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550 1049 Quality of Biotechnological Products: Stability / General Information USP 35

1049 QUALITY OF
II. SCOPE OF THE ANNEX (2)

BIOTECHNOLOGICAL The guidance stated in this annex to “Stability Testing of

New Drug Substances and Products” applies to well-charac-
PRODUCTS: STABILITY TESTING terized proteins and polypeptides, their derivatives and
products of which they are components, and which are iso-
OF BIOTECHNOLOGICAL/ lated from tissues, body fluids, cell cultures, or produced
using recombinant deoxyribonucleic acid (r-DNA) technol-
BIOLOGICAL PRODUCTS1 ogy. Thus, the document covers the generation and submis-
sion of stability data for products such as cytokines (interfer-
ons, interleukins, colony-stimulating factors, tumor necrosis
factors), erythropoietins, plasminogen activators, blood
plasma factors, growth hormones and growth factors, insu-
lins, monoclonal antibodies, and vaccines consisting of well-
I. INTRODUCTION (1) characterized proteins or polypeptides. In addition, the
guidance outlined in the following sections may apply to
The guidance stated in the ICH harmonized tripartite other types of products, such as conventional vaccines, after
guideline entitled “Stability Testing of New Drug Substances consultation with the appropriate regulatory authorities. The
and Products” (issued by ICH on October 27, 1993) applies document does not cover antibiotics, allergenic extracts,
in general to biotechnological/biological products. However, heparins, vitamins, whole blood, or cellular blood
biotechnological/biological products have distinguishing components.
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 prod- III. TERMINOLOGY (3)
ucts in which the active components are typically proteins
and/or polypeptides, maintenance of molecular conforma- For the basic terms used in this annex, the reader is re-
tion and, hence, of biological activity, is dependent on non- ferred to the “Glossary” in “Stability Testing of New Drug
covalent as well as covalent forces. The products are particu- Substances and Products.” However, because manufacturers
larly sensitive to environmental factors such as temperature of biotechnological/biological products sometimes use tradi-
changes, oxidation, light, ionic content, and shear. To en- tional terminology, traditional terms are specified in paren-
sure maintenance of biological activity and to avoid degra- theses to assist the reader. A supplemental glossary is also
dation, stringent conditions for their storage are usually included that explains certain terms used in the production
necessary. of biotechnological/biological products.
The evaluation of stability may necessitate complex ana-
lytical methodologies. Assays for biological activity, where
applicable, should be part of the pivotal stability studies. IV. SELECTION OF BATCHES (4)
Appropriate physicochemical, biochemical, and immu-
nochemical methods for the analysis of the molecular entity A. Drug Substance (Bulk Material) (4.1)
and the quantitative detection of degradation products Where bulk material is to be stored after manufacture, but
should also be part of the stability program whenever purity before formulation and final manufacturing, stability data
and molecular characteristics of the product permit use of should be provided on at least three batches for which
these methodologies. manufacture and storage are representative of the manufac-
With these concerns in mind, the applicant should de- turing scale of production. A minimum of 6 months stability
velop the proper supporting stability data for a biotechno- data at the time of submission should be submitted in cases
logical/biological product and consider many external con- where storage periods greater than 6 months are requested.
