Q6 Bstep 4

INTERNATIONAL CONFERENCE ON HARMONISATION OF TECHNICAL
REQUIREMENTS FOR REGISTRATION OF PHARMACEUTICALS FOR HUMAN USE
ICH HARMONISED TRIPARTITE GUIDELINE
SPECIFICATIONS: TEST PROCEDURES AND ACCEPTANCE CRITERIA FOR

BIOTECHNOLOGICAL/BIOLOGICAL PRODUCTS
Q6B
Current Step 4 version

Dated 10 March 1999
This Guideline has been developed by the appropriate ICH Expert Working
Group and has been subject to consultation by the regulatory parties, in
accordance with the ICH Process. At Step 4 of the Process the final draft is
recommended for adoption to the regulatory bodies of the European Union,
Japan and USA.
Q6B
Document History
New
First Codification
History Date
Codification November
2005
Q6B Approval by the Steering Committee under Step 27 Q6B

2 and release for public consultation. February
1998
Current Step 4 version
Q6B Approval by the Steering Committee under Step 4 10 Q6B

and recommendation for adoption to the three March
ICH regulatory bodies. 1999
ICH Harmonised Tripartite Guideline
Having reached Step 4 of the ICH Process at the ICH Steering Committee
meeting
on 10 March 1999, this guideline is recommended for
adoption to the three regulatory parties to ICH
TABLE OF CONTENTS
1. INTRODUCTION............................................................... .............1
1.1 Objective..............................................................................................1
1.2 Background..........................................................................................1
1.3 Scope...................................................................................................1
2. PRINCIPLES FOR CONSIDERATION IN SETTING SPECIFICATIONS......2
2.1 Characterization...................................................................................2
2.1.1Physicochemical properties.......................................................2
2.1.2Biological activity......................................................................3
2.1.3 Immunochemical properties...................................................4
2.1.4 Purity, impurities and contaminants........................................4

2.1.5 Quantity...................................................................................5
2.2 Analytical Considerations.....................................................................6

2.2.1 Reference standards and reference materials..........................6
2.2.2Validation of analytical procedures...........................................6
2.3 Process Controls...................................................................................6

2.3.1 Process-related considerations.................................................6
2.3.2 In-process acceptance criteria and action limits......................7
2.3.3 Raw materials and excipient specifications..............................8
2.4 Pharmacopoeial Specifications............................................................8

2.5 Release Limits vs. Shelf-life Limits.......................................................8
2.6 Statistical Concepts..............................................................................8
3. JUSTIFICATION OF THE SPECIFICATION..........................................8

4. SPECIFICATIONS.................................. .......................................9
4.1 Drug Substance Specification.............................................................11
4.1.1 Appearance and description..................................................11
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Test Procedures and Acceptance Criteria for Biotechnological/Biological Products
4.1.2 Identity............................................................................. ......11
4.1.3 Purity and impurities..............................................................11

4.1.4 Potency..................................................................................11
4.1.5 Quantity.................................................................................12
4.2 Drug Product Specification.................................................................12

4.2.1 Appearance and description..................................................12
4.2.2 Identity............................................................................. ......12
4.2.3 Purity and impurities..............................................................12

4.2.4 Potency..................................................................................13
4.2.5 Quantity.................................................................................13
4.2.6 General tests..........................................................................13
4.2.7 Additional testing for unique dosage forms...........................13
5. GLOSSARY............................................. ...................................13
6. APPENDICES....................................................... ......................16
6.1 Appendix for Physicochemical Characterization.................................16
6.1.1 Structural characterization and confirmation.........................16
6.1.2 Physicochemical properties....................................................17
6.2 Appendix for Impurities......................................................................18

6.2.1 Process-related impurities and contaminants........................18
6.2.2 Product-related impurities including degradation products. . .19
ii
1. INTRODUCTION
1.1 Objective
This guidance document provides general principles on the setting and
justification, to the extent possible, of a uniform set of international
specifications for biotechnological and biological products to support new
marketing applications.
1.2 Background
A specification is defined as a list of tests, references to analytical procedures,
and appropriate acceptance criteria which are numerical limits, ranges, or
other criteria for the tests described. It establishes the set of criteria to which
a drug substance, drug product or materials at other stages of its manufacture
should conform to be considered acceptable for its intended use.
“Conformance to specification” means that the drug substance and drug
product, when tested according to the listed analytical procedures, will meet
the acceptance criteria. Specifications are critical quality standards that are
proposed and justified by the manufacturer and approved by regulatory
authorities as conditions of approval.
Specifications are one part of a total control strategy designed to ensure

product quality and consistency. Other parts of this strategy include thorough
product characterization during development, upon which many of the
specifications are based, adherence to Good Manufacturing Practices, a
validated manufacturing process, raw materials testing, in-process testing,
stability testing, etc.
Specifications are chosen to confirm the quality of the drug substance and
drug product rather than to establish full characterization and should focus on
those molecular and biological characteristics found to be useful in ensuring
the safety and efficacy of the product.
1.3 Scope
The principles adopted and explained in this document apply to proteins and
polypeptides, their derivatives, and products of which they are components
(e.g., conjugates). These proteins and polypeptides are produced from
recombinant or non-recombinant cell-culture expression systems and can be
highly purified and characterized using an appropriate set of analytical
procedures.
The principles outlined in this document may also apply to other product types
such as proteins and polypeptides isolated from tissues and body fluids. To
determine applicability, manufacturers should consult with the appropriate
regulatory authorities.
This document does not cover antibiotics, synthetic peptides and polypeptides,
heparins, vitamins, cell metabolites, DNA products, allergenic extracts,
conventional vaccines, cells, whole blood, and cellular blood components. A
separate ICH Guideline, ”Specifications: Test Procedures and Acceptance
Criteria for New Drugs Substances and New Drug Products: Chemical
1
Substances” addresses specifications, and other criteria for chemical

substances.
This document does not recommend specific test procedures or specific
acceptance criteria nor does it apply to the regulation of preclinical and/or
clinical research material.
2. PRINCIPLES FOR CONSIDERATION IN SETTING SPECIFICATIONS

