Guideline for Industry

Quality of Biotechnological

Products: Stability Testing of

Biotechnological/Biological

Products

July 1996

ICH Q5C
i

Table of Contents

I. INTRODUCTION (1) .................................................. 1

II. SCOPE OF THE ANNEX (2) ............................................ 2

III. TERMINOLOGY (3) .................................................. 2

IV. SELECTION OF BATCHES (4) .......................................... 2

A. Drug Substance (Bulk Material) (4.1) ................................. 2

B. Intermediates (4.2) ............................................... 3

C. Drug Product (Final Container Product) (4.3) ........................... 3

D. Sample Selection (4.4) ............................................ 4

V. STABILITY-INDICATING PROFILE (5) .................................. 4

A. Protocol (5.1) ................................................... 5

B. Potency (5.2) ................................................... 5

C. Purity and Molecular Characterization (5.3) ............................ 6

D. Other Product Characteristics (5.4) ................................... 6

VI. STORAGE CONDITIONS (6) ........................................... 7

A. Temperature (6.1) ............................................... 7

B. Humidity (6.2) .................................................. 7

C. Accelerated and Stress Conditions (6.3) ............................... 7

D. Light (6.4) ..................................................... 8

E. Container/Closure (6.5) ........................................... 8

F. Stability after Reconstitution of Freeze-Dried Product (6.6) ................ 8

VII. TESTING FREQUENCY (7) ............................................ 8

VIII. SPECIFICATIONS (8) ................................................. 9

IX. LABELING (9) ....................................................... 9

X. GLOSSARY (10) .................................................... 10
This guideline was developed within the Expert Working Group (Quality) of the International Conference on 1

Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) and has been

subject to consultation by the regulatory parties, in accordance with the ICH process. 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

draft is recommended for adoption to the regulatory bodies of the European Union, Japan and the USA. This guideline

was published in the Federal Register on July 10, 1996 (61 FR 36466) and is applicable to drug and 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 industry, it does represent the agency’s current thinking on stability testing of biotechnological/biological

products. For additional copies of this guideline, contact the Drug Information Branch, HFD-210, CDER, FDA, 5600

Fishers Lane, Rockville, MD 20857 (Phone: 301-827-4573) or the Manufacturers Assistance and Communication Staff

(HFM-42), CBER, FDA, 1401 Rockville Pike, Rockville, MD 20852-1448. Send one self-addressed adhesive label to

assist the offices in processing your request. An electronic version of this guidance is also available via Internet using

the World Wide Web (WWW) (connect to the CDER Home Page at http://www.fda.gov/cder and go to the “Regulatory

Guidance” section).

GUIDELINE FOR INDUSTRY 1

QUALITY OF BIOTECHNOLOGICAL PRODUCTS: STABILITY

TESTING OF BIOTECHNOLOGICAL/BIOLOGICAL

PRODUCTS

I. INTRODUCTION (1)

The guidance stated in the ICH harmonized tripartite guideline entitled “Stability Testing of New

Drug Substances and Products” (issued by ICH on October 27, 1993) applies in general to

biotechnological/biological products. However, biotechnological/biological products 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. 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

physicochemical, 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 these concerns in mind, the applicant should develop the proper supporting stability data for

a biotechnological/biological product and consider many external conditions that can affect the
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product's potency, purity, and quality. Primary data to support a requested storage period for

either drug substance or drug product should be based on long-term, real-time, real-condition

stability studies. Thus, the development of a proper long-term 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.

II. SCOPE OF THE ANNEX (2)

The guidance stated in this annex to “Stability Testing of New Drug Substances and Products”

applies to well-characterized 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 recombinant deoxyribonucleic acid (r-DNA) technology. Thus, the document covers the

generation and submission of stability data for products such as cytokines (interferons,

interleukins, colony-stimulating factors, tumor necrosis factors), erythropoietins, plasminogen

activators, blood plasma factors, growth hormones and growth factors, insulins, monoclonal

antibodies, and vaccines consisting of well-characterized proteins or polypeptides. In addition,

the guidance outlined in the following sections may apply to other types of products, such as

conventional vaccines, after consultation with the appropriate regulatory authorities. The

document does not cover antibiotics, allergenic extracts, heparins, vitamins, whole blood, or

cellular blood components.

III. TERMINOLOGY (3)

For the basic terms used in this annex, the reader is referred to the “Glossary” in “Stability Testing

of New Drug Substances and Products.” However, because 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.

IV. SELECTION OF BATCHES (4)

A. Drug Substance (Bulk Material) (4.1)

Where bulk material is to be stored after manufacture, but before formulation and final

manufacturing, stability data should be provided on at least three batches for which

manufacture and storage are representative of the manufacturing scale of production. A

minimum of 6 months stability data at the time of submission should be submitted in cases

where storage periods greater than 6 months are requested. For drug substances with

storage periods of less than 6 months, the minimum amount of stability data in the initial

submission should be determined on a case-by-case basis. Data from pilot-plant scale

batches of drug substance produced at a reduced scale of fermentation and purification
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may be provided at the time the dossier is submitted to the regulatory agencies with a

commitment to place the first three manufacturing scale batches into the long-term

stability program after approval.