ditions that can affect the product’s potency, purity, and For drug substances with storage periods of less than 6
quality. Primary data to support a requested storage period months, the minimum amount of stability data in the initial
for either drug substance or drug product should be based submission should be determined on a case-by-case basis.
on long-term, real-time, real-condition stability studies. Data from pilot-plant scale batches of drug substance pro-
Thus, the development of a proper long-term stability pro- duced at a reduced scale of fermentation and purification
gram becomes critical to the successful development of a may be provided at the time the dossier is submitted to the
commercial product. The purpose of this document is to regulatory agencies with acommitment to place the first
give guidance to applicants regarding the type of stability three manufacturing scale batches into the long-term stabil-
studies that should be provided in support of marketing ap- ity program after approval.
plications. It is understood that during the review and evalu- The quality of the batches of drug substance placed into
ation process, continuing updates of initial stability data the stability program should be representative of the quality
may occur. of the material used in preclinical and clinical studies and of
1This guideline was developed within the Expert Working Group (Quality) of
the quality of the material to be made at manufacturing
the International Conference on Harmonisation of Technical Requirements for scale. In addition, the drug substance (bulk material) made
Registration of Pharmaceuticals for Human Use (ICH) and has been subject to at pilot-plant scale should be produced by a process and
consultation by the regulatory parties, in accordance with the ICH process. stored under conditions representative of that used for the
This document has been endorsed by the ICH Steering Committee at Step 4
of the ICH process, November 20, 1995. At Step 4 of the process, the final manufacturing scale. The drug substance entered into the
draft is recommended for adoption to the regulatory bodies of the European stability program should be stored in containers that prop-
Union, Japan and the USA. This guideline was published in the Federal erly represent the actual holding containers used during
Register on July 10, 1996 (61 FR 36466) and is applicable to drug and manufacture. Containers of reduced size may be acceptable
biological products. Although this guideline does not create or confer any
rights for or on any person and does not operate to bind FDA or the for drug substance stability testing provided that they are
industry, it does represent the agency’s current thinking on stability testing of constructed of the same material and use the same type of
biotechnological/biological products. For additional copies of this guideline, container/closure system that is intended to be used during
contact the Drug Information Branch, HFD-210, CDER, FDA, 5600 Fishers
Lane, Rockville, MD 20857 (Phone: 301-827-4573) or the Manufacturers manufacture.
Assistance and Communication Staff (HFM-42), CBER, FDA, 1401 Rockville B. Intermediates (4.2)
Pike, Rockville, MD 20852-1448. Send one self-addressed adhesive label to During manufacture of biotechnological/biological prod-
assist the offices in processing your request. An electronic version of this
guidance is also available via Internet using the World Wide Web (WWW) ucts, the quality and control of certain intermediates may be
(connect to the CDER Home Page at http://www.fda.gov/cder and go to the critical to the production of the final product. In general,
“Regulatory Guidance” section). the manufacturer should identify intermediates and generate