2.1 Characterization
Characterization of a biotechnological or biological product (which includes the
determination of physicochemical properties, biological activity,
immunochemical properties, purity and impurities) by appropriate techniques
is necessary to allow relevant specifications to be established. Acceptance
criteria should be established and justified based on data obtained from lots
used in preclinical and/or clinical studies, data from lots used for
demonstration of manufacturing consistency and data from stability studies,
and relevant development data.
Extensive characterization is performed in the development phase and, where

necessary, following significant process changes. At the time of submission,
the product should have been compared with an appropriate reference
standard, if available. When feasible and relevant, it should be compared with
its natural counterpart. Also, at the time of submission, the manufacturer
should have established appropriately characterized in-house reference
materials which will serve for biological and physicochemical testing of
production lots. New analytical technology and modifications to existing
technology are continually being developed and should be utilized when
appropriate.
2.1.1 Physicochemical properties

A physicochemical characterization program will generally include a
determination of the composition, physical properties, and primary structure of
the desired product. In some cases, information regarding higher-order
structure of the desired product (the fidelity of which is generally inferred by
its biological activity) may be obtained by appropriate physicochemical
methodologies.
An inherent degree of structural heterogeneity occurs in proteins due to the
biosynthetic processes used by living organisms to produce them; therefore,
the desired product can be a mixture of anticipated post-translationally
modified forms (e.g., glycoforms). These forms may be active and their
presence may have no deleterious effect on the safety and efficacy of the
product (section 2.1.4). The manufacturer should define the pattern of
heterogeneity of the desired product and demonstrate consistency with that of
the lots used in preclinical and clinical studies. If a consistent pattern of
product heterogeneity is demonstrated, an evaluation of the activity, efficacy
and safety (including immunogenicity) of individual forms may not be
necessary.
Heterogeneity can also be produced during manufacture and/or storage of the

drug substance or drug product. Since the heterogeneity of these products
defines their quality, the degree and profile of this heterogeneity should be
characterized, to assure lot-to-lot consistency. When these variants of the
desired product have properties comparable to those of the desired product
2
with respect to activity, efficacy and safety, they are considered product-
related substances. When process changes and degradation products result in
heterogeneity patterns which differ from those observed in the material used
during preclinical and clinical development, the significance of these
alterations should be evaluated.
Analytical methods to elucidate physicochemical properties are listed in

Appendix 6.1. New analytical technology and modifications to existing
technology are continually being developed and should be utilized when
appropriate.
For the purpose of lot release (section 4), an appropriate subset of these
methods should be selected and justified.
2.1.2 Biological activity

Assessment of the biological properties constitutes an equally essential step in
establishing a complete characterization profile. An important property is the
biological activity that describes the specific ability or capacity of a product to
achieve a defined biological effect.
A valid biological assay to measure the biological activity should be provided
by the manufacturer. Examples of procedures used to measure biological
activity include:
• Animal-based biological assays, which measure an organism's biological

response to the product;
• Cell culture-based biological assays, which measure biochemical or

physiological response at the cellular level;
• Biochemical assays, which measure biological activities such as enzymatic

reaction rates or biological responses induced by immunological
interactions.
Other procedures such as ligand and receptor binding assays, may be
acceptable.
Potency (expressed in units) is the quantitative measure of biological activity
based on the attribute of the product which is linked to the relevant biological
properties, whereas, quantity (expressed in mass) is a physicochemical
measure of protein content. Mimicking the biological activity in the clinical
situation is not always necessary. A correlation between the expected clinical
response and the activity in the biological assay should be established in
pharmacodynamic or clinical studies.
The results of biological assays should be expressed in units of activity

calibrated against an international or national reference standard, when
available and appropriate for the assay utilized. Where no such reference
standard exists, a characterized in-house reference material should be
established and assay results of production lots reported as in-house units.
Often, for complex molecules, the physicochemical information may be
extensive but unable to confirm the higher-order structure which, however,
can be inferred from the biological activity. In such cases, a biological assay,
with wider confidence limits, may be acceptable when combined with a
specific quantitative measure. Importantly, a biological assay to measure the
3
biological activity of the product may be replaced by physicochemical tests

only in those instances where:
• sufficient physicochemical information about the drug, including higher-

order structure, can be thoroughly established by such physicochemical
methods, and relevant correlation to biologic activity demonstrated; and
• there exists a well-established manufacturing history.
Where physicochemical tests alone are used to quantitate the biological

activity (based on appropriate correlation), results should be expressed in
mass.
For the purpose of lot release (section 4), the choice of relevant quantitative
assay (biological and/or physicochemical) should be justified by the
manufacturer.
2.1.3 Immunochemical properties