The quality of the batches of drug substance placed into the stability program should be

representative of the quality of the material used in preclinical and clinical studies and of

the quality of the material to be made at manufacturing scale. In addition, the drug

substance (bulk material) made at pilot-plant scale should be produced by a process and

stored under conditions representative of that used for the manufacturing scale. The drug

substance entered into the stability program should be stored in containers that properly

represent the actual holding containers used during manufacture. Containers of reduced

size may be acceptable for drug substance stability testing provided that they are

constructed of the same material and use the same type of container/closure system that is

intended to be used during manufacture.

B. Intermediates (4.2)

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. Although the use of

pilot-plant scale data is permissible, the manufacturer should establish the suitability of

such data using the manufacturing scale process.

C. Drug Product (Final Container Product) (4.3)

Stability information should be provided on at least three batches of final container

product representative of that which will be used at manufacturing scale. Where possible,

batches of final container product included in stability testing should be derived from

different batches of bulk material. A minimum of 6 months data at the time of submission

should be submitted in cases where storage periods greater than 6 months are requested. 

For drug products with storage periods of less than 6 months, the minimum amount of

stability data in the initial submission should be determined on a case-by-case basis. 

Product expiration dating should be based upon the actual data submitted in support of the

application. Because dating is based upon the real-time/real-temperature data submitted

for review, continuing updates of initial stability data should occur during the review and

evaluation process. The quality of the final container product placed on stability studies

should be representative of the quality of the material used in the preclinical and clinical

studies. Data from pilot-plant scale batches of drug product may be provided at the time

the dossier is submitted to the regulatory agencies with a commitment to place the first

three manufacturing scale batches into the long-term stability program after approval. 

Where pilot-plant scale batches were submitted to establish the dating for a product and,

in the event that the product produced at manufacturing scale does not meet those
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long-term stability specifications throughout the dating period or is not representative of

the material used in preclinical and clinical studies, the applicant should notify the

appropriate regulatory authorities to determine a suitable course of action.

D. Sample Selection (4.4)

Where one product is distributed in batches differing in fill volume (e.g., 1 milliliter (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 drug 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.

V. STABILITY-INDICATING PROFILE (5)

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 emphasized in the following

subsections are not intended to be all-inclusive, but represent product characteristics that should

typically be documented to demonstrate product stability adequately.

A. Protocol (5.1)
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The dossier accompanying the application for marketing authorization should include a

detailed protocol for the assessment of the stability of both drug substance and drug

product in support of the proposed storage conditions and expiration dating periods. The

protocol should include all necessary information that 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.

B. Potency (5.2)

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 in vivo or in vitro

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 recognized standards. Where no

national or international reference standards exist, the assay results may be reported in

in-house derived units using a characterized reference material.

In some biotechnological/biological products, potency is dependent upon the conjugation

of the active ingredient(s) to a second moiety or binding to an adjuvant. Dissociation of

the active ingredient(s) from the carrier used in conjugates or adjuvants should be

examined in real-time/real-temperature studies (including conditions encountered during

shipment). The assessment of the stability of such products may be difficult because, in

some cases, in vitro tests for biological activity and physicochemical characterization are

impractical or provide inaccurate results. Appropriate strategies (e.g., testing the product

before 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.

C. Purity and Molecular Characterization (5.3)

For the purpose of stability testing of the products described in this guideline, purity is a

relative term. Because of the effect of glycosylation, deamidation, or other

heterogeneities, the absolute purity of a biotechnological/biological product is extremely
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difficult to determine. Thus, the purity 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 testing, tests for purity should focus on

methods for determination of degradation products.

The degree of purity, as well as the individual and total amounts of degradation products

of the biotechnological/biological product entered into the stability studies, should be

reported and documented whenever possible. Limits of acceptable degradation should be

derived from the analytical profiles of batches of the drug substance and drug product

used in the preclinical and clinical studies.

The use of relevant physicochemical, biochemical, and immunochemical analytical

methodologies should permit a comprehensive characterization of the drug substance

and/or drug product (e.g., molecular size, charge, hydrophobicity) and the accurate

detection of degradation changes that may result from deamidation, oxidation,

sulfoxidation, aggregation, or fragmentation during storage. As examples, methods that

may contribute to this include electrophoresis (SDS09Page, immunoelectrophoresis,

Western blot, isoelectrofocusing), high-resolution chromatography (e.g., reversed-phase

chromatography, gel filtration, ion exchange, affinity chromatography), and peptide

mapping.

Wherever significant qualitative or quantitative changes indicative of degradation product

formation are detected during long-term, accelerated, and/or stress stability studies,

consideration should be given to potential hazards and to the need for characterization and

quantification of degradation products within the long-term stability program. Acceptable

limits should be proposed and justified, taking into account the levels observed in material

used in preclinical and clinical studies.