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USP 35 General Information / 1049 Quality of Biotechnological Products: Stability 551

in-house data and process limits that assure their stability provides assurance that changes in the identity, purity, and
within the bounds of the developed process. Although the potency of the product will be detected.
use of pilot-plant scale data is permissible, the manufacturer At the time of submission, applicants should have vali-
should establish the suitability of such data using the manu- dated the methods that comprise the stability-indicating
facturing scale process. profile, and the data should be available for review. The
C. Drug Product (Final Container Product) (4.3) determination of which tests should be included will be
Stability information should be provided on at least three product-specific. The items emphasized in the following
batches of final container product representative of that subsections are not intended to be all-inclusive, but repre-
which will be used at manufacturing scale. Where possible, sent product characteristics that should typically be docu-
batches of final container product included in stability test- mented to demonstrate product stability adequately.
ing should be derived from different batches of bulk mate- A. Protocol (5.1)
rial. A minimum of 6 months data at the time of submission The dossier accompanying the application for marketing
should be submitted in cases where storage periods greater authorization should include a detailed protocol for the as-
than 6 months are requested. For drug products with stor- sessment of the stability of both drug substance and drug
age periods of less than 6 months, the minimum amount of product in support of the proposed storage conditions and
stability data in the initial submission should be determined expiration dating periods. The protocol should include all
on a case-by-case basis. Product expiration dating should be necessary information that demonstrates the stability of the
based upon the actual data submitted in support of the biotechnological/biological product throughout the pro-
application. Because dating is based upon the real-time/real- posed expiration dating period including, for example, well-
temperature data submitted for review, continuing updates defined specifications and test intervals. The statistical meth-
of initial stability data should occur during the review and ods that should be used are described in the tripartite
evaluation process. The quality of the final container prod- guideline on stability.
uct placed on stability studies should be representative of B. Potency (5.2)
the quality of the material used in the preclinical and clinical When the intended use of a product is linked to a defina-
studies. Data from pilot-plant scale batches of drug product ble and measurable biological activity, testing for potency
may be provided at the time the dossier is submitted to the should be part of the stability studies. For the purpose of
regulatory agencies with a commitment to place the first stability testing of the products described in this guideline,
three manufacturing scale batches into the long-term stabil- potency is the specific ability or capacity of a product to
ity program after approval. Where pilot-plant scale batches achieve its intended effect. It is based on the measurement
were submitted to establish the dating for a product and, in of some attribute of the product and is determined by a
the event that the product produced at manufacturing scale suitable in vivo or in vitro quantitative method. In general,
does not meet those long-term stability specifications potencies of biotechnological/biological products tested by
throughout the dating period or is not representative of the different laboratories can be compared in a meaningful way
material used in preclinical and clinical studies, the applicant only if expressed in relation to that of an appropriate refer-
should notify the appropriate regulatory authorities to deter- ence material. For that purpose, a reference material cali-
mine a suitable course of action. brated directly or indirectly against the corresponding na-
D. Sample Selection (4.4) tional or international reference material should be included
Where one product is distributed in batches differing in in the assay.
fill volume (e.g., 1 milliliter (mL), 2 mL, or 10 mL), unitage Potency studies should be performed at appropriate inter-
(e.g., 10 units, 20 units, or 50 units), or mass (e.g., 1 milli- vals as defined in the stability protocol and the results
gram (mg), 2 mg, or 5 mg), samples to be entered into the should be reported in units of biological activity calibrated,
stability program may be selected on the basis of a matrix whenever possible, against nationally or internationally rec-
system and/or by bracketing. ognized standards. Where no national or international refer-
Matrixing, i.e., the statistical design of a stability study in ence standards exist, the assay results may be reported in
which different fractions of samples are tested at different in-house derived units using a characterized reference
sampling points, should only be applied when appropriate material.
documentation is provided that confirms that the stability of In some biotechnological/biological products, potency is
the samples tested represents the stability of all samples. dependent upon the conjugation of the active ingredient(s)
The differences in the samples for the same drug product to a second moiety or binding to an adjuvant. Dissociation
should be identified as, for example, covering different of the active ingredient(s) from the carrier used in conju-
batches, different strengths, different sizes of the same clo- gates or adjuvants should be examined in real-time/real-
sure, and, possibly, in some cases, different container/clo- temperature studies (including conditions encountered dur-
sure systems. Matrixing should not be applied to samples ing shipment). The assessment of the stability of such prod-
with differences that may affect stability, such as different ucts may be difficult because, in some cases, in vitro tests
strengths and different containers/closures, where it cannot for biological activity and physicochemical characterization
be confirmed that the products respond similarly under stor- are impractical or provide inaccurate results. Appropriate
age conditions. strategies (e.g., testing the product before conjugation/
Where the same strength and exact container/closure sys- binding, assessing the release of the active compound from
tem is used for three or more fill contents, the manufacturer the second moiety, in vivo assays) or the use of an appropri-
may elect to place only the smallest and largest container ate surrogate test should be considered to overcome the
size into the stability program, i.e., bracketing. The design inadequacies of in vitro testing.
of a protocol that incorporates bracketing assumes that the C. Purity and Molecular Characterization (5.3)
stability of the intermediate condition samples are repre- For the purpose of stability testing of the products de-
sented by those at the extremes. In certain cases, data may scribed in this guideline, purity is a relative term. Because of
be needed to demonstrate that all samples are properly rep- the effect of glycosylation, deamidation, or other heteroge-
resented by data collected for the extremes. neities, the absolute purity of a biotechnological/biological
product is extremely difficult to determine. Thus, the purity
V. STABILITY-INDICATING PROFILE (5) of a biotechnological/biological product should be typically
assessed by more than one method and the purity value
derived is method-dependent. For the purpose of stability
On the whole, there is no single stability-indicating assay testing, tests for purity should focus on methods for deter-
or parameter that profiles the stability characteristics of a mination of degradation products.
biotechnological/biological product. Consequently, the man- The degree of purity, as well as the individual and total
ufacturer should propose a stability-indicating profile that amounts of degradation products of the biotechnological/