When an antibody is the desired product, its immunological properties should
be fully characterized. Binding assays of the antibody to purified antigens and
defined regions of antigens should be performed, as feasible, to determine
affinity, avidity and immunoreactivity (including cross-reactivity). In addition,
the target molecule bearing the relevant epitope should be biochemically
defined and the epitope itself defined, when feasible.
For some drug substances or drug products, the protein molecule may need to
be examined using immunochemical procedures (e.g., ELISA, Western-blot)
utilizing antibodies which recognize different epitopes of the protein molecule.
Immunochemical properties of a protein may serve to establish its identity,
homogeneity or purity, or serve to quantify it.
If immunochemical properties constitute lot release criteria, all relevant
information pertaining to the antibody should be made available.
2.1.4 Purity, impurities and contaminants
• Purity
The determination of absolute, as well as relative purity, presents considerable
analytical challenges, and the results are highly method-dependent.
Historically, the relative purity of a biological product has been expressed in
terms of specific activity (units of biological activity per mg of product) which
is also highly method-dependent. Consequently, the purity of the drug
substance and drug product is assessed by a combination of analytical
procedures.
Due to the unique biosynthetic production process and molecular

characteristics of biotechnological and biological products, the drug substance
can include several molecular entities or variants. When these molecular
entities are derived from anticipated post-translational modification, they are
part of the desired product. When variants of the desired product are formed
during the manufacturing process and/or storage and have properties
comparable to the desired product, they are considered product-related
substances and not impurities (section 2.1.1).
Individual and/or collective acceptance criteria for product-related substances

should be set, as appropriate.
4
For the purpose of lot release, (section 4), an appropriate subset of methods
should be selected and justified for determination of purity.
• Impurities
In addition to evaluating the purity of the drug substance and drug product,
which may be composed of the desired product and multiple product-related
substances, the manufacturer should also assess impurities which may be
present. Impurities may be either process or product-related. They can be of
known structure, partially characterized, or unidentified. When adequate
quantities of impurities can be generated, these materials should be
characterized to the extent possible and, where possible, their biological
activities should be evaluated.
Process-related impurities encompass those that are derived from the

manufacturing process, i.e., cell substrates (e.g., host cell proteins, host cell
DNA), cell culture (e.g., inducers, antibiotics, or media components), or
downstream processing (see “Appendix”, section 6.2.1). Product-related
impurities (e.g., precursors, certain degradation products) are molecular
variants arising during manufacture and/or storage, which do not have
properties comparable to those of the desired product with respect to activity,
efficacy, and safety.
Further, the acceptance criteria for impurities should be based on data

obtained from lots used in preclinical and clinical studies and manufacturing
consistency lots.
Individual and/or collective acceptance criteria for impurities (product-related
and process-related) should be set, as appropriate. Under certain
circumstances, acceptance criteria for selected impurities may not be
necessary (section 2.3).
Examples of analytical procedures which may be employed to test for the
presence of impurities are listed in Appendix 6.2. New analytical technology
and modifications to existing technology are continually being developed and
should be utilized when appropriate.
For the purpose of lot release (section 4), an appropriate subset of these
methods should be selected and justified.
• Contaminants
Contaminants in a product include all adventitiously introduced materials not
intended to be part of the manufacturing process, such as chemical and
biochemical materials (e.g., microbial proteases), and/or microbial species.
Contaminants should be strictly avoided and/or suitably controlled with
appropriate in-process acceptance criteria or action limits for drug substance
or drug product specifications (section 2.3). For the special case of
adventitious viral or mycoplasma contamination, the concept of action limits is
not applicable, and the strategies proposed in ICH Harmonised Tripartite
Guidelines “Quality of Biotechnological/Biological Products: Viral Safety
Evaluation of Biotechnology Derived Products Derived from Cell Lines of
Human or Animal Origin” and “Quality of Biotechnological/Biological Products:
Derivation and Characterization of Cell Substrates Used for Production of
Biotechnological/Biological Products“ should be considered.
2.1.5 Quantity
5
Quantity, usually measured as protein content, is critical for a biotechnological

and biological product and should be determined using an appropriate assay,
usually physicochemical in nature. In some cases, it may be demonstrated
that the quantity values obtained may be directly related to those found using
the biological assay. When this correlation exists, it may be appropriate to use
measurement of quantity rather than the measurement of biological activity
in manufacturing processes, such as filling.
2.2 Analytical Considerations

2.2.1 Reference standards and reference materials
For drug applications for new molecular entities, it is unlikely that an
international or national standard will be available. At the time of submission,
the manufacturer should have established an appropriately characterized in-
house primary reference material, prepared from lot(s) representative of
production and clinical materials. In-house working reference material(s) used
in the testing of production lots should be calibrated against this primary
reference material. Where an international or national standard is available
and appropriate, reference materials should be calibrated against it. While it
is desirable to use the same reference material for both biological assays and
physicochemical testing, in some cases, a separate reference material may be
necessary. Also, distinct reference materials for product-related substances,
product-related impurities and process-related impurities, may need to be
established. When appropriate, a description of the manufacture and/or
purification of reference materials should be included in the application.
Documentation of the characterization, storage conditions and formulation
supportive of reference material(s) stability should also be provided.
2.2.2 Validation of analytical procedures

