts,
days,
etc.
5.
The lowest amount of analyte in a sample which can be detected but not quantitated as an
exact value. The LOD is mostly a parameter of limit tests.
6.
The lowest amount of analyte in a sample which can be quantitatively determined with
defined precision and accuracy under the stated experimental conditions.
7.
LINEARITY
The linearity of a test procedure is its ability (within a given range) to obtain test results
directly proportional to the concentration (amount) of analyte in the sample.
8.
RANGE
The range of the test procedure is the interval between the upper and lower levels of analyte
(including these levels) for which the procedure has been demonstrated as suitable with
precision, accuracy and linearity using the method as written.
9.
SENSITIVITY
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DE
EN
Exactitude
Richtigkeit
Accuracy
Fidlit
Przision
Precision
Spcificit
Spezifizitt
Specificity
Slectivit
Selektivitt
Selectivity
Linarit
Linearitt
Linearity
Intervalle linaire
Linearer Bereich
Linear range
Seuil de dtection
Nachweisgrenze
Seuil de quantification
Bestimmungsgrenze
Sensibilit
Empfindlichkeit
Sensitivity
Valeur moyenne
Mittelwert
Mean value
Standard deviation
Coefficient of variation
Coefficient de variation
Variationskoeffizient
Intervalle de confiance de la
valeur moyenne
Vertrauensbereich des
Mittelwertes
Rptabilit
Wiederholprzision
Repeatability
Reproductibilit
Vergleichsprzision
Reproducibility
Mthode analytique
Analysenmethode
Analytical method
Procdure d'analyse
Prfverfahren
Test procedure
Rsultat
Ermittlungsergebnis
Result of determination
Erreur systmatique
Systematische
Ergebnisunsicherheit
Zufllige
Ergebnisunsicherheit
Valeur conventionnellement
vraie
Richtiger Wert
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PT
NL
Accuratezza
Rigor, exactido
Nauwkeurigheid
Precisione
Preciso
Precisie
Specificit
Especificidade
Specificiteit
Selettivit
Selectividade
Selectiviteit
Linearita
Linearidade
Lineariteit
Intervallo lineare
Intervalo linear
Lineair interval
Limite di rilevazione
Limite de deteco
Detectiegrens
Limite di determinazione
Limite de quantificao
Bepalingsgrens
Sensibilit
Sensibilidade
Gevoeligheid
Valore medio
Valor mdio
Gemiddelde waarde
Deviazione standard
Desvio padro
Standaardafwijking
Coeficiente de variao
Variatiecofficint
Coefficiente di variazione
Relat,
standaardafwijking
Betrouwbaarheidsinterval
van de gemiddelde waarde
Ripetibilit
Repetibilidade
Herhaalbaarheid
Riproducibilit
Reprodutibilidade
Reproduceerbaarheid
Metodo di analisi
Mtodo analitico
Analysemethode
Procedimento
Procedimento analitico
Proefopzet
Risultato dell'analisi
Resultado
Errore sistematico
Errore casuale
Valore reale
Conventioneel ware
Valor verdadeiro
convencional /Valor nominal waarde
Toevallige
afwijking
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DA
Exactitud
Njagtighed
Precisin
Prcision
Especificidad
Specificitet
Selectividad
Selektivitet
Linealidad
Linearitet
Intervalo lineal
Lineari tetsomrde
Limite de deteccin
Detektionsgrnse
Limite de cuantificacin
Bestemmlsesgrnse (kvantitativ)
Sensibilidad
Flsomhed
Valor medio
Middelvrdi
Desviacin estndar
Spredning
Coeficiente de variacin
Variationskoefficient
Relativ standardafvigelse
Konfidensinterval for
middelvrdi
Repetibilidad
Repetrbarhed
Reproductibilidad
Reproducerbarhed
Mtodo analtico
Analysemetode
Procedimiento analtico
Afprvningsmetode
Resultado de analisis
Resultat
Systematisk fejl
Tilfldig fejl
Valor verdadero
convencional
Sand vrdi
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Guideline Title
Legislative basis
CONTENTS
1.
INTRODUCTION
2.
SOURCE MATERIALS
3.
MANUFACTURE
4.
QUALITY CONTROL
5.
VALIDATION STUDIES
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INTRODUCTION
Council Directive 89/381EC extends the scope of Directives 65/65/EEC and 75/319/EEC to
medicinal products derived from human blood or human plasma.
Human blood and plasma contain many proteins, the extraction and purification of which
are of great medical importance. The therapeutic use of blood and blood products goes back to
the turn of the century. Two important advances since then have led to a surge in the use of
blood products. These were the discovery of blood groups and the development of methods to
fractionate plasma into components of medical value.
Improvements in protein purification and molecular separation technology over recent years
have made available a wide variety of products with medical applications covering a large
and growing field. However, blood can harbour many viruses, and the use of medicinal
products derived from human blood or plasma has led to the transmission of severe viral
diseases, including hepatitis , and C and AIDS caused by the human immunodeficiency
virus (HIV). Other microbial contaminants have also been the cause of serious accidents.
Thus, measures designed to prevent the transmission of pathogens are essential to ensure
the general safety of products derived from human blood and plasma.
Products derived from human blood and plasma can roughly be divided into two groups:
The first group covers products derived from single donations or from small pools of source
material (< 12 donors). These products, for example, cell concentrates and cryoprecipitates
are commonly made and distributed by blood donor centres and used in transfusion
medicine. They are either subjected to one or a few separation procedures. Their quality and
safety are almost exclusively dependent on the careful selection and control of donors, on the
screening of donations and on measures taken to minimise contamination during
processing.
The second group is represented by derivatives of plasma produced on an industrial scale
from pools of source material through various manufacturing procedures. They may be used
as therapeutic medicines or as excipients. The quality and safety of these products rely both
on the selection and screening of source materials and on the choice of the manufacturing
processes, including processes which inactivate or remove microbial contaminants.
Directive 89/381/EEC covers only products belonging to the second group. These include:
This note for guidance covers medicinal products derived from plasma and focuses on
specific aspects relating to the manufacture and control of these products, paying particular
attention to the steps taken to minimise the risks of microbial contamination of the finished
product. It does not cover cellular blood products although many parts contained in this
document may be pertinent.
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2.
the document from the Council of Europe, entitled "Guide to the preparation, use and
quality assurance of blood components (1995)", which addresses the collection,
preparation and use of blood. This document deals primarily with the requirements for
blood and blood components for use in blood transfusion and in immunohaematology;
3.
2.
SOURCE MATERIALS
2.1 CLASSIFICATION
Several types of source materials currently used in the manufacture of medicinal products
derived from human plasma are specified by the European Pharmacopoeia and by the WHO
requirements. Their origin and means of collection differ in several respects.
Two types of these source materials are relevant in the context of this note for guidance:
a)
Whole blood donations are collected at blood donor centres. This material is used to
prepare products made from single donations for direct transfusion while much of the
plasma is used for fractionation on an industrial scale;
b)
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clotting factors, which can be affected by the treatment and preparation of the source
materials after collection.
General
The means of collecting source materials and its control are major factors in the quality
assurance of the manufacture of biological medicinal products. Measures taken to reduce
risks include the meticulous control of source materials and their origin.
Collecting centres should be inspected and approved by a competent authority. Manufacturers
should provide documentary information on the acceptability of the centres, and on the
procedures for collecting, storage and shipping of source materials.
2.3.2 A Quality
Assurance
System for
collection
Recommendations given in the document from the Council of Europe "Guide to the
preparation, use and quality assurance of blood components (1995)" and from the forty-third
report of the WHO Expert Committee on Biological Standardisation (WHO Technical Report
Series 840, 1994) with later amendments should be followed.
Each collection/transfusion centre should establish, document and maintain an effective
quality assurance system. The main requirements are summarised below:
the preparation of standard operating procedures;
the establishment of records so that donations can be traced, e.g. date of collection,
quality control tests undertaken, with results etc., should be included;
specifications for source plasma for further industrial processing into medicinal
products;
control of labelling, storage and transportation of donations;
establishment of quality audits/review;
appropriate premises.
a)
b)
it is found subsequent to donations that the donor did not meet the current donor
selection criteria;
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it is discovered that testing for viral markers has not been carried out according
to agreed procedures;
the donor develops an infectious disease caused by a transmissible agent (see
section 2.2);
the recipient develops post transfusion infection which implicates or can be
traced back to the donor.
3.
MANUFACTURE
b)
viruses may be introduced by reagents during manufacture (e.g. enzymes from tissue
extracts or monoclonal antibodies used for affinity chromatography);
c)
d)
methods of manufacture may modify the product resulting in adverse consequences for
recipients, for example by the formation of neo-antigens or by compromising the
biological activity of the active component. This is true for steps introduced to
inactivate viral contamination which may affect the quality or yield of products.
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3.3.1 Fractionation/purification
a)
procedures
Precipitation methods
Physical methods:
Cryoprecipitation is most often used as the initial step for the production of Factor VIII
concentrates.