For substances that cannot be properly characterized or products for which an exact

analysis of the purity cannot be determined through routine analytical methods, the

applicant should propose and justify alternative testing procedures.

D. Other Product Characteristics (5.4)

The following product characteristics, though not specifically relating to

biotechnological/biological products, should be monitored and reported for the drug

product in its final container:

Visual appearance of the product (color and opacity for solutions/suspensions; color,

texture, and dissolution time for powders), visible particulates in solutions or after the

reconstitution of powders or lyophilized cakes, pH, and moisture level of powders and

lyophilized products.
7

Sterility testing or alternatives (e.g., container/closure integrity testing) should be

performed at a minimum initially and at the end of the proposed shelf life.

Additives (e.g., stabilizers, preservatives) or excipients may degrade during the dating

period of the drug product. If there is any indication during preliminary stability studies

that reaction or degradation of such materials adversely affect the quality of the drug

product, these items may need to be monitored during the stability program.

The container/closure has the potential to affect the product adversely and should be

carefully evaluated (see below).

VI. STORAGE CONDITIONS (6)

A. Temperature (6.1)

Because 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.

B. Humidity (6.2)

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.

 

C. Accelerated and Stress Conditions (6.3)

As previously noted, the expiration dating should be based on real-time/real-temperature

data. However, it is strongly suggested that studies be conducted on the drug substance

and drug product under accelerated and stress conditions. Studies under accelerated

conditions may provide useful support data for establishing the expiration date, provide

product stability information or future product development (e.g., preliminary assessment

of proposed manufacturing changes such as change in formulation, scale-up), assist in

validation of analytical methods for the stability program, or generate information that may

help elucidate the degradation profile of the drug substance or drug product. Studies

under stress conditions may be useful in determining whether accidental exposures to

conditions other than those proposed (e.g., during transportation) are deleterious to the

product and also for evaluating which specific test parameters may be the best indicators

of product stability. Studies of the exposure of the drug substance or drug product to

extreme conditions may help to reveal patterns of degradation; if so, such changes should

be monitored under proposed storage conditions. Although the tripartite guideline on
8

stability describes the conditions of the accelerated and stress study, the applicant should

note that those conditions may not be appropriate for biotechnological/biological

products. Conditions should be carefully selected on a case-by-case basis.

D. Light (6.4)

Applicants should consult the appropriate regulatory authorities on a case-by-case basis to

determine guidance for testing.

E. Container/Closure (6.5)

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 ampules), 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 labeling should

be in accordance with relevant national/regional requirements.

F. Stability after Reconstitution of Freeze-Dried Product (6.6)

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 labeling should be in accordance with relevant national/regional

requirements.

VII. TESTING FREQUENCY (7)

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 5 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.
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When shelf lives of 1 year or less are proposed, the real-time stability studies should be conducted

monthly for the first 3 months and at 3 month intervals thereafter. For products with proposed

shelf lives of greater than 1 year, the studies should be conducted every 3 months during the first

year of storage, every 6 months during the second year, and annually thereafter.

While the testing intervals listed above may be appropriate in the preapproval or prelicense 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 that supports elimination of

specific test intervals (e.g., 9-month testing) for postapproval/postlicensure, long-term studies.

VIII. SPECIFICATIONS (8)

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 respect to distinct release and end of shelf life specifications. 

Recommendations for maximum acceptable losses of activity, limits for physicochemical changes,

or degradation during the proposed shelf life have not been developed for individual types or

groups of biotechnological/biological products but are considered on a case-by-case basis. Each

product should retain its specifications within established limits for safety, purity, and potency

throughout its proposed shelf life. These specifications and limits should be derived from all

available information using the appropriate statistical methods. The use of different specifications

for release 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.

IX. LABELING (9)

For most biotechnological/biological drug substances and drug products, precisely defined storage

temperatures are recommended. Specific recommendations should be stated, particularly for drug

substances and drug products that cannot tolerate freezing. These conditions, and where

appropriate, recommendations for protection against light and/or humidity, should appear on

containers, packages, and/or package inserts. Such labeling should be in accordance with relevant

national and regional requirements.

X. GLOSSARY (10)

Conjugated Product

A conjugated product is made up of an active ingredient (e.g., peptide, carbohydrate)

bound covalently or noncovalently to a carrier (e.g., protein, peptide, inorganic mineral)

with the objective of improving the efficacy or stability of the product.

Degradation Product
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A molecule resulting from a change in the drug 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 drug substance (bulk material) or drug product (final container

product) that is not the chemical entity defined as the drug substance, an excipient, or

other additives to the drug product.

Intermediate

For biotechnological/biological products, a material produced during a manufacturing

process that is not the drug substance or the drug product but for which manufacture is

critical to the successful production of the drug substance or the drug product. Generally,

an intermediate will be quantifiable and specifications will be established to determine the

successful completion of the manufacturing step before continuation of the manufacturing

process. This includes material that may undergo further molecular modification or be

held for an extended period before 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 drug substance or drug 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.