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552 1049 Quality of Biotechnological Products: Stability / General Information USP 35

biological product entered into the stability studies, should strongly suggested that studies be conducted on the drug
be reported and documented whenever possible. Limits of substance and drug product under accelerated and stress
acceptable degradation should be derived from the analyti- conditions. Studies under accelerated conditions may pro-
cal profiles of batches of the drug substance and drug prod- vide useful support data for establishing the expiration date,
uct used in the preclinical and clinical studies. provide product stability information or future product de-
The use of relevant physicochemical, biochemical, and im- velopment (e.g., preliminary assessment of proposed manu-
munochemical analytical methodologies should permit a facturing changes such as change in formulation, scale-up),
comprehensive characterization of the drug substance and/ assist in validation of analytical methods for the stability pro-
or drug product (e.g., molecular size, charge, hydrophobic- gram, or generate information that may help elucidate the
ity) and the accurate detection of degradation changes that degradation profile of the drug substance or drug product.
may result from deamidation, oxidation, sulfoxidation, ag- Studies under stress conditions may be useful in determin-
gregation, or fragmentation during storage. As examples, ing whether accidental exposures to conditions other than
methods that may contribute to this include electrophoresis those proposed (e.g., during transportation) are deleterious
(SDS09Page, immunoelectrophoresis, Western blot, isoelec- to the product and also for evaluating which specific test
trofocusing), high-resolution chromatography (e.g., re- parameters may be the best indicators of product stability.
versed-phase chromatography, gel filtration, ion exchange, Studies of the exposure of the drug substance or drug prod-
affinity chromatography), and peptide mapping. uct to extreme conditions may help to reveal patterns of
Wherever significant qualitative or quantitative changes degradation; if so, such changes should be monitored under
indicative of degradation product formation are detected proposed storage conditions. Although the tripartite guide-
during long-term, accelerated, and/or stress stability studies, line on stability describes the conditions of the accelerated
consideration should be given to potential hazards and to and stress study, the applicant should note that those con-
the need for characterization and quantification of degrada- ditions may not be appropriate for biotechnological/biologi-
tion products within the long-term stability program. Ac- cal products. Conditions should be carefully selected on a
ceptable limits should be proposed and justified, taking into case-by-case basis.
account the levels observed in material used in preclinical D. Light (6.4)
and clinical studies. Applicants should consult the appropriate regulatory au-
For substances that cannot be properly characterized or thorities on a case-by-case basis to determine guidance for
products for which an exact analysis of the purity cannot be testing.
determined through routine analytical methods, the appli- E. Container/Closure (6.5)
cant should propose and justify alternative testing Changes in the quality of the product may occur due to
procedures. the interactions between the formulated biotechnological/
D. Other Product Characteristics (5.4) biological product and container/closure. Where the lack of
The following product characteristics, though not specifi- interactions cannot be excluded in liquid products (other
cally relating to biotechnological/biological products, should than sealed ampules), stability studies should include sam-
be monitored and reported for the drug product in its final ples maintained in the inverted or horizontal position (i.e.,
container: in contact with the closure), as well as in the upright posi-
Visual appearance of the product (color and opacity for tion, to determine the effects of the closure on product
solutions/suspensions; color, texture, and dissolution time quality. Data should be supplied for all different container/
for powders), visible particulates in solutions or after the re- closure combinations that will be marketed.
constitution of powders or lyophilized cakes, pH, and mois- In addition to the standard data necessary for a conven-
ture level of powders and lyophilized products. tional single-use vial, the applicant should demonstrate that
Sterility testing or alternatives (e.g., container/closure in- the closure used with a multiple-dose vial is capable of with-
tegrity testing) should be performed at a minimum initially standing the conditions of repeated insertions and with-
and at the end of the proposed shelf life. drawals so that the product retains its full potency, purity,
Additives (e.g., stabilizers, preservatives) or excipients may and quality for the maximum period specified in the instruc-
degrade during the dating period of the drug product. If tions-for-use on containers, packages, and/or package in-
there is any indication during preliminary stability studies serts. Such labeling should be in accordance with relevant
that reaction or degradation of such materials adversely af- national/regional requirements.
fect the quality of the drug product, these items may need F. Stability after Reconstitution of Freeze-Dried Product (6.6)
to be monitored during the stability program. The stability of freeze-dried products after their reconstitu-
The container/closure has the potential to affect the prod- tion should be demonstrated for the conditions and the
uct adversely and should be carefully evaluated (see below). maximum storage period specified on containers, packages,
and/or package inserts. Such labeling should be in accor-
VI. STORAGE CONDITIONS (6) dance with relevant national/regional requirements.