At the time the application is submitted to the regulatory authorities,
applicants should have validated the analytical procedures used in the
specifications in accordance with the ICH Harmonised Tripartite Guidelines
“Validation of Analytical Procedures: Definitions and Terminology” and
“Validation of Analytical Procedures: Methodology", except where there are
specific issues for unique tests used for analyzing biotechnological and
biological products.
2.3 Process Controls

2.3.1 Process-related considerations
Adequate design of a process and knowledge of its capability are part of the
strategy used to develop a manufacturing process which is controlled and
reproducible, yielding a drug substance or drug product that meets
specifications. In this respect, limits are justified based on critical information
gained from the entire process spanning the period from early development
through commercial scale production.
For certain impurities, testing of either the drug substance or the drug product
may not be necessary and may not need to be included in the specifications if
efficient control or removal to acceptable levels is demonstrated by suitable
studies. This testing can include verification at commercial scale in
accordance with regional regulations. It is recognized that only limited data
may be available at the time of submission of an application. This concept
6
may, therefore, sometimes be implemented after marketing authorization, in

accordance with regional regulations.
2.3.2 In-process acceptance criteria and action limits

In-process tests are performed at critical decision making steps and at other
steps where data serve to confirm consistency of the process during the
production of either the drug substance or the drug product. The results of in-
process testing may be recorded as action limits or reported as acceptance
criteria. Performing such testing may eliminate the need for testing of the
drug substance or drug product (section 2.3.1). In-process testing for
adventitious agents at the end of cell culture is an example of testing for
which acceptance criteria should be established.
The use of internal action limits by the manufacturer to assess the consistency
of the process at less critical steps is also important. Data obtained during
development and validation runs should provide the basis for provisional
action limits to be set for the manufacturing process. These limits, which are
the responsibility of the manufacturer, may be used to initiate investigation or
further action. They should be further refined as additional manufacturing
experience and data are obtained after product approval.
7
2.3.3 Raw materials and excipient specifications

The quality of the raw materials used in the production of the drug substance
(or drug product) should meet standards, appropriate for their intended use.
Biological raw materials or reagents may require careful evaluation to
establish the presence or absence of deleterious endogenous or adventitious
agents. Procedures which make use of affinity chromatography (for example,
employing monoclonal antibodies), should be accompanied by appropriate
measures to ensure that such process-related impurities or potential
contaminants arising from their production and use do not compromise the
quality and safety of the drug substance or drug product. Appropriate
information pertaining to the antibody should be made available.
The quality of the excipients used in the drug product formulation (and in
some cases, in the drug substance), as well as the container/closure systems,
should meet pharmacopoeial standards, where available and appropriate.
Otherwise, suitable acceptance criteria should be established for the non-
pharmacopoeial excipients.
2.4 Pharmacopoeial Specifications

Pharmacopoeias contain important requirements pertaining to certain
analytical procedures and acceptance criteria which, where relevant, are part
of the evaluation of either the drug substance or drug product. Such
monographs, applicable to biotechnological and biological products, generally
include, but are not limited to tests for sterility, endotoxins, microbial limits,
volume in container, uniformity of dosage units and particulate matter. With
respect to the use of pharmacopoeial methods and acceptance criteria, the
value of this guidance is linked to the extent of harmonisation of the analytical
procedures of the pharmacopoeias. The pharmacopoeias are committed to
developing identical or methodologically equivalent test procedures and
acceptance criteria.
2.5 Release Limits vs. Shelf-life Limits

The concept of release limits vs. shelf-life limits may be applied where
justified. This concept pertains to the establishment of limits which are tighter
for the release than for the shelf-life of the drug substance or drug product.
Examples where this may be applicable include potency and degradation
products. In some regions, the concept of release limits may only be
applicable to in-house limits and not to the regulatory shelf-life limits.
2.6 Statistical Concepts

Appropriate statistical analysis should be applied, when necessary, to
quantitative data reported. The methods of analysis, including justification
and rationale, should be described fully. These descriptions should be
sufficiently clear to permit independent calculation of the results presented.
3. JUSTIFICATION OF THE SPECIFICATION

The setting of specifications for drug substance and drug product is part of an
overall control strategy which includes control of raw materials and excipients,
in-process testing, process evaluation or validation, adherence to Good
Manufacturing Practices, stability testing, and testing for consistency of lots.
When combined in total, these elements provide assurance that the
appropriate quality of the product will be maintained. Since specifications are
chosen to confirm the quality rather than to characterize the product, the
8
manufacturer should provide the rationale and justification for including and/or
excluding testing for specific quality attributes. The following points should be
taken into consideration when establishing scientifically justifiable
specifications.
• Specifications are linked to a manufacturing process.
Specifications should be based on data obtained from lots used to

demonstrate manufacturing consistency. Linking specifications to a
manufacturing process is important, especially for product-related substances,
product-related impurities and process-related impurities. Process changes
and degradation products produced during storage may result in heterogeneity
patterns which differ from those observed in the material used during
preclinical and clinical development. The significance of these alterations
should be evaluated.
• Specifications should account for the stability of drug substance and drug
product.
Degradation of drug substance and drug product, which may occur during
storage, should be considered when establishing specifications. Due to the
inherent complexity of these products, there is no single stability-indicating
assay or parameter that profiles the stability characteristics. Consequently,
the manufacturer should propose a stability-indicating profile. The result of this
stability-indicating profile will then provide assurance that changes in the
quality of the product will be detected. The determination of which tests
should be included will be product-specific. The manufacturer is referred to
the ICH Harmonised Tripartite Guideline: “Stability Testing of
Biotechnological/Biological Products”.
• Specifications are linked to preclinical and clinical studies.
Specifications should be based on data obtained for lots used in pre-clinical