Subsequent purification techniques, such as precipitation or chromatographic
separation as well as procedures for viral inactivation are used to obtain the finished
products. Cryoprecipitate-depleted plasma can be used for the preparation of other
coagulation factors or plasmaprotein solutions.
Physical/chemical methods:
Among these methods, the ethanol fractionation procedures derived from the Cohn
method are the most widely used for albumin and immunoglobulins. They commonly
incorporate several steps, in each of which compliance with specific requirements is
decisive for product quality; under proper conditions some of these steps may also
contribute to effective reduction of potential viral contaminants. Therefore, clear
specifications for ethanol and protein concentration, temperature, pH and ionic
strength, and time of treatment, with data on acceptable tolerance as well as the means
of controlling them should exist.
Appropriate data should also be provided for methods relying on other chemical agents
such as ethylacridin-lactate, methanol, ammonium sulphate, polyethylene glycol,
cationic detergents, which are sometimes used in the preparation of certain plasma
derivatives, as a rule in combination with other purification procedures. Some of these
substances may have an impact on viral safety, for others information is still scarce.
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b)
Chromatographie methods
Three basic types of procedures play an increasing role in the processing of plasma
derivatives, as a rule in combination with precipitation procedures and often with each
other:
-
The selectivity of the procedures and the yields depend critically on the quality of the
material as well as on factors like the capacity of the column, nature and
concentration of proteins in the product, ionic strength and the pH of buffers, flow rate
and temperature. Therefore, all appropriate specifications and accepted tolerances
should be stated, and control data documented.
Several other compounds like charcoal, bentonite, colloidal silica are sometimes used
for clearing various impurities like pigments, lipoproteins etc. Details on the
characteristics of the compounds, on their decontamination and on the operating
conditions should be provided.
The conditions of storage of the columns, preservation and elution of preservatives,
and methods of regeneration should also be described. Details should be given of
clarification and sterile, dia- or ultra-filtration procedures used.
c)
Complementary procedures
Immunoglobulins intended for intramuscular administration may cause adverse
reactions upon intravenous administration. Therefore, the production process for
immunoglobulins intended for intravenous administration includes various
procedures which can substantially reduce such reactions. The materials and the
procedures used should be described and their suitability justified and documented.
procedures
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Stabilisation to protect proteins can also protect virus from inactivation. Careful
validation is, therefore, needed for each product ensuring that the validation includes
worst case conditions.
b)
c)
Solvent/detergent treatment
Treatment with a solvent such as tri-n-butyl-phosphate (TNBP) combined with' a nonionic detergent such as Triton X-100 or Tween 80 can inactivate enveloped viruses.
Prior to such treatment, in-process solutions should be free from gross aggregates that
may harbour virus and protect it from the treatment. This can be achieved by filtration
which should be done prior to addition of the solvent/detergent or if done after, the
filters should be demonstrated not to alter the levels of these additives in the incubation
solution. Physical validation must demonstrate that mixing achieves a homogeneous
mixture for the duration of the defined incubation time. In-process checks should be
carried out to confirm that the correct amount of solvent and detergent have been
added. Validation experiments should investigate the range of key process variables
and in-process limits should be set accordingly. Since lipid content can affect the
efficacy of inactivation, inactivation should be confirmed under worst case conditions
for lipid content. Residual levels of solvent and detergent should be minimised by
processing and carefully monitored in the final product. Non-enveloped viruses will
not be inactivated by this process.
d)
e)
Low pH
Low pH (approximately 4) can inactivate certain viruses. The reduction factors that
have been demonstrated depend on the exact conditions used in manufacturing (e.g.
pH value, time and temperature of treatment, composition of the solution, etc.). Each
process therefore has to be carefully validated.
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3.4 C o n s i s t e n c y of p r o d u c t i o n
The manufacturer should demonstrate consistency of the specifications of the product for at
least 3 full scale production batches.
4.
QUALITY CONTROL
5.
VALIDATION STUDIES
Validation studies should be carried out by each manufacturer for the specific processes used
and, unless otherwise justified, for each production site. Moreover, if studies involve
modelling the process on a reduced scale, they should be capable of mimicking satisfactorily
the conditions of full scale production and the accuracy of the modelling should be
demonstrated.
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The studies should be designed to justify the selected operating conditions and the acceptable
tolerances, including worst case conditions, and to document their adequacy in achieving
the expected process performances.
When chromatographic columns are used, conditions leading to overloading as well as
leaching from the gels, particularly in the case of affinity chromatography with potentially
harmful ligands, should be carefully investigated. Attention should also be paid to the
cleaning and regeneration of the columns and to the effective removal of residues from the
previous run or of preservatives added for storage, particularly if mild treatments are used.
5.2 V i r u s I n a c t i v a t i o n / R e m o v a l
52.1 Manufacturing process design
General principles concerning the incorporation of virus inactivation/removal steps in the
manufacture of biological products are outlined in the note for guidance Virus Validation
Studies: The Design, Contribution and Interpretation of Studies Validating the Inactivation and
Removal of Viruses. This section contains further guidance relevant to plasma derivatives.
The principles in both guidelines should be taken into account when designing
manufacturing processes or modifying processes to give further assurance of viral safety.
a)
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c)
d)
e)
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iii)
studies
General guidance on choice of viruses is given in the CPMP guideline Virus Validation
Studies: The Design, Contribution and Interpretation of Studies Validating the Inactivation and
Removal of Viruses. Viruses to be used in validation studies on plasma-derived medicinal
products should include at least:
i)
Hrv-i
It is not necessary to carry out additional studies with TTTV-2 as it is similarly affected
by inactivation procedures.
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ii)
iii)
Non-enveloped viruses
The package of validation studies on non-enveloped viruses should establish the range
of viruses susceptible to the inactivation/removal processes and identify the limits of
the process. For example, a heat inactivation step used in the manufacture of a
coagulation factor might be effective against hepatitis A virus but ineffective against
another non-enveloped virus.
Hepatitis A transmission has been associated with certain coagulation factors.
However, there are insufficient data to identify appropriate models for hepatitis A at
the present time. HAV should be used for validation studies for coagulation factors as
it is thought to be significantly different to other picornaviruses. Consideration should
be given to the possible interfering effects of antibodies.
Validation studies for coagulation factors should also include an appropriate model for
the parvovirus B19. Models that have been used include canine, porcine, murine and
bovine parvoviruses. Studies using HAV and B19 are not required for
immunoglobulins if the presence of protective levels of antibodies in the product can be
assured. However, studies with non-enveloped viruses for which antibodies are
unlikely to be present should be performed to evaluate the ability of the process to
inactivate/remove possible unknown non-enveloped viruses.
iv)
5.2.3 Difficulties
studies.
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toxic for cell cultures used for virus detection as is the presence in intermediary products of
chemicals such as ethanol and ethylacridinlactate. Therefore, assays may have to be
preceded by procedures designed to counteract these effects, such as dilution, dialysis, etc. In
addition, the product itself or chemicals used to prepare or to treat it may change the
properties of viruses, for example leading to their coating and/or aggregation, which may
result in difficulties in reliable quantification of residual infectivity.
5.3 Revalidation
New validation studies are required when relevant changes in the manufacturing process or
in individual steps are being considered.
Validation experiments have many limitations. Any virus transmission seen in clinical
use should result in an evaluation of available data by manufacturers and regulatory
authorities so that appropriate action can be taken.
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ADDENDUM
STRATEGY FOR INTRODUCING ADDITIONAL PROCESS STEPS FOR THE
INACTIVATION/REMOVAL OF VIRUSES
Specific virus inactivation/removal steps are included in the manufacturing processes for
many plasma derivatives. However, recent transmissions of enveloped and non-enveloped
viruses by certain products have highlighted the need for a strategy to further increase the
assurance of viral safety of plasma derivatives. The objectives are set out in the guideline.
This Addendum sets out the priorities for action and the strategy to be followed.
Manufacturers should, as a matter of urgency, validate their processes for the
inactivation/removal of enveloped and non-enveloped viruses where they have not already
done so and, where the current process is not effective in inactivation/removal, develop and
validate additional virus inactivation/removal steps in order to improve safety. The priority
order is, starting from the highest: coagulation factors, intravenous immunoglobulins,
intramuscular immunoglobulins and albumin.
Marketing authorisation holders and applicants are required to set and justify timetables for
such developments; to submit a programme of process/product improvements to Member
States for their agreement and to commit themselves to providing 6 monthly reports to
Member States on their progress. Timescales for introduction of process changes should
reflect the manufacturer's best efforts. In the meantime, product literature needs to be looked
at critically and, where necessary, amended to provide relevant and specific information to
enable clinicians to make an informed choice of product.
Member States will keep the progress of process improvements under review. It will not be
acceptable to keep on the market products that have fallen behind general developments for
their class of products.