A. Temperature (6.1) VII. TESTING FREQUENCY (7)

Because most finished biotechnological/biological prod-
ucts need precisely defined storage temperatures, the stor- The shelf lives of biotechnological/biological products may
age conditions for the real-time/real-temperature stability vary from days to several years. Thus, it is difficult to draft
studies may be confined to the proposed storage uniform guidelines regarding the stability study duration
temperature. and testing frequency that would be applicable to all types
B. Humidity (6.2) of biotechnological/biological products. With only a few ex-
Biotechnological/biological products are generally distrib- ceptions, however, the shelf lives for existing products and
uted in containers protecting them against humidity. There- potential future products will be within the range of 0.5 to
fore, where it can be demonstrated that the proposed con- 5 years. Therefore, the guidance is based upon expected
tainers (and conditions of storage) afford sufficient pro- shelf lives in that range. This takes into account the fact that
tection against high and low humidity, stability tests at dif- degradation of biotechnological/biological products may not
ferent relative humidities can usually be omitted. Where hu- be governed by the same factors during different intervals
midity-protecting containers are not used, appropriate sta- of a long storage period.
bility data should be provided. When shelf lives of 1 year or less are proposed, the real-
C. Accelerated and Stress Conditions (6.3) time stability studies should be conducted monthly for the
As previously noted, the expiration dating should be first 3 months and at 3 month intervals thereafter. For prod-
based on real-time/real-temperature data. However, it is ucts with proposed shelf lives of greater than 1 year, the

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USP 35 General Information / 1050 Viral Safety Evaluation 553