and clinical studies. The quality of the material made at commercial scale
should be representative of the lots used in preclinical and clinical studies.
• Specifications are linked to analytical procedures.
Critical quality attributes may include items such as potency, the nature and
quantity of product-related substances, product-related impurities, and
process-related impurities. Such attributes can be assessed by multiple
analytical procedures, each yielding different results. In the course of product
development, it is not unusual for the analytical technology to evolve in
parallel with the product. Therefore, it is important to confirm that data
generated during development correlate with those generated at the time the
marketing application is filed.
4. SPECIFICATIONS
Selection of tests to be included in the specifications is product specific. The
rationale used to establish the acceptable range of acceptance criteria should
be described. Acceptance criteria should be established and justified based on
data obtained from lots used in preclinical and/or clinical studies, data from
lots used for demonstration of manufacturing consistency, and data from
stability studies, and relevant development data.
In some cases, testing at production stages rather than at the drug substance
or drug product stages may be appropriate and acceptable. In such
9
circumstances, test results should be considered as in-process acceptance

criteria and included in the specification of drug substance or drug product in
accordance with the requirements of the regional regulatory authorities.
10
4.1 Drug Substance Specification

Generally, the following tests and acceptance criteria are considered
applicable to all drug substances (for analytical procedures see section 2.2.2).
Pharmacopoeial tests (e.g., endotoxin detection) should be performed on the
drug substance, where appropriate. Additional drug substance specific
acceptance criteria may also be necessary.
4.1.1 Appearance and description

A qualitative statement describing the physical state (e.g., solid, liquid) and
color of a drug substance should be provided.
4.1.2 Identity
The identity test(s) should be highly specific for the drug substance and should
be based on unique aspects of its molecular structure and/or other specific
properties. More than one test (physicochemical, biological and/or
immunochemical) may be necessary to establish identity. The identity test(s)
can be qualitative in nature. Some of the methods typically used for
characterization of the product as described in section 2.1 and in Appendix 6.1
may be employed and/or modified as appropriate for the purpose of
establishing identity.
4.1.3 Purity and impurities

The absolute purity of biotechnological and biological products is difficult to
determine and the results are method-dependent (section 2.1.4.).
Consequently, the purity of the drug substance is usually estimated by a
combination of methods. The choice and optimization of analytical procedures
should focus on the separation of the desired product from product-related
substances and from impurities.
The impurities observed in these products are classified as process-related and

product-related:
• Process-related impurities (section 2.1.4) in the drug substance may

include cell culture media, host cell proteins, DNA, monoclonal antibodies
or chromatographic media used in purification, solvents and buffer
components. These impurities should be minimized by the use of
appropriate well-controlled manufacturing processes.
• Product-related impurities (section 2.1.4) in the drug substance are

molecular variants with properties different from those of the desired
product formed during manufacture and/or storage.
For the impurities, the choice and optimization of analytical procedures should
focus on the separation of the desired product and product-related substances
from impurities. Individual and/or collective acceptance criteria for impurities
should be set, as appropriate. Under certain circumstances, acceptance
criteria for selected impurities may not be required (section 2.3).
4.1.4 Potency
A relevant, validated potency assay (section 2.1.2) should be part of the
specifications for a biotechnological or biological drug substance and/or drug
product. When an appropriate potency assay is used for the drug product
(section 4.2.4), an alternative method (physicochemical and/or biological) may
11
suffice for quantitative assessment at the drug substance stage. In some

cases, the measurement of specific activity may provide additional useful
information.
4.1.5 Quantity
The quantity of the drug substance, usually based on protein content (mass),
should be determined using an appropriate assay. The quantity determination
may be independent of a reference standard or material. In cases where
product manufacture is based upon potency, there may be no need for an
alternate determination of quantity.
4.2 Drug Product Specification

Generally, the following tests and acceptance criteria are considered
applicable to all drug products. Each section (4.2.1 - 4.2.5) is cross-referenced
to respective sections (4.1.1 - 4.1.5) under Drug Substance. Pharmacopoeial
requirements apply to the relevant dosage forms. Typical tests found in the
pharmacopoeia include, but are not limited to sterility, endotoxin, microbial
limits, volume in container, particulate matter, uniformity of dosage units, and
moisture content for lyophilized drug products. If appropriate, testing for
uniformity of dosage units may be performed as in-process controls and
corresponding acceptance criteria set.
4.2.1 Appearance and description

A qualitative statement describing the physical state (e.g., solid, liquid), color,
and clarity of the drug product should be provided.
4.2.2 Identity
The identity test(s) should be highly specific for the drug product and should
be based on unique aspects of its molecular structure and for other specific
properties. The identity test(s) can be qualitative in nature. While it is
recognized that in most cases, a single test is adequate, more than one test
(physicochemical, biological and/or immunochemical) may be necessary to
establish identity for some products. Some of the methods typically used for
characterization of the product as described in section 2.1 and in Appendix 6.1
may be employed and/or modified as appropriate for the purpose of
establishing identity.
4.2.3 Purity and impurities