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ANNEX I
PUBLISHED MONOGRAPHS ON BLOOD PRODUCTS
Current version
(date)
Implementation
date
1995
1/1/96
1995
1/1/96
1994
1/V95
1994
1/1/95
1987
1/1/88
1994
1/1/95
1995
1/1/96
1991
1/1/92
1995
1/V96
1994
1/7/94
1994
1/7/94
1995
1/1/96
1995
1/V96
1995
1/1/96
1995
1/1/96
1995
1/1/96
1985
1/1/86
1995
1/1/96
Revision
Implementation
of revision
1996
1/1/97
1996
1/1/97
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ANNEX II
GENERAL METHODS
Current version
(date)
Implementation
date
1994
1/1/95
1980
1980
Fe function of immunoglobulin
(V.2.2.10)
1995
1/1/96
1995
1/7/96
1994
1/7/94
350
Revision
Implementation
of revision
3AB13a
Guideline Title
Legislative basis
Date of first adoption
Date of entry into
force
Status
Previous titles/other
references
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1.
With regard to blood products as well as with other medicinal products, improvement
of testing is a continuous process, evolving with the state of the art. The introduction of a new
test or test method does not automatically mean that the formerly produced batches are
unsafe.
2.
Following the specific requests for comments on plasma pool testing by
manufacturers and/or control authorities during the consultation period on the batch release
documents for medicinal products derived form human blood or plasma, plasma pool testing
was extensively discussed by the Biotechnology/Pharmacy Working Party.
3.
Plasma pool testing is seen as one of a number of steps which, taken together, provide
assurance of the virological safety of blood products. Individual donations of blood/plasma
for manufacture of blood derivatives must be tested and found negative for viral markers
(anti-HrV 1 and 2, hepatitis surface antigen and anti-hepatitis C antibody). Because errors
in testing and/or pooling can occur, manufacturers of blood derivatives should, in addition,
introduce testing of their plasma pools for the above-mentioned viral markers.
4.
Test procedures must be the most up-to-date and must be validated for specificity and
sensitivity for testing plasma pools;
Pools which are confirmed positive for any of the above markers must be rejected as
well as any intermediates or products produced therefrom;
Manufacturers who are not already implementing plasma pool testing as described
above, should introduce plasma pool testing as from 1 November 1994.
5.
Five Member States require batch release for some or all medicinal products derived
from human blood or plasma.
Currently the United Kingdom requires and operates plasma pool testing within the batch
release procedure. The same is envisaged in the near future in Germany.
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Guideline Title
Legislative basis
Date of first adoption
Date of entry into
force
Status
Previous titles/other
references
Additional Notes
CONTENTS
A.
B.
C.
D.
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INTRODUCTION
1.1
Directive 89/342/EEC relating to immunological products (consisting of vaccines,
toxins or serums and allergens) provides in article 4.3 that, where a Member State considers
it necessary in the interests of public health, it may require that samples from each batch be
submitted for examination by a State laboratory or a designated laboratory for the following
medicinal products:
-
live vaccines;
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2.
2.1
Protocol submission
The manufacturer's detailed protocol of production and tests carried out according to the
European Pharmacopoeia monograph on influenza vaccines shall be approved by the control
authority for each vaccine batch. The protocol should be based upon the WHO summary
protocol for influenza vaccine (inactivated) (WHO Technical Report Series 638, 1979) an
example of which is illustrated in paragraph 5. Manufacturers should submit full details of
test results; it is insufficient to indicate only "pass" or "fail".
a)
b)
endotoxin content;
2.2.3
a)
2.2.4 Tests to be performed on the first 5 lots of monovalent purified subunit vaccine
following the introduction of a new influenza strain:
a)
3.
Additional tests from the EP monograph on influenza vaccines may be necessary for batch
release in special circumstances:
-
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4.
RELEASE CERTIFICATE
A release certificate for each vaccine batch shall be presented to the manufacturer after
approval when the results of testing are satisfactory. The release certificate must give details
of:
4.1
4.2
4.3
Batch number
4.4
Number of containers
4.5
4.6
Type of container
4.7
4.8
Date of expiry
5.
The following summary protocol is an example of the type of information required for batch
release. The data submitted should be in accordance with the current EP monograph on
influenza vaccines.
Name of product:
Marketing authorisation:
Name and address of manufacturer:
Batch number:
Filling lot number:
Date of manufacture:
Date of expiry:
Type of container:
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Number of doses:
Dose volume:
Composition:
e.g. strain 1
strain 2
strain 3
15 pg HA/0.5 ml
15 pg HA/0.5 ml
15 ug HA/0.5 ml
Statement of quality:
e.g.
I certify that lot number
of this product satisfies the requirements of the European
monograph on influenza vaccines.
Signature:
Name (typed):
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Primary seed
Primary seed
H3N2
HINI
Lotn :
Lotn:
Lotn:
Working seed
Working seed
Working seed
H3N2
HINI
Lotn:
Lotn:
Lotn:
Monovalent bulk
Monovalent bulk
Monovalent bulk
H3N2
HINI
Lotn:
Lotn:
Lotn :
Trivalent bulk
Lotn 0 :
Final product
Filling lot N:
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Seed Virus
1.
Information on manufacture
1.1
Virus strain:
1.2
1.3
1.4
Date of receipt:
1.5
Comments:
1.6
Storage conditions:
1.7
1.8
1.9
Added antibiotics:
2.1
Sterility
method:
Date of test:
Volume tested:
Test results:
2.2
2.3
Identity
a)
Haemagglutinin
Date oftest:
Test results:
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e.g.
HI Titre
Antigen
Antiserum
Shang/1 Sich/2/87
1/87
Taiw/1/86
B/Yam/16/88
A/Shang/11/87
(H3N2) Ref.
A/Sich/2/87
(H3N2) Ref.
A/Taiw/1/86
(HINI) Ref.
A/Sang/11/87
Working seed lot n
b)
Neuraminidase
Date oftest:
Test results:
e.g.
NT Titre
Antiserum
Antigen
anti-N2NA
A/Shang/11/87
(H3N2) Ref.
A/Sich/2/87
(H3N2) Ref.
A/Taiw/1/86
(HINI) Ref.
B/Yam/16/88
Ref.
A/Sang/11/87
Working seed lot n ...
2.4
Infectivity titre:
Date of tests:
Test results:
364
anti-NlNA
anti-BNA
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Information on manufacture
Name and address of manufacturer:
1.1
Virus strain:
12
Lot numbers):
1.3
1.4
Date of inoculation:
1.5
Date of harvesting:
1.6
Method of inactivation:
1.7
Date of inactivation:
1.8
Concentration/purification procedure:
1.9
Added antibiotics:
2.1
2.2
2.3
Identity of haemagglutinin
Method:
Date oftest:
Test results:
2.4
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Bulk Vaccine
Date of test:
Test results:
1.
Information on manufacture
Name and address of manufacturer:
1.1
Lot number:
1.2
1.3
1.4
Date of blending:
2.
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Finished Product
1.
Information on manufacture
Name and address of manufacturer:
1.1
Lot number:
1.2
Date of filling:
1.3
Type of container:
1.4
Volume in container:
1.5
2.
2.1
2.2
Sterility
Method:
Date of test:
Test results:
2.3
2.4
2.5
Abnormal toxicity
Method:
Date of test:
Test results:
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2.6
2.7
Endotoxin
Method:
(e.g. type of limulus kit)
Date oftest:
Test results:
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INTRODUCTION
When a new application for marketing authorisation for an influenza vaccine is made, full
clinical trial data should be submitted with the application. Such clinical trials are outside
the scope of this note for guidance. However, the strain composition of influenza vaccines i s
modified periodically to take account of the changes in the prevalent viruses causing
influenza and manufacturers should apply for yearly licensing to accommodate strain
changes.
Vaccine manufacturers are required to be involved in ongoing clinical trials of influenza
vaccines and to present the results to the competent authorities. The results of the trials are
not part of the yearly licensing procedure. Guidance for performing these clinical trials is
given in this section.
The purpose of such trials is to verify:
-
the immunogenicity of the haemagglutinin of the vaccine strains, i.e. the titre and
frequency of anti-HA antibody responses;
Whenever the characteristics of a new strain incorporated into the vaccine or the
susceptibility of the population to the new strain requires adjustment of the doses, various
doses of antigens need to be tested to confirm the adequacy of 15 pg HA per strain and per
dose.
The yearly clinical trials on influenza vaccine shall be carried out in accordance with the
note for guidance on Good Clinical Practice.
The clinical trials are carried out by the manufacturers, who will forward the results, as
soon as they have been obtained, to the competent authorities and in any event before the next
influenza season.
2.
GENERAL REQUIREMENTS
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pregnant women.
