July 26, 2001

VETERINARY SERVICES MEMORANDUM NO. 800.300

Subject: Stability Testing of New Biotechnological/Biological

Veterinary Medicinal Products

To: Biologics Licensees, Permittees, and Applicants

Directors, Center for Veterinary Biologics

This guideline provides information and recommendations about the stability testing of

veterinary medicinal products (both drugs and biologics). It is the result of a collaborative

initiative by regulatory authorities and industry associations known as International

Cooperation on Harmonization of Technical Requirements for the Registration of

Veterinary Medicinal Products (VICH). One objective of VICH is to promote

harmonization of regulatory requirements for veterinary medicinal products by reducing the

differences in technical requirements for such products among regulatory agencies in

different countries. As a VICH member, APHIS is committed to seeking scientifically

based harmonized technical requirements for veterinary biological products. VICH has 

adopted this guideline as an international standard for the generation and submission of

stability data for veterinary drugs and biologics.

APHIS will consider the information and recommendations in this guideline as a basis for

the approval of stability studies conducted to establish and extend expiration dates for

applicable veterinary biological products under 9 CFR 114.13 and 114.4. Although this

guideline represents the agency’s current thinking on a standard for stability testing of

veterinary biologics as defined in this guideline, section II, scope, it does not confer rights

for, or on, any person and does not operate to bind APHIS or the public. An alternative

approach may be used if such approach satisfies the requirements of the applicable statute,

regulations, or both.

I. PREAMBLE (1)

The guidance stated in the VICH harmonized tripartite guidelines entitled “Stability Testing

of New Veterinary Drug Substances and Medicinal Products,” (GL3) applies in general to

new 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 well-characterized

proteins and/or polypeptides, maintenance of molecular conformation and, hence, of

biological activity, is dependent on nonequivalent 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 
Veterinary Services Memorandum No. 800.300 2

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 new biotechnological/biological product and consider many external conditions

that can affect the 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 applies to products composed of well-characterized

proteins and polypeptides, and their derivatives, 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, growth hormones and growth factors, insulins, monoclonal

antibodies, and those vaccines which consist of well-characterized proteins or

polypeptides even when chemically synthesized. This document does not cover

antibiotics, heparins, vitamins, cell metabolites, DNA products, allergenic extracts,

conventional vaccines, cells, whole blood, and 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 Veterinary Drug Substances and Medicinal 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. 
 Veterinary Services Memorandum No. 800.300 3

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 manufacture 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 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 overall quality of the batches of drug substance placed on formal stability studies

should be representative of the quality of the material used in pre clinical 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 (Finished Product) (4.3)

Stability information should be provided on at least three batches of finished product

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

of finished product included in stability testing should be derived from different batches of

bulk material. A minimum of six months data at the time of submission should be 
Veterinary Services Memorandum No. 800.300 4

submitted in cases where storage periods greater than six months are requested. For drug

products with storage periods of less than six 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 finished product placed on stability studies should be

representative of the quality of the material used in the pre clinical 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 authorities 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 long-term

stability specifications throughout the dating period or is not representative of the material

used in pre clinical 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.
Veterinary Services Memorandum No. 800.300 5

E. Container/Closure (4.5)

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 ampoules), stability

studies should include samples maintained in the inverted or horizontal position (i.e., in

contact with the closure), as well as in the upright position, to determine the effects of the

closure on product quality. Data should be supplied for all different container/closure

combinations that will be marketed.

V. STABILITY-INDICATING PROFILE (5)

On the whole, there is no single stability-indicating assay or parameter that profiles the

stability characteristic 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 that 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)

The dossier accompanying the application for marketing authorization should include a

detailed protocol for the assessment of the stability of both drug substance, when

applicable, 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 quantitative method. In general,

potencies of biotechnological/biological products tested by different laboratories 
Veterinary Services Memorandum No. 800.300 6

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 if possible.

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. In many cases, the validated in vivo potency

test will indicate that there has been no significant dissociation.

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 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 and necessary. Limits of acceptable

degradation should be derived from the analytical profiles of batches of the drug substance

and drug product used in the pre clinical 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 
Veterinary Services Memorandum No. 800.300 7

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 (SDS-polyacrylamide gel electrophoresis,

immunoelectrophoresis, Western Blot, isoelctrofocusing), 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 pre clinical 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.

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 affects 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.
Veterinary Services Memorandum No. 800.300 8

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 for 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 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.
Veterinary Services Memorandum No. 800.300 9

VII. USAGE CONDITIONS (7) 

A. Stability after First Opening or Reconstitution of Freeze-Dried Product (7.1)

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.

B. Multiple-Dose Vials (7.2)

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 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.

VIII. TESTING FREQUENCY (8)

The shelf lives of biotechnological/biological product 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.

When shelf lives of less than 1 year are expected, the real-time stability studies should be

conducted monthly for the first 3 months and at 3-month intervals thereafter. For products

with expected 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 pre-approval or

pre-license stage, reduced testing may be appropriate after approval or licensure where

data are available that demonstrate adequate stability. Where data exist that indicate the

stability of a product is not compromised, the applicant is encouraged to submit a protocol

that supports elimination of specific test intervals (e.g., 9-month testing) for

post-approval/post-licensure, long-term studies. If in vivo potency tests are part of the

stability protocol, the omission of some testing points for those tests should be justified.
Veterinary Services Memorandum No. 800.300 10

IX. SPECIFICATIONS (9)

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.

X. LABELING (10)

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.

XI. GLOSSARY (11)

A. 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.

B. Degradation Product

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.
Veterinary Services Memorandum No. 800.300 11

C. Impurity

Any component of the drug substance (bulk material) or drug product (finished product)

that is not the chemical entity defined as the drug substance, an excipient, or other additives

to the drug product.

D. Intermediate

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

process that is not the drug substance or the drug product but whose 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 the manufacturing process is

continued. This includes material that may undergo further molecular modification to be

held for an extended period before further processing.

E. Manufacturing Scale Production

Manufacture at the scale typically encountered in a facility intended for product production

for marketing.

F. 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.

 /s/

Alfonso Torres

Deputy Administrator

Veterinary Services