Impurities may be generated or increased during manufacture and/or storage
of the drug product. These may be either the same as those occurring in the
drug substance itself, process-related, or degradation products which form
specifically in the drug product during formulation or during storage. If
impurities are qualitatively and quantitatively (i.e., relative amounts and/or
concentrations) the same as in the drug substance, testing is not necessary. If
impurities are known to be introduced or formed during the production and/or
storage of the drug product, the levels of these impurities should be
determined and acceptance criteria established.
Acceptance criteria and analytical procedures should be developed and
justified, based upon previous experience with the drug product, to measure
changes in the drug substance during the manufacture and/or storage of the
drug product.
12
The choice and optimization of analytical procedures should focus on the

separation of the desired product and product-related substances from
impurities including degradation products, and from excipients.
4.2.4 Potency
A relevant, validated potency assay (section 2.1.2) should be part of the
specifications for a biotechnological and biological drug substance and/or drug
product. When an appropriate potency assay is used for the drug substance,
an alternative method (physicochemical and/or biological) may suffice for
quantitative assessment of the drug product. However, the rationale for such a
choice should be provided.
4.2.5 Quantity
The quantity of the drug substance in the drug product, usually based on
protein content (mass), should be determined using an appropriate assay. In
cases where product manufacture is based upon potency, there may be no
need for an alternate determination of quantity.
4.2.6 General tests

Physical description and the measurement of other quality attributes is often
important for the evaluation of the drug product functions. Examples of such
tests include pH and osmolarity.
4.2.7 Additional testing for unique dosage forms

It should be recognized that certain unique dosage forms may need additional
tests other than those mentioned above.
5. GLOSSARY
Acceptance Criteria
Numerical limits, ranges, or other suitable measures for acceptance of the
results of analytical procedures which the drug substance or drug product or
materials at other stages of their manufacture should meet.
Action Limit
An internal (in-house) value used to assess the consistency of the process at
less critical steps.
Biological Activity
The specific ability or capacity of the product to achieve a defined biological
effect. Potency is the quantitative measure of the biological activity.
Contaminants
Any adventitiously introduced materials (e.g., chemical, biochemical, or
microbial species) not intended to be part of the manufacturing process of the
drug substance or drug product.
Degradation Products
Molecular variants resulting from changes in the desired product or product-
related substances brought about over time and/or by the action of, e.g., light,
temperature, pH, water, or by reaction with an excipient and/or the immediate
13
container/closure system. Such changes may occur as a result of manufacture

and/or storage (e.g., deamidation, oxidation, aggregation, proteolysis).
Degradation products may be either product-related substances, or product-
related impurities.
Desired Product
(1) The protein which has the expected structure, or (2) the protein which is
expected from the DNA sequence and anticipated post-translational
modification (including glycoforms), and from the intended downstream
modification to produce an active biological molecule.
Drug Product (Dosage Form; Finished Product)

A pharmaceutical product type that contains a drug substance, generally, in
association with excipients.
Drug Substance (Bulk Material)

The material which is subsequently formulated with excipients to produce the
drug product. It can be composed of the desired product, product-related
substances, and product- and process-related impurities. It may also contain
excipients including other components such as buffers.
Excipient
An ingredient added intentionally to the drug substance which should not have
pharmacological properties in the quantity used.
Impurity
Any component present in the drug substance or drug product which is not the
desired product, a product-related substance, or excipient including buffer
components. It may be either process- or product-related.
In-house Primary Reference Material

An appropriately characterized material prepared by the manufacturer from a
representative lot(s) for the purpose of biological assay and physicochemical
testing of subsequent lots, and against which in-house working reference
material is calibrated.
In-house Working Reference Material

A material prepared similarly to the primary reference material that is
established solely to assess and control subsequent lots for the individual
attribute in question. It is always calibrated against the in-house primary
reference material.
Potency
The measure of the biological activity using a suitably quantitative biological
assay (also called potency assay or bioassay), based on the attribute of the
product which is linked to the relevant biological properties.
Process-Related Impurities
Impurities that are derived from the manufacturing process. They may be
derived from cell substrates (e.g., host cell proteins, host cell DNA), cell culture
14
(e.g., inducers, antibiotics, or media components), or downstream processing

(e.g., processing reagents or column leachables).
Product-Related Impurities
Molecular variants of the desired product (e.g., precursors, certain degradation
products arising during manufacture and/or storage) which do not have
properties comparable to those of the desired product with respect to activity,
efficacy, and safety.
15
Product-Related Substances
Molecular variants of the desired product formed during manufacture and/or
storage which are active and have no deleterious effect on the safety and
efficacy of the drug product. These variants possess properties comparable to
the desired product and are not considered impurities.
Reference Standards
Refer to international or national standards.
Specification
A specification is defined as a list of tests, references to analytical procedures,
and appropriate acceptance criteria which are numerical limits, ranges, or
other criteria for the tests described. It establishes the set of criteria to which
a drug substance, drug product or materials at other stages of its manufacture
should conform to be considered acceptable for its intended use.
“Conformance to specification” means that the drug substance and drug
product, when tested according to the listed analytical procedures, will meet
the acceptance criteria. Specifications are critical quality standards that are
proposed and justified by the manufacturer and approved by regulatory
authorities as conditions of approval.
6. APPENDICES
6.1 Appendix for Physicochemical Characterization