Just prior to vaccination, a 10 ml venous blood sample shall be taken from each trial
subject, for base-line titration of circulating anti-HA antibodies;
b)
immediately thereafter, each subject shall receive 1 dose of vaccine (0.5 ml) by
intramuscular or subcutaneous injection into the upper arm. The injection shall be
given into the opposite arm from which blood was drawn;
c)
d)
Trial subjects shall receive, at the time of vaccination, a standardised form to complete
and give to the investigator when they come for the post-vaccination blood sampling;
b)
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for the purposes of calculation, any HI result < 10 (= undetectable) shall be expressed as
5 and any negative SRH result shall be expressed as 4 mm 2 * ;
b)
c)
d)
a significant increase in antibody titre, i.e. at least a fourfold increase in' titre;
number of seroconversions;
e)
clinical tolerance: frequency, mean time of appearance and duration of all local and
general side-effects shall be calculated.
3.
3.1
Serological data
a)
The following serological assessments should be considered for each strain in adult
subjects, aged between 18 and 60:
-
increase
in
antihaemagglutinin
In most SRH test systems, a zone area of 25 mm 2 is approximately equivalent to an HI titre of 1:40. However,
this relationship can be affected by experimental conditions and should be re-examined in each laboratory so
as to calibrate the test system adequately.
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b)
The following serological assessments should be considered for each strain in the
group of subjects aged over 60:
-
increase
in
antihaemagglutinin
local reactions:
b)
indurations larger than 50 mm diameter and persisting for more than 3 days;
ecchymosis;
general symptoms:
-
malaise;
shivering.
References
Aymard, M., Million, J., Kessier, N.: Diagnostic srologique rapide de la grippe par la
mthode d'hmolyse radiale modifie et volution des anticorps. Path. Biol. 1980, 28, n 8,
535-539.
Palmer D.F., Dowie, W.R., Coleman M.T. et Schild G.C.: Advanced laboratory technicals for
immunological diagnostic. U.S. Dept. Hith. Ed. Welfare, P.H.S. Atlanta. Immunology ser.
Nr. 6, Procedural guide. Part 2: haemagglutination - inhibition test, 1975, 25-62.
Schild G.C., Pereira, M.S. Chakraverty, P.: Single radial haemolysis: a new method for the
assay of antibody to influenza haemagglutinin. Bull. WHO. 1975, 52, 43-50.
In most SRH test systems, a zone area of 25 mm 2 is approximately equivalent to an HI titre of 1:40. However,
this relationship can be affected by experimental conditions and should be re-examined in each laboratory so
as to calibrate the test system adequately.
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ALLERGEN PRODUCTS
Guideline Title
Legislative basis
Date of first adoption
Date of entry into
force
Status
Previous titles/other
references
Additional Notes
Allergen Products
Directive 89/342/EEC, Directive 75/318 EEC as amended
May 1992
This version revised May 1996
November 1996
Last revised May 1996
None
This revision consists of a clarification related to chapter
4, control of starting material, first paragraph, last
sentence of point 4.2.1.
This note for guidance only refers to industrially
produced allergen products placed on the market as
medicinal products for the purpose of in vivo diagnosis
or for treatment of allergic disease.
CONTENTS
1.
INTRODUCTION
2.
3.
4.
5.
6.
7.
STABILITY
8.
SAFETY TESTING
9.
EFFICACY TESTING
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ALLERGEN PRODUCTS
L
INTRODUCTION
b)
This note for guidance only refers to industrially produced allergen products placed on the
market as medicinal products for the purpose of in vivo diagnosis or for treatment of
allergic disease (point a) above).
2.
Qualitative Particulars
The name (scientific name, e.g. genus and species as well as any common name), and type
(e.g. pelt, dander, saliva) of the allergenic source material(s) should be stated. In the case of
modified or adsorbed allergen products, this and the agents used in the modification
procedures should be described. For all the excipients, the names, grades and quantities
should be given. This also includes the composition of any separate packaged dilution or
reconstitution fluid to be used with the product.
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Quantitative Particulars
Whenever possible, the potency of the active ingredient should be expressed in units of
biological activity, and the unit system used should be unambiguously indicated, in order to
avoid confusion with similar unit systems currently in use on the market.
3.
The manufacturing process within the meaning of this section is the preparation of the
finished product from the starting materials. The description should include details of any
process employed, in particular sterilisation, filling, freeze-drying, addition of any
preservative and of any stabiliser. Whenever materials of human or animal origin are
present in the finished product as excipients, particular attention should be given to safety
from transmission of infectious diseases. The use of materials and/or excipients known to
give rise to sensitisation should be avoided or justified.
4.
General Requirements
The allergenic source material should be described in as much detail as possible. Details
concerning collection, pre-treatment and storage should be supplied for each separate
allergen. The name and the address of the supplier shall be stated. Specifications and
control methods for the source material(s), applied by the supplier, should be included. The
specifications should ensure that the qualitative and quantitative composition of the material
is as uniform as possible from one delivery to another. They should encompass
requirements and control methods relating to identity and purity. Quality control of source
materials should be documented. The source materials should be stored under controlled
conditions.
Additional Requirements for Specific Source Materials
42.1
Pollens
Nature of the fields, seeds used, field characteristics and treatment, visual control, manner
of collection and random sampling should be described. The manner of collection of pollens
should be stated. Tests for content of foreign pollens, spores, extraneous plant material from
the same species and non-related contamination should be included. The content of
representative pesticides and lead should be measured on a limited number of pollen
batches, in order to demonstrate that their level is kept at a minimum.
The pollen content from other species should be limited to 1% of mixed pollens and 0.5% of a
single pollen as determined by a microscopic particle count. Detectable spores should not
exceed 1%. Contamination by particles of plant origin, other than pollen, should not exceed a
total of 10% in terms of microscopic count. Justification should be given if these standards
cannot be compiled with.
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4.2.2
Moulds
The strain or strains of moulds should be specified. The cultivation method should be
described. Details of the composition of the cultivation medium should be submitted.
Strains which produce mycotoxins such as anatoxins or ochratoxins should not be used
unless justified. In this case, the source material should be tested for mycotoxins; the source
materials should be tested for mutagenicity before processing unless the removal of
mycotoxins has been validated.
Synthetic and consequently allergen-free media shall preferably be used. Morphological
characteristics (mycelium and spores / spores only / mycelium only) as well as the
cultivation method in the isolated raw material should be specified. Conditions of culture
must be validated to provide evidence that mycotoxins are not produced.
42.3
Mites
The cultivation method and the composition of the cultivation medium should be described.
When substrates of human origin are used in the culture medium, the absence of risk of
transmission of infectious diseases should be demonstrated. To this end, the manner of
collection of these substrates should be described in detail. Any allergenicity of the medium
should be as low as possible in order not to produce any unspecific reactions of the finished
product. Therefore, the use of animal dander or any animal protein in the culture medium
should be avoided. It should be indicated whether for further processing, mites only or the
whole mite culture is used.
4.2.4 Animal
allergens
The species of the animal should be stated. An account should be given of the health
examination of the animals from which the raw materials are collected. Materials should be
collected from animals that do not exhibit overt infections at the time of collection. The
collector should certify that the animals used are overtly healthy and have not recently been
treated with antiparasitics or other medicinal products.
When killed animals are used, the epidermals should be collected within a few hours. The
animal must be stored in conditions ensuring that post-mortem decomposition processes do
not affect the epithelium; these storage conditions should be described. Collection of hair and
dander must take place using methods which provide a good epithelial harvest without
injuring the skin of the animal. Methods employing the grinding of whole skin/pelts must
not be used. The composition of the final source material (e.g. hair, dander, pelt, saliva,
urinary fluid) should be indicated.
4.2.5 Hymenoptera
venom
The method of collection of venom from the venom sacs of hymenoptera species should be
described and should be such as to ensure that the raw material is of a proper quality.
Description of the Production Process
The production process should be described, step by step with a diagram (flow-chart)
indicating the principles of the process, accompanied by an explanatory text. The different
stages of the production process, such as grinding, extraction, filtration, clarification,
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4.4.1 Characterisation
(IHR)
The IHR shall be characterised using available relevant methods and its specific allergenic
activity shall be established, and data should be provided on protein and, whenever possible,
carbohydrate composition. Some of the following methods should be applied: Crossedimmunoelectrophoresis (CIE), isolectricfocusing, electrophoresis in Polyacrylamide gel,
determination of the distribution of molecular weight by SDS-PAGE analysis, HPLC, gel
electrophoresis and quantitative determination of total protein. Information regarding the
allergenic specificity of the proteins in the IHR may be obtained from experiments
involving combinations of electrophoretic methods and immunoblotting techniques or
Crossed-Radio-immuno-Electrophoresis (CRIE). Sensitivity spectra (allergograms) derived
from such basic documentation, thus identifying the major, intermediate and minor
allergens. The presence of all relevant allergens in the IHR shall have been demonstrated
in comparative studies involving several batches of extract. As far as possible, the
individual allergens should be identified using internationally accepted nomenclature or
the correspondence with allergens described in the scientific literature should be given,
including literature references. The potency of this IHR should be judged by immuno-assays
(e.g. IgE-inhibition, ELISA-techniques, immunofluorescence techniques) or/and Skin Prick
Test and expressed in terms of Units of Biological Activity. When a product consists of one
or a few well characterised allergenic components, potency can be assayed by means of
alternative relevant techniques, such as single radial immunodiffusion, quantitative
Immunoelectrophoresis or other quantitative techniques. All these methods and the
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5.