studies should be conducted every 3 months during the first specifications will be established to determine the suc-
year of storage, every 6 months during the second year, cessful completion of the manufacturing step before
and annually thereafter. continuation of the manufacturing process. This in-
While the testing intervals listed above may be appropri- cludes material that may undergo further molecular
ate in the preapproval or prelicense stage, reduced testing modification or be held for an extended period before
may be appropriate after approval or licensure where data further processing.
are available that demonstrate adequate stability. Where • Manufacturing Scale Production—Manufacture at the
data exist that indicate the stability of a product is not com- scale typically encountered in a facility intended for
promised, the applicant is encouraged to submit a protocol product production for marketing.
that supports elimination of specific test intervals (e.g., 9- • Pilot-Plant Scale—The production of the drug substance
month testing) for postapproval/postlicensure, long-term or drug product by a procedure fully representative of
studies. and simulating that to be applied at manufacturing
scale. The methods of cell expansion, harvest, and
product purification should be identical except for the
VIII. SPECIFICATIONS (8) scale of production.
Although biotechnological/biological products may be
subject to significant losses of activity, physicochemical
changes, or degradation during storage, international and
national regulations have provided little guidance with re-
spect to distinct release and end of shelf life specifications.
Recommendations for maximum acceptable losses of activ-
ity, limits for physicochemical changes, or degradation dur- 1050 VIRAL SAFETY
ing the proposed shelf life have not been developed for in-
dividual types or groups of biotechnological/biological
EVALUATION OF
products but are considered on a case-by-case basis. Each
product should retain its specifications within established
BIOTECHNOLOGY PRODUCTS
limits for safety, purity, and potency throughout its pro-
posed shelf life. These specifications and limits should be
DERIVED FROM CELL LINES OF
derived from all available information using the appropriate
statistical methods. The use of different specifications for re- HUMAN OR ANIMAL ORIGIN
lease and expiration should be supported by sufficient data
to demonstrate that the clinical performance is not affected,
as discussed in the tripartite guideline on stability.
I. INTRODUCTION
IX. LABELING (9)
This document is concerned with testing and evaluation
For most biotechnological/biological drug substances and of the viral safety of biotechnology products derived from
drug products, precisely defined storage temperatures are characterized cell lines of human or animal origin (i.e.,
recommended. Specific recommendations should be stated, mammalian, avian, insect), and outlines data that should be
particularly for drug substances and drug products that can- submitted in the marketing application/registration package.
not tolerate freezing. These conditions, and where appropri- For the purposes of this document, the term virus excludes
ate, recommendations for protection against light and/or nonconventional transmissible agents like those associated
humidity, should appear on containers, packages, and/or with Bovine Spongiform Encephalopathy (BSE) and scrapie.
package inserts. Such labeling should be in accordance with Applicants are encouraged to discuss issues associated with
relevant national and regional requirements. BSE with the regulatory authorities.
The scope of the document covers products derived from
cell cultures initiated from characterized cell banks. It covers
X. GLOSSARY (10) products derived from in vitro cell culture, such as interfer-
ons, monoclonal antibodies, and recombinant deoxy-
• Conjugated Product—A conjugated product is made up ribonucleic acid (DNA)-derived products including recombi-
of an active ingredient (e.g., peptide, carbohydrate) nant subunit vaccines, and also includes products derived
bound covalently or noncovalently to a carrier (e.g., from hybridoma cells grown in vivo as ascites. In this latter
protein, peptide, inorganic mineral) with the objective case, special considerations apply and additional information
of improving the efficacy or stability of the product. on testing cells propagated in vivo is contained in Appendix
• Degradation Product—A molecule resulting from a 1. Inactivated vaccines, all live vaccines containing self-repli-
change in the drug substance (bulk material) brought cating agents, and genetically engineered live vectors are
about over time. For the purpose of stability testing of excluded from the scope of this document.
the products described in this guideline, such changes The risk of viral contamination is a feature common to all
could occur as a result of processing or storage (e.g., biotechnology products derived from cell lines. Such con-
by deamidation, oxidation, aggregation, proteolysis). tamination could have serious clinical consequences and can
For biotechnological/biological products, some degrada- arise from the contamination of the source cell lines them-
tion products may be active. selves (cell substrates) or from adventitious introduction of
• Impurity—Any component of the drug substance (bulk virus during production. To date, however, biotechnology
material) or drug product (final container product) that products derived from cell lines have not been implicated in
is not the chemical entity defined as the drug sub- the transmission of viruses. Nevertheless, it is expected that
stance, an excipient, or other additives to the drug the safety of these products with regard to viral contamina-
product. tion can be reasonably assured only by the application of a
• Intermediate—For biotechnological/biological products, virus testing program and assessment of virus removal and
a material produced during a manufacturing process inactivation achieved by the manufacturing process, as out-
that is not the drug substance or the drug product but lined below.
for which manufacture is critical to the successful pro- Three principal, complementary approaches have evolved
duction of the drug substance or the drug product. to control the potential viral contamination of biotechnol-
Generally, an intermediate will be quantifiable and ogy products:

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