This appendix provides examples of technical approaches which might be
considered for structural characterization and confirmation, and evaluation of
physicochemical properties of the desired product, drug substance and/or drug
product. The specific technical approach employed will vary from product to
product and alternative approaches, other than those included in this
appendix, will be appropriate in many cases. New analytical technology and
modifications to existing technology are continuously being developed and
should be utilized when appropriate.
6.1.1 Structural characterization and confirmation

a) Amino acid sequence
The amino acid sequence of the desired product should be determined
to the extent possible using approaches such as those described in
items b) through e) and then compared with the sequence of the amino
acids deduced from the gene sequence of the desired product.
b) Amino acid composition
The overall amino acid composition is determined using various

hydrolytic and analytical procedures, and compared with the amino acid
composition deduced from the gene sequence for the desired product,
or the natural counterpart, if considered necessary. In many cases
amino acid composition analysis provides some useful structural
information for peptides and small proteins, but such data are generally
less definitive for large proteins. Quantitative amino acid analysis data
can also be used to determine protein content in many cases.
16
c) Terminal amino acid sequence
Terminal amino acid analysis is performed to identify the nature and

homogeneity of the amino- and carboxy-terminal amino acids. If the
desired product is found to be heterogeneous with respect to the
terminal amino acids, the relative amounts of the variant forms should
be determined using an appropriate analytical procedure. The sequence
of these terminal amino acids should be compared with the terminal
amino acid sequence deduced from the gene sequence of the desired
product.
d) Peptide map
Selective fragmentation of the product into discrete peptides is
performed using suitable enzymes or chemicals and the resulting
peptide fragments are analyzed by HPLC or other appropriate analytical
procedure. The peptide fragments should be identified to the extent
possible using techniques such as amino acid compositional analysis, N-
terminal sequencing, or mass spectrometry. Peptide mapping of the
drug substance or drug product using an appropriately validated
procedure is a method that is frequently used to confirm desired product
structure for lot release purposes.
e) Sulfhydryl group(s) and disulfide bridges

If, based on the gene sequence for the desired product, cysteine
residues are expected, the number and positions of any free sulfhydryl
groups and/or disulfide bridges should be determined, to the extent
possible. Peptide mapping (under reducing and non-reducing
conditions), mass spectrometry, or other appropriate techniques may be
useful for this evaluation.
f) Carbohydrate structure
For glycoproteins, the carbohydrate content (neutral sugars, amino

sugars, and sialic acids) is determined. In addition, the structure of the
carbohydrate chains, the oligosaccharide pattern (antennary profile) and
the glycosylation site(s) of the polypeptide chain is analyzed, to the
extent possible.
6.1.2 Physicochemical properties

a) Molecular weight or size
Molecular weight (or size) is determined using size exclusion
chromatography, SDS-polyacrylamide gel electrophoresis (under
reducing and/or non-reducing conditions), mass spectrometry, and other
appropriate techniques.
b) Isoform pattern
This is determined by isoelectric focusing or other appropriate

techniques.
c) Extinction coefficient (or molar absorptivity)

In many cases it will be desirable to determine the extinction coefficient
(or molar absorptivity) for the desired product at a particular UV/visible
wavelength (e.g., 280 nm). The extinction coefficient is determined
17
using UV/visible spectrophotometry on a solution of the product having

a known protein content as determined by techniques such as amino
acid compositional analysis, or nitrogen determination, etc. If UV
absorption is used to measure protein content, the extinction coefficient
for the particular product should be used.
d) Electrophoretic patterns
Electrophoretic patterns and data on identity, homogeneity and purity
can be obtained by polyacrylamide gel electrophoresis, isoelectric
focusing, SDS-polyacrylamide gel electrophoresis, Western-blot,
capillary electrophoresis, or other suitable procedures.
e) Liquid chromatographic patterns
Chromatographic patterns and data on the identity, homogeneity, and

purity can be obtained by size exclusion chromatography, reverse-phase
liquid chromatography, ion-exchange liquid chromatography, affinity
chromatography or other suitable procedures.
f) Spectroscopic profiles
The ultraviolet and visible absorption spectra are determined as
appropriate. The higher-order structure of the product is examined
using procedures such as circular dichroism, nuclear magnetic
resonance (NMR), or other suitable techniques, as appropriate.
6.2 Appendix for Impurities

This appendix lists potential impurities, their sources and examples of relevant
analytical approaches for detection. Specific impurities and technical
approaches employed, as in the case of physicochemical characterization, will
vary from product to product and alternative approaches, other than those
listed in this appendix will be appropriate in many cases. New analytical
technology and modifications to existing technology are continuously being
developed, and should be applied when appropriate.
6.2.1 Process-related impurities and contaminants