Control tests carried out at intermediate stages of manufacture should be defined. When
certain control tests cannot be applied to the finished product, for instance in the case of
chemically modified, precipitated or adsorbed allergen preparations, quality specifications
should be defined for the product just prior to the modification, dilution, etc.
6.
7.
STABILITY
The note for guidance Stability tests of Active Ingredients and Finished Products should be
followed. However, in some circumstances, It is impossible or difficult to fully evaluate the
allergenic potency and other characteristics of the finished product. In these cases, such as
adsorbed-modified or adsorbed-unmodified allergens, it would be acceptable to carry out
stability tests just before applying the modifying treatment.
In addition, the stability of adsorption should be monitored over the proposed shelf life, since
free allergens can cause immediate (anaphylactic) reaction.
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For stability data, the concept of taxonomie family may be applied and data obtained on one
member of such a family may be extrapolated within that same family. This extrapolation
should be discussed and justified. In the case of mixtures of members of different taxonomie
families, extrapolation is not acceptable.
No less than 30% of the stated allergenic activity should be maintained at the end of shelf
life.
8.
SAFETY TESTING
Details of the safety testing undertaken should be provided. Safety testing may have to be
adapted to individual products and, if so, any omissions with regard to the requirements
laid down in Part 3 of the Annex to Directive 91/507/EEC should be justified.
For safety testing, the concept of taxonomie family may be applied and data obtained on one
member of the family may be extrapolated to another member of that family providing the
manufacturing procedures applied are the same. In the case of mixtures of members of
different taxonomie families, extrapolation is not acceptable.
9.
EFFICACY TESTING
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Guideline Title
CONTENTS
1.
INTRODUCTION
2.
3.
APPENDIX 1
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INTRODUCTION
Before, 1960, plasma was the only agent generally available for the treatment of the
hereditary coagulation disorders. Several plasma product concentrates are now available for
this purpose which have been purified and virally inactivated in various ways. With respect
to factor VIII deficiency the replacement therapy consists of factor VIII concentrates of
different purity. Random reports of a relatively high incidence of inhibitors after
administration of factor VIII products together with reports of viral infections (hepatitis C,
hepatitis A and 7) after administration of human plasma derived coagulation factors,
has made the regulatory authorities more aware of the potential risks of these products. As
one of several measures aimed at improving viral safety of blood products, it is necessary to
demand adequate clinical investigation before a marketing approval is granted. Clinical
trials are necessary addressing efficacy, safety with respect to transmission of viral
infections and immunogenicity for FVIII and FDC concentrates. In addition in applications
for FDC concentrates thrombogenicity should be addressed.
In view of outbreaks of hepatitis A among haemophiliacs treated with a solvent detergent
factor VIII in 1992 the Committee on Proprietary Medicinal Products (CPMP) approved the
position paper of the biotechnology working party (IH/5830/93 Final) on blood products and
non-enveloped viruses. It was recommended that the manufacturing process should include
viral inactivation/removal which is also effective against non-enveloped virus. As a virus
inactivation/removal step (or any change in the purification process) may alter the structure
of the coagulation factor and/or induce loss of activity, efficacy and immunogenicity data
are clearly needed. This guideline describes the clinical trials required for authorisation
with respect to two different human plasma derived factor VIII and Factor TX. products:
1)
2)
Authorised products in which a change in the manufacturing process has been made
(e.g. additional viral inactivation step).
The clinical trials should be performed according to the guidelines on Good Clinical
Practice.
Recombinant DNA products are excluded from this guideline.
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2.
2.1 Efficacy
In clinically evaluating a new human plasma derived coagulation factor for the treatment
of haemophilia A or patients, the initial trial is typically one that examines the
pharmacokinetics of the principal active factor. Half life and recovery data are the most
important (surrogate) endpoints for efficacy of a new factor VIII or Factor DC product. The
guideline below is according to the guidelines proposed by the International Society of
Thrombosis and Haemostasis (ISTH) (Thrombosis and Haemostasis 1991; 66 (3):384-386).
2.2 Safety
Safety aspects of a new factor VIII or Factor DC product refer to viral safety, immunogenicity
and any other adverse events. For factor DC products thrombogenicity should also be
considered as a safety issue.
22.1 Adverse
events
All adverse events after infusion of the new product should be reported.
22.2 Viral
Safety
Immunogenicity
2.2.3.1 Factor
VIIIproducts
The occurrence of antibodies against factor VIII is one of the major possible complications of
haemophilia treatment. The risk of inhibitor occurrence is higher in patients with severe
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haemophilia A than in patients with moderate and mild disease. Inhibitor risk may be
associated with commencing or changing treatment or where the antigenicity of the product
has been altered due to changes in the manufacturing process. Prior to authorisation
immunogenicity of a new factor VIII product should be investigated first in previously
treated patients (PTPs) and then, depending on the claimed indication, in previously
untreated patients (PUPs). The Summary of Product Characteristics (SPC) should include a
section stating the experience with respect to immunogenicity in PUPs or state that there is
no such experience.
2.2.32 Factor IX
products
(factor IX
products)
Treatment with Factor DC concentrates containing Factors II, VII and X has been associated
with a risk of thrombosis. Purified Factor DC products have been shown to be associated with
a lower thrombogenic risk. For all new factor DC concentrates testing for markers of
activation of coagulation should be carried out in the non-bleeding state.
2.3
2.3.1
2.3.1.1
Pre-Authorisation
Efficacy
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23.12
Safety
Clinical safety will be assessed in all patients receiving the new factor VIII product.
In hospitalised patients, such as patients included in the pharmacokinetic trial, for
blood pressure, heart rate, temperature, respiratory rate and adverse events.
In out-patients for adverse events.
2.3.1.2.1 FTP (Previously treated patient) study
A minimum of 30 immunocompetent (CD4 lymphocytes > 400/ml) previously treated patients
with severe haemophilia A with at least 10 exposure days to the new factor VIII product and a
follow up of at least 6 months for all patients must be included prospectively.
Immunogenicity
The factor VIII inhibitor titre will be determined every 3 months. An interim analysis will
be performed when 30 patients not having undergone surgery have been treated for 6 months
with a minimum of 10 exposure days each. The titre of the inhibitor should be reported i n
Bethesda Units (BU) as well as the clinical relevance the cumulative incidence and the
number of exposure days (also in relation to development of inhibitors).
Viral safety
According to EC Good Clinical Practice the PTP patients should be followed up for viral
safety markers. Full baseline data for markers of viral infection (aminotransferase, HIV
1+2 ab, HCV ab, HBV antigen and ab and HAV ab) should be provided according to the table
in Appendix 1. All patients negative for these markers should have regular testing
according to the schedule in the Table. In patients who are Parvovirus 19 antibody negative
at entry a sample should be tested using gene amplification methods at one week after the
first treatment. Serum samples should be stored at -70C whenever the patient is sampled, for
possible future testing. No claims can be made in the SPC on viral safety of the product with
respect to parvovirus 19 transmission.
2.3.1.2.2 PUP (previously untreated patient) study
PUP studies should be carried out, or at least initiated. The SPC should include a section
stating the experience with respect to inhibitor development in PUPs or state that there is no
such experience. A PUP study should be done only after results of the PTP trial are available
and according to the guideline.
An open label uncontrolled multicentre trial in previously untreated severe haemophilia A
patients should include at least 20 patients. These patients should be tested for viral safety
and inhibitor development.
Viral safety
Viral safety data (serum aminotransferases, 7, hepatitis C) should be monitored at 3
month intervals for at least 2 years and be reported at 6 month intervals. In patients who are
Parvovirus B19 antibody negative at entry a sample should be tested using gene
amplification methods at one week after the first treatment. Serum samples should be stored
at -70C whenever the patient is sampled, for possible future testing.
Immunogenicity
The patients should be tested every 3 months for at least 100 exposure days or 5 years
whichever comes first. The titre of the inhibitors should be reported (Bethesda Units = BU) as
well as the clinical relevance, the cumulative incidence and the number of exposure days.
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2.3.2
Post-Authorisation
2.3.2.1 PTP
After authorisation laboratory parameters (according to the table in the appendix) from at
least 50 PTPs treated for at least 2 years should be included in the Periodic Safety Update.
2.3.22 PUP
Immunogenicity and viral safety data (including parvovirus B19 seroconversion) on any
PUPs treated with the product should be included in the Periodic Safety Update as described
in the note for guidance Clinical Safety Data Management: Periodic Safety Update Reports
for Marketed Medicinal Products.
2.4
C l i n i c a l t r i a l s w i t h f a c t o r LX p r o d u c t s
2.4.1
2.4.1.1
Pre-Authorisation
Efficacy
Safety
In addition to the requirements for factor VIII products, tests for activation of coagulation
after administration of the product should be carried out. This should be determined in the
patients of the pharmacokinetic trial as well in a minimum of 5 patients undergoing at least
10 surgical procedures. In the surgical patients clinical evaluation of thrombosis should be
undertaken by safe, objective means. Due to the lower incidence of haemophilia as
compared to haemophilia A, the number of previously treated patients followed up for viral
safety and immunogenicity should be lower than for factor VIII products: 12 for viral safety
and 20 patients for immunogenicity. If a PUP study is done, it should include 15 patients.
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2.4.2 Post-Authorisation
2.4.2.1 PTP
After authorisation, laboratory parameters (according to the table in the appendix in the
appendix) from at least 30 PTPs treated for at least 2 years should be included in the
Periodic Safety Update.
2.42.2 PUP
Immunogenicity and viral safety data (including parvovirus 19 seroconversion) on any
PUPs treated with the produce should be included in the Periodic Safety Update.
3.
3.1
Efficacy
As a virus inactivation/removal step (or any change in the purification process) may alter
the structure of the coagulation factor and/or induce loss of activity, pharmacokinetic
studies, measuring half-life and recovery are clearly needed.
3.2 Safety
Viral safety
If the additional viral reduction process concerns reduction of non-enveloped viruses only,
and the original manufacturing process has already been validated for the removal of
enveloped viruses, then it may be assumed that the original factor VIII or Factor DC product
is safe with respect to removal/inactivation of hepatitis virus, H P / 1+2 and hepatitis C
virus and that the additional viral removal/inactivation step is designed to remove nonenveloped viruses such as hepatitis A and parvovirus 19. As most haemophilia patients are
vaccinated against hepatitis A, clinical follow-up of these patients is difficult. The high
incidence of parvovirus B19 seroconversion in all age groups makes it very difficult to
assess the infectivity of the product with respect to parvovirus 19. No clinical trials testing
parvovirus 19 seroconversion are mandatory before authorisation. After authorisation it is
recommended that parvovirus 19 seronegative patients should be tested by gene
amplification methods one week after the first treatment. Serum should be stored at -70C for
future testing.
Immunogenicity
The currently available factor VIII preparations differ with respect to purity and method of
viral removal/inactivation. Until recently, the evidence supporting the notion that a
particular production process is associated with a higher than normal risk of inhibitor
induction has been very limited. Nevertheless some products do cause a higher incidence
inhibitors than others. The clusters of inhibitors after infusion of factor VIII CPS-P in
previously treated patients in 1991, illustrate the necessity to perform immunogenicity trials.
Such inhibitors will be demonstrable in previously treated patients.
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3.3
3.3.1
3.3.1.1
Pre-Authorisation
Efficacy
Safety
Post-Authorisation
PTP
After authorisation laboratory parameters (according to the table in the appendix) from at
least 50 PTPs treated for at least 2 years should be included in the Periodic Safety Update.
3.322
PUP
Immunogenicity and viral safety data (including parvovirus B19 seroconversion) on any
PUPs treated with the product should be included in the Periodic Safety Update.
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Pre-Authorisation
3.4.1.1 Efficacy
A comparative pharmacokinetic trial, measuring half-life and recovery, should be
performed in at least 10 subjects with severe haemophilia (factor DC < 2%) patients should
be at least 12 years of age and should not have received an infusion of concentrate in at least
the past 4 days (if possible 7 days). As comparative product the currently authorised factor DC
product should be used. Samples for factor DC activity determination should be taken before
injection of 50-75 IU/kg of the new factor DC product and 30 minutes, 1, 3, 6, 9, 12, 24, 30, 36
and 50 hours after the infusion. A minimum period of 4 days (if possible 7 days) should be
maintained between infusions. At least 3 different lots should be employed in the trial.
Recovery should be determined from the peak factor DC activity in the first four samples time
periods post-infusion.
Patients who participated in the pharmacokinetic trial should continue treatment with the
product for 6 months and at least 5 patients should be tested again for half life and recovery
after 3-6 months.
After administration of the factor DC product in any patient treated with surgical procedures,
adverse events and response will be determined by the physician, including achievement of
haemostasis, loss of blood and requirement for transfusions and occurrence of
thromboembolic episodes.
3.4.12
Safety
3.4.2
Post-Authorisation
3.4.2.1 PTP
After authorisation laboratory parameters (according to the table in the appendix) from at
least 30 PTPs treated for at least 2 years should be included in the Periodic Safety Update.
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3 AB 16a
3.4.22 PUP
Immunogenicity and viral safety date (including parvovirus B19 seroconversion) on any
PUPs treated with the product should be included in the Periodic Safety Update report.
391
3AB16a
APPENDLX 1
BIOCHEMICAL AND SEROLOGIC SURVEBLLANCE EV
HAEMOPHBLIAC PATB3NTS COMMENCING TREATMENT WITH THE
FACTOR VIH AND FACTOR LX CONCENTRATE UNDER STUDY
Month after commencing new concentrate
12
18
24
36
48
ALT
HIV I + II
HCV
HBV
HAV
Parvo Virus B 19
392
3AB17a
CONTENTS
1.
INTRODUCTION
2.
3.
393
3AB17a
INTRODUCTION
ii)
The clinical trials described in these Guidelines should be performed according to the note
for guidance on Good Clinical Practice.
11
Efficacy
with
severe
secondary
iii)
Kawasaki disease
iv)
395
3AB17a
Other indications
Where the mechanism of action is essentially unknown, relevant clinical data are
required. The trials should be carried out with reference to the Notice to Applicants and
all relevant EC Guidelines for clinical studies of medicinal products.
1.2
Safety
1.2.1 Immediate
adverse
events
safety
safety
issues
2.
Biological and pharmacokinetic data are the key elements to evaluate activity and safety of
IVIg preparations with reference to physiological immunoglobulins.
2.1
2.1.1 Biological
(cross-reference
to relevant
data
Part IT)
396
3AB17a
Most of the data are provided in Part II of the dossier and follow the European
Pharmacopoeia monograph 918, Xth of January l8t, 95. However, specific data are needed to
support the pharmacodynamic and therapeutic activities as well as the safety profile of the
P7Ig preparation. These data should thus appear along with the cross-reference to Part II, i n
Part TV of the dossier.
For the values not defined in the European Pharmacopoeia 918, ranges and/or limits are to
be defined.
i)
Biological characteristics
-
General:
Molecular size distribution: quantification of monomers + dimers, fragments
and polymers + aggregates
ii)
Biological
IgA content
Anti-complementary activity
Anti- and anti-B isoglutinins
Anti-D antibodies
Prekallikrein activator,
activity
397
3AB17a
Pharmacokinetics
Pharmacokinetic data are essential to support the pharmacological activity and efficacy of a
particular PTIg preparation, and may differentiate one from another. Therefore, they must be
provided in each application dossier. Pharmacokinetic data should be derived from patients
with hypo- or agammaglobulinemia.
Half-life should be studied in 15 patients with primary immunodeficiency due to primary
immunodeficiency syndromes and possibly in myeloma or chronic lymphatic leukaemia
with severe secondary hypogammaglobulinemia and recurrent infections. Patients, of whom
at least 10 should have primary immunodeficiency, may already be stabilised. The
pharmacokinetics should be assessed over a period of 6 months (6.5 times the expected halflife). No cross-over study is necessary.
The IgG concentration should be determined before injection of the recommended dose of the
DTIg. Pharmacokinetic profile should be assessed by repeated sampling during the first
infusion, and followed by trough levels (measured before the next injection). In patients
nave to PTIg, the Time to reach Steady State (Tss) could be determined.
2.2
Efficacy
22.1 Replacement
therapy
in primary
immunodeficiency
syndromes
Clinical data should include an open study comparing historical data with reference IVIg in
at least 15 patients, whatever the primary immunodeficiency syndrome. Evaluation criteria
would be: number of days out of school/work, number of days of hospitalisation. Trough
levels of IgG and T s s when possible, should be documented over 6 months. Trough levels
should be no less than 4 to 6g/L.
The results regarding efficacy would apply to all types of primary
syndrome due to deficiency of functional IgG.
immunodeficiency
2.2.2 Replacement
therapy in myeloma or chronic lymphatic leukaemia
with
severe secondary hypogammaglobulinemia
and recurrent
infections
Indication in myeloma or chronic lymphatic leukaemia with severe secondary
hypogammaglobulinemia and recurrent infections would be granted, as long as efficacy has
been proven in primary immunodeficiency syndromes.
2.2.3 Replacement
therapy
in congenital
infections
ITP
TVLg is used for the treatment of ITP in children or adults, at high risk of bleeding, or prior
to surgery to correct the platelet count.
398
3AB17a
There are no data to support the equivalence of different P7Ig preparations, especially with
regard to immunomodulatory activities. Thus a clinical efficacy study is required to
establish efficacy of the preparation in this indication.
-
Clinical efficacy data should include an open study comparing historical data with
reference rVIg, performed over a few days in acute phase on at least 15 adult chronic
ITP patients, with a platelets count below 20 109/L.