These are derived from the manufacturing process (section 2.1.4) and are
classified into three major categories: cell substrate-derived, cell culture-
derived and downstream-derived.
a) Cell substrate-derived impurities include, but are not limited to, proteins
derived from the host organism, nucleic acid (host cell genomic, vector,
or total DNA). For host cell proteins, a sensitive assay e.g.,
immunoassay, capable of detecting a wide range of protein impurities is
generally utilized. In the case of an immunoassay, a polyclonal antibody
used in the test is generated by immunization with a preparation of a
production cell minus the product-coding gene, fusion partners, or other
appropriate cell lines. The level of DNA from the host cells can be
detected by direct analysis on the product (such as hybridization
techniques). Clearance studies, which could include spiking
experiments at the laboratory scale, to demonstrate the removal of cell
substrate-derived impurities such as nucleic acids and host cell proteins
may sometimes be used to eliminate the need for establishing
acceptance criteria for these impurities.
18
b) Cell culture-derived impurities include, but are not limited to, inducers
antibiotics, serum, and other media components.
c) Downstream-derived impurities include, but are not limited to, enzymes,
chemical and biochemical processing reagents (e.g., cyanogen bromide,
guanidine, oxidizing and reducing agents), inorganic salts (e.g., heavy
metals, arsenic, non metallic ion), solvents, carriers, ligands (e.g.,
monoclonal antibodies), and other leachables.
For intentionally introduced, endogenous and adventitious viruses, the ability

of the manufacturing process to remove and/or inactivate viruses should be
demonstrated as described in ICH Harmonised Tripartite Guideline “Viral
Safety Evaluation of Biotechnology Products Derived From Cell Lines of Human
or Animal Origin”
6.2.2 Product-related impurities including degradation products

The following represents the most frequently encountered molecular variants
of the desired product and lists relevant technology for their assessment.
Such variants may need considerable effort in isolation and characterization in
order to identify the type of modification(s). Degradation products arising
during manufacture and/or storage in significant amounts should be tested for
and monitored against appropriately established acceptance criteria.
a) Truncated forms: Hydrolytic enzymes or chemicals may catalyze the

cleavage of peptide bonds. These may be detected by HPLC or SDS-
PAGE. Peptide mapping may be useful, depending on the property of
the variant.
b) Other modified forms: Deamidated, isomerized, mismatched S-S linked,

oxidized or altered conjugated forms (e.g., glycosylation,
phosphorylation) may be detected and characterized by
chromatographic, electrophoretic and/or other relevant analytical
methods (e.g., HPLC, capillary electrophoresis, mass spectroscopy,
circular dichroism).
c) Aggregates: The category of aggregates includes dimers and higher
multiples of the desired product. These are generally resolved from the
desired product and product-related substances, and quantitated by
appropriate analytical procedures (e.g., size exclusion chromatography,
capillary electrophoresis).
19

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INTERNATIONAL CONFERENCE ON HARMONISATION OF TECHNICAL

REQUIREMENTS FOR REGISTRATION OF PHARMACEUTICALS FOR HUMAN USE

ICH HARMONISED TRIPARTITE GUIDELINE

SPECIFICATIONS: TEST PROCEDURES AND ACCEPTANCE CRITERIA FOR

Current Step 4 version

Q6B Approval by the Steering Committee under Step 27 Q6B

Current Step 4 version

Q6B Approval by the Steering Committee under Step 4 10 Q6B

2.1.4 Purity, impurities and contaminants........................................4

2.2 Analytical Considerations.....................................................................6

2.2.2Validation of analytical procedures...........................................6

2.3 Process Controls...................................................................................6

2.3.3 Raw materials and excipient specifications..............................8

2.4 Pharmacopoeial Specifications............................................................8

3. JUSTIFICATION OF THE SPECIFICATION..........................................8

4.1.2 Identity............................................................................. ......11

4.1.3 Purity and impurities..............................................................11

4.2 Drug Product Specification.................................................................12

4.2.3 Purity and impurities..............................................................12

4.2.7 Additional testing for unique dosage forms...........................13

6.2 Appendix for Impurities......................................................................18

6.2.2 Product-related impurities including degradation products. . .19

Specifications are one part of a total control strategy designed to ensure

Substances” addresses specifications, and other criteria for chemical

2. PRINCIPLES FOR CONSIDERATION IN SETTING SPECIFICATIONS

Extensive characterization is performed in the development phase and, where

2.1.1 Physicochemical properties

Heterogeneity can also be produced during manufacture and/or storage of the

Analytical methods to elucidate physicochemical properties are listed in

2.1.2 Biological activity

• Animal-based biological assays, which measure an organism's biological

• Cell culture-based biological assays, which measure biochemical or

• Biochemical assays, which measure biological activities such as enzymatic

The results of biological assays should be expressed in units of activity

biological activity of the product may be replaced by physicochemical tests

• sufficient physicochemical information about the drug, including higher-

• there exists a well-established manufacturing history.

Where physicochemical tests alone are used to quantitate the biological

2.1.3 Immunochemical properties

2.1.4 Purity, impurities and contaminants

Due to the unique biosynthetic production process and molecular

Individual and/or collective acceptance criteria for product-related substances

Process-related impurities encompass those that are derived from the

Further, the acceptance criteria for impurities should be based on data

Quantity, usually measured as protein content, is critical for a biotechnological

2.2 Analytical Considerations

2.2.2 Validation of analytical procedures

2.3 Process Controls

may, therefore, sometimes be implemented after marketing authorization, in

2.3.2 In-process acceptance criteria and action limits

2.3.3 Raw materials and excipient specifications

2.4 Pharmacopoeial Specifications

2.5 Release Limits vs. Shelf-life Limits

2.6 Statistical Concepts

3. JUSTIFICATION OF THE SPECIFICATION

• Specifications are linked to a manufacturing process.

Specifications should be based on data obtained from lots used to

• Specifications are linked to preclinical and clinical studies.

Specifications should be based on data obtained for lots used in pre-clinical