Standard doses should be studied (lg/kg b.w./day for 2 days, or 0.4g/kg b.wVday for 5
days) Other dosage regimens should be documented.
22.5 Kawasaki
disease
It is no longer ethical to conduct placebo controlled trial in this indication and it can be
granted by reference to the literature, providing that efficacy in primary immunodeficiency
syndromes and in ITP has been established for the relevant PTIg.
2.2.6 A l l o g e n i c B o n e M a r r o w T r a n s p l a n t a t i o n
rVIg effect in BMT requires both substitution and immunomodulatory activities. In
reference to this indication, specific data are not required as long as efficacy has been
proven in primary immunodeficiency syndromes and in ITP for the relevant rVIg.
22.7 Other
indications
Other possible indications can not be granted without relevant clinical data. Biological and
pharmacokinetic data alone are not sufficient to support clinical efficacy.
Controlled clinical trials comparing the DTIg preparation with placebo or with an established
therapy are thus required to substantiate marketing authorisation in other indications. These
trials should be carried out with reference to the Notice to Applicants and all relevant EC
Guidelines for clinical studies of medicinal products.
2.3. Safety
2.3.1 Immediate
safety
events
All adverse events in clinical studies should be recorded in all patients treated, whatever the
indication, and reported in reference with the note for guidance on Structure and Content of
Clinical Study Reports. Data from at least 30 patients or 180 infusions are required.
Safety evaluation should include monitoring of short term tolerance (blood pressure, heart
rate, temperature, respiratory rate, and monitoring of other adverse events) at 30 minutes
intervals for 4 hours and at 12 and 24 hours following the injection of the new product i
patients, such as patients included in the pharmacokinetic studies or patients included i n
clinical studies for efficacy. Renal function should be monitored.
399
3AB17a
2.32 Viral
safety
For new products, some viral safety data should be provided as part of the marketing
application. These data should be derived from all patients having entered efficacy clinical
trials and for a minimum of 3 batches of the P7Ig preparation. Each patient should preferably
be treated with one batch only. The company should continue to follow-up patients treated with
the product in the long term as a post-marketing surveillance for viral markers. Updated
data should be provided on a yearly basis after the marketing authorisation has been
granted.
Blood sampling for the viral safety study should include: one pre-infusion sample for every
parameter, and appropriately thereafter for up to 6 months following exposure to any new
batch of the preparation. Sera from these points should be stored at -70C as well as being
tested as set out below for each patient group.
The monitoring of viral safety depends on the category of patients receiving the new product
(immunocompetent or non-immunocompetent patients). Non-immunocompetent patients who
are primary recipients of rVIg may not have formed detectable antibodies against human
viruses which may be transmitted through rVIg. All safety data obtained in various
populations receiving the new product should be presented together but identifying
immunocompetent and non-immunocompetent patients. This will provide a basis for an
overall safety assessment for all clinical studies.
Passive transmission of HBs, HBc, HAV and parvovirus B19 antibodies by the IVIg
preparation may be difficult to differentiate from transmission of infection, and this should
be discussed.
i)
Immunocompetent patients
-
Seroconversion for:
HAV Ab (IgM); HIV 1-2 Ab: at weeks 12 and 24
HBsAg; HBc Ab and HCV Ab: at weeks 16 and 24
Parvovirus 19: a sample before and at week 1 after the first infusion must
be taken.
If the pre-treatment sample has no anti-B19 Ab (IgM or IgG), the week 1-sample
should be tested with gene amplification method.
In patients parvovirus 19 seropositive in the pre-treatment sample, no further
investigation is required.
ii)
Non-immunocompetent patients
-
400
3AB17a
HCV: nucleic acid amplification method before the first infusion and at
weeks 8 and 16 after the first infusion.
Parvovirus B19: before the first infusion and at week 1 after the first
infusion using either nucleic acid amplification method or Ag detection.
3.
Appropriate viral removal/inactivation steps in the manufacturing process for PTIg are
mandatory, to increase the viral safety of these products.
Little data exist on the effect of some viral inactivation steps or other purification steps on
IgG integrity and function, or on PTIg immunomodulatory activity. Thus, it is important to
include full data on antibody integrity and function, in Part II and crossrefer to this in Part
TV of the dossier as for new products. Data on pharmacokinetics and on immediate safety
should also be provided with the application.
3.1
Pharmacokinetics
Pharmacokinetic data must be provided in each application dossier, from patients with
primary immunodeficiency syndromes
Halflife should be studied in 15 patients with primary immunodeficiency due to primary
immunodeficiency syndromes and possibly in myeloma or chronic lymphatic leukaemia
with severe secondary hypogammaglobulinemia and recurrent infections. Patients, of whom
at least 10 should have primary immunodeficiency, may already be stabilised. The
pharmacokinetics should be assessed over a period of 6 months (6.5 expected halflife). No
crossover study is necessary.
The IgG concentration should be determined before injection of the recommended dose of the
DTIg. Pharmacokinetic profile should be assessed by repeated sampling during the first
infusion, and followed by trough levels (measured before the next injection). In patients
nave to P7Ig, the Time to reach Steady State (T ss ) could be determined.
3.2 I m m e d i a t e s a f e t y
Immediate safety for modified products should be the same as required for a new product,
that is: all adverse events in clinical studies should be recorded in all patients treated,
whatever the indication, and reported in reference with the note for guidance on Structure
and Conte nt of Clinical Study Re ports. Data from at least 30 patients or 180 infusions are
required.
Safety evaluation should include monitoring of short term tolerance (blood pressure, heart
rate, temperature, respiratory rate, and other adverse events) at 30 minutes intervals for 4
hours and at 12 and 24 hours following the injection of the modified product in patients, such
as patients included in the pharmacokinetic studies or patients included in clinical studies
for efficacy. Renal function should be monitored.
401
3AB17a
3.3
Efficacy
safety
data
Where biological data, pharmacokinetics and immediate safety profile show identity to the
parent product, further efficacy data will be required with the application in order to verify
efficacy of the modified preparation.
Requirements on efficacy data will be as follow:
3.3.1.1 Replacement therapy in primary
immunodeficiency
syndromes
immunodeficiency
due to
Primary
with
severe
infections
regression of haemorrhages
3.3.1.5 Kawasaki
disease
This indication can be granted by reference to the literature, providing that biological data,
pharmacokinetics and immediate safety data have been provided and show identity to the
parent product, and that efficacy has been established in ITP for the modified product.
3.3.1.6 Bone Marrow
Transplantation
No further clinical trial would be required, providing biological data, pharmacokinetics and
immediate safety data have been provided and show identity to the parent product, and that
efficacy has been established in ITP for the modified product.
3.3.1.7 Other
indications
For modified preparations only the four established indications, foreseen in the core SPC,
would be granted, unless relevant clinical data was submitted, either with the modified or
with the parent preparation.
Providing that the modified product satisfies the above requirements, no further clinical
trial would be required for the other indications.
402
3AB17a
or immediate
safety
If the biological data, pharmacokinetics or immediate safety data are different from the
parent preparation, the product is then considered as a new product and, as such, should
comply with the requirements defined in section 2.2.
Any new indication would have to be supported by full efficacy and safety data, as for a new
product.
403
INDEX
batch, 5; 7; 14; 15; 25; 26; 27; 29; 34; 35; 37; 38; 42;
50; 53; 59; 60; 61; 62; 69; 71; 81; 86; 87; 88; 89; 90
92; 93; 94; 98; 99; 100; 102; 130; 132; 133; 134;
135; 136; 138; 139; 140; 141; 159; 162; 163; 166,
170; 171; 172; 173; 179; 183; 184; 188; 189; 193
194; 210; 213; 214; 215; 216; 229; 231; 232; 234
235; 247; 248; 250; 251; 252; 262; 266; 267; 269
283; 284; 285; 293; 342; 353; 355; 358; 359; 360
369; 376; 378; 379; 396; 400
batch analysis, 215; 252
batch to batch consiste ncy, 378
bioburden, 16; 17; 26; 27; 28
bracketing, 135; 137; 267
BSE, 291; 317; 318; 319; 322
bulk harvest, 216; 248; 250; 251; 257; 262
bulk product, 25; 30; 87; 88; 93; 94; 214; 231; 250;
284; 378
consistency, 50; 53; 74; 177; 188; 189; 208; 211; 214;
216; 219; 225; 226; 229; 231; 244; 247; 248; 251;
252; 261; 280; 283; 284; 291; 292; 294; 342; 378;
379; 396
control of starting mate rials, 34; 179
cross-contamination, 188; 209; 211; 229; 242; 243;
282; 318; 340
cytokine, 223; 225; 226; 227; 230; 231; 232; 233; 234;
235; 246; 265; 281; 293
D
delayed re le ase , 170
Development che mistry, 52
Development Pharmaceutics, 3; 14; 15; 16; 69; 87; 90;
178
dissolution te st, 7; 111; 121; 170; 171; 172; 173
Dosage form, 80
Dose Mapping, 29
Dosimeter, 29
Drug Master File, 41; 44; 47; 49; 50; 51; 53; 54
DMF, 41; 44; 47; 49; 50; 51; 53; 54
E
electrophoresis, 213; 230; 250; 269; 378
end-of-shelf life , 16; 17; 18
endotoxins, 208; 226
enhancers, 220
evidence of structure, 35; 52
excipient, 5; 6; 13; 14; 67; 69; 70; 71; 72; 73; 85; 90; 97;
98; 100; 101; 102; 110; 137; 171; 173; 174; 201;
234; 269; 273; 297; 318; 327; 328; 335; 345; 375;
376
Excipients, 5; 67; 69; 70; 73; 90
Expert Report, 35; 37; 53
expiry date, 183; 193; 245; 338
expression construct, 209; 210; 217; 219; 220; 221;
222; 277; 279
factorisation, 14; 90
Finished product, 173; 215; 216; 253
flavouring age nts, 69
405
Index
H
harvest, 199; 210; 211; 214; 216; 222; 227; 229; 234;
246; 247; 248; 250; 251; 257:261; 262; 273; 283;
293; 365; 377
herbal remedies, 197
murine, 187; 188; 228; 237; 239; 240; 241; 242; 245;
248; 255; 298; 299; 300; 346
N
new active substance, 6; 31; 33; 35; 41; 57; 59; 71; 72;
88; 97; 99; 127; 129; 143; 155; 159
nomenclature, 33; 378
Notice to Applicants, 50; 51; 69; 77; 92; 169; 290; 396;
399
nucleic acid analysis, 219; 221
nucleic acid techniques, 219
O
Immediate packaging, 75; 78; 137; 166
impurities, 31; 34; 36; 37; 42; 43; 49; 50; 51; 52; 55;
57; 59; 6; 61; 62; 63; 64; 65; 70; 73; 85; 86; 97; 98;
99; 100; 101; 109; 110; 112; 113; 121; 123; 124;
125; 179; 189; 190; 208; 212; 214; 226; 230; 231;
249; 251; 252; 280; 284; 293; 327; 340
impurity profile, 43; 55; 60; 62; 63; 64; 65; 98; 99; 101;
102;110
in vitro, 7; 66; 105; 169; 170; 171; 172; 173; 174; 190;
220; 221; 233; 239; 246; 253; 258; 268; 279; 280;
284; 291; 293; 297; 325; 397
in vivo, 7; 170; 171; 172; 173; 174; 180; 182; 187; 190;
192; 213; 237; 239; 240; 247; 248; 253; 268; 277;
285; 297; 299; 325; 373; 375; 397
inducer, 227
in-process controls, 14; 15; 16; 17; 18; 88
integration site, 221; 280; 292
interactions, 162; 164; 182; 192; 201; 271; 340
Intermediate product, 172
isoelectric focusing, 213; 250
isomer, 34; 35; 36; 52
lactation, 292
letter of access, 53; 54
ligand, 180; 212; 338; 340; 343
linearity, 109; 111; 112; 125; 173; 329
M
manufacturing formula, 13; 14
markers, 200; 210; 221; 222; 228; 243; 244; 245; 298;
336; 338; 342; 353; 385; 386; 396; 400
master cell bank, 188; 216; 228; 261
matrixing, 135
Mean Kinetic Temperature, 138
modified release, 7; 9; 88; 155; 170
mould, 29; 282; 283; 308; 377
406
packaging material, 18; 25; 29; 30; 75; 77; 78; 79; 80
81; 135; 184; 194
parametric release, 16; 179
periodic tests, 88; 94
pharmaceutical development studies, 6
physical characteristics, 33; 37; 162; 163; 213; 232
physico-chemical properties, 35; 72; 212; 230; 246;
250; 300; 304; 344
pilot plant scale, 130
pollen, 376
polymorphism, 292
post translational modifications, 294
post-translational modifications, 213; 219; 230
potency, 37; 93; 123; 124; 134; 189; 190; 208; 214;
215; 216; 226; 231; 232; 233; 241; 251; 252; 262;
265; 268; 271; 272; 284; 285; 307; 327; 357; 376;
378; 379
potential toxicology, 52
precision, 36; 55; 90; 109; 111; 112; 113; 114; 115;
123; 124; 125; 138; 173; 307; 328; 329
preservative
preservatives, 6; 7; 80; 85; 87; 91; 133; 134; 202;
233; 269; 340; 343; 376
process validation, 9; 16; 17; 18; 49; 69; 77
production, 14; 15; 16; 18; 25; 29; 30; 37; 86; 87; 88,
89; 90; 92; 93; 130; 132; 133; 135; 139; 172; 173
177; 178; 181; 187; 188; 189; 202; 207; 208; 209
210; 211; 212; 213; 214; 216; 217; 219; 220; 221
222; 225; 226; 227; 228; 229; 230; 231; 234; 235.
239; 240; 241; 242; 243; 244; 245; 246; 247; 248,
249; 250; 251; 252; 253; 254; 255; 256; 257; 261
262; 266; 273; 278; 279; 280; 281; 282; 283; 284,
285; 289; 290; 291; 292; 293; 294; 297; 298; 299
300; 301; 302; 303; 304; 305; 309; 318; 321; 336,
339; 340; 341; 342; 343; 357; 359; 377; 378; 384,
388
Index
trace elements, 36
transgenic animal, 287; 289; 290; 291; 293
transgenic animals, 289; 291
R
reagent, 34; 36; 124; 189; 190; 207; 220; 226; 241;
279; 326; 327; 328; 329; 338; 379
reference material, 79; 109; 110; 112; 190; 215; 232;
235; 252; 268; 326; 359
reference standards, 215
reprocessing, 211; 249
re-test, 129; 130; 131; 132; 138; 139; 141; 215; 252
routine tests, 52; 53; 69; 70; 78; 86; 87; 325
vaccines, 13; 208; 266; 279; 297; 355; 357; 358; 359;
360; 361; 365; 369; 375
validated limit of quantitation, 61; 65
validation, 9; 16; 17; 18; 27; 42; 49; 51; 52; 61; 69; 77;
85; 86; 87; 88; 91; 109; 112; 121; 122; 130; 133;
162; 173; 178; 179; 188; 189; 199; 211; 212; 220;
246; 249; 252; 254; 270; 290; 293; 295; 296; 297;
298; 299; 300; 301; 302; 303; 304; 305; 309; 311;
313; 318; 325; 326; 327; 333; 336; 341; 343; 344;
345; 346; 347; 384
variations, 15; 63; 113; 116; 125; 129; 171; 172; 303;
329; 379; 393; 395
vector, 207; 208; 209; 210; 211; 214; 216; 219; 220;
222; 226; 227; 228; 229; 231; 244; 251; 261; 275;
277; 278; 279; 280; 281; 282; 283; 284; 285; 291;
297
vegetable substance, 41; 42; 197; 198; 199; 200; 202
virus removal, 290; 299; 302; 304; 311; 341; 344
w
working cell bank, 188; 210; 222; 228; 245; 261; 282;
283
407
European Commission
The rules governing medicinal products in the European Union
Guidelines - Medicinal products for human use
Quality and biotechnology
Volume 3 A
Luxembourg: Office for Official Publications of the European Communities
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Fax (27-11)883 65 69
SOUTH KOREA
Kyowa Book Company
1 F1. Phyung Hwa Bldg
411-2 Hap Jeong Dong, Mapo Ku
121-220 Seoul
Tel. (82-2) 322 67 80/1
Fax (82-2) 322 67 82
E-mail: kyowa2@ktnet.co.kr.
THALANDE
EBIC Thailand
Vanissa Building 8th Floor
29 Soi Chidlom
Ploenchit
10330 Bangkok
Tel. (66-2)655 06 27
Fax (66-2) 655 06 28
E-mail: ebtcbkk@ksc15.th.com
UNITED STATES OF AMERICA
EGYPT
The Middle East Observer
41. Sherif Street
Cairo
Tel. (20-2) 393 97 32
Fax (20-2) 393 97 32
HRVATSKA
Mediatrade Ltd
Pavia u.U.-j 1
HR-10000 Zagreb
Tel (385-1)43 03 92
Fax (385-1) 43 03 92
Beman Associates
4611 -F Assembly Drive
MD20706 Lanham
Tel. (800) 274 44 47 (toll tree telephone)
Fax (800) 865 34 50 (toll free fax)
E-mail: query beman com
URL: http://www.bernan.com
ANDERE LANDER/OTHER COUNTRIES/
AUTRES PAYS
Bitte wenden Sie sich an ein Bro Ihrer
Wahl / Please contact the sales office of
your choice / Veuillez vous adresser au
bureau de vente de votre choix
Price (excluding VAT) in Luxembourg: ECU 122 (Volumes 3A, 3B and 3C)
op
ISBN
iS-aBfi-EM37-3
9 789282"824375