Guidance for Industry

Q6B Specifications: Test Procedures

and Acceptance Criteria for

Biotechnological/Biological

Products

U.S. Department of Health and Human Services

Food and Drug Administration

Center for Drug Evaluation and Research (CDER)

Center for Biologics Evaluation and Research (CBER)

August 1999

ICH
Guidance for Industry

Q6B Specifications: Test Procedures

and Acceptance Criteria for

Biotechnological/Biological

Products

Additional copies are available from:

Office of Training and Communications

Division of Drug Information, HFD-240

Center for Drug Evaluation and Research

Food and Drug Administration

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Rockville, MD 20857

(Tel) 301-827-4573

http://www.fda.gov/cder/guidance/index.htm

or

 Office of Communication, Training and

Manufacturers Assistance, HFM-40

Center for Biologics Evaluation and Research

 Food and Drug Administration

1401 Rockville Pike, Rockville, MD 20852-1448

 http://www.fda.gov/cber/guidelines.htm

Fax: 1-888-CBERFAX or 301-827-3844

Phone: the Voice Information System at 800-835-4709 or 301-827-1800

U.S. Department of Health and Human Services

Food and Drug Administration

Center for Drug Evaluation and Research (CDER)

Center for Biologics Evaluation and Research (CBER)

August 1999

ICH
TABLE OF CONTENTS

I. OBJECTIVES OF THIS DOCUMENT (1.0)..................................................................... 1

A. Objective (1.1).......................................................................................................................1

B. Background (1.2) ..................................................................................................................1

C. Scope (1.3) ............................................................................................................................2

II. PRINCIPLES FOR CONSIDERATION IN SETTING SPECIFICATIONS (2.0)........ 2

A. Characterization (2.1)...........................................................................................................2

B. Analytical Considerations (2.2).............................................................................................7

C. Process Controls (2.3) ...........................................................................................................8

D. Pharmacopoeial Specifications (2.4)......................................................................................9

E. Release Limits Versus Shelf-Life Limits (2.5)........................................................................9

F. Statistical Concepts (2.6).....................................................................................................10

III. JUSTIFICATION OF THE SPECIFICATION (3.0)...................................................... 10

IV. SPECIFICATIONS (4.0).................................................................................................... 11

A. Drug Substance Specification (4.1)......................................................................................11

B. Drug Product Specification (4.2).........................................................................................13

GLOSSARY (5.0)........................................................................................................................ 15

APPENDICES (6.0) .................................................................................................................... 17

A. Appendix for Physiochemical Characterization (6.1)..........................................................17

B. Appendix for Impurities (6.2)..............................................................................................20
1

GUIDANCE FOR INDUSTRY1

Q6B Specifications: Test Procedures and Acceptance Criteria for

Biotechnological/Biological Products

This guidance represents the Food and Drug Administration's (FDA's) current thinking on this topic. It

does not create or confer any rights for or on any person and does not operate to bind FDA or the public.

An alternative approach may be used if such approach satisfies the requirements of the applicable statutes

and regulations.

I. OBJECTIVES OF THIS DOCUMENT (1.0)2

A. Objective (1.1)

This guidance document provides guidance on general principles for 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.

B. Background (1.2)

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.

 

1

 This guidance was developed within the Expert Working Group (Quality) of the International Conference on

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, March 10, 1999. At Step 4 of the process,

the final draft is recommended for adoption to the regulatory bodies of the European Union, Japan, and the United

States. This guidance was published in the Federal Register on August 18, 1999 (62 FR 44928), and is applicable to

drug and biological products.

2

 Arabic numbers reflect the organizational breakdown in the document endorsed by the ICH Steering Committee at

Step 4 of the ICH process, March 10, 1999.
2

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, inprocess 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.

C. Scope (1.3)

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

nonrecombinant 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, deoxyribonucleic acid (DNA) products, allergenic extracts,

conventional vaccines, cells, whole blood, and cellular blood components. A separate

ICH draft guidance, Q6A Specifications: Test Procedures and Acceptance Criteria for

New Drug Substances and New Drug Products: Chemical 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.

II. PRINCIPLES FOR CONSIDERATION IN SETTING SPECIFICATIONS (2.0)

A. Characterization (2.1)

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, data from stability

studies, and relevant development data.
3

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 inhouse 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.

1. Physicochemical Properties (2.1.1)

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 II.A.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 ensure lot-to-lot consistency. When these variants of the desired product have

properties comparable to those of the desired product with respect to activity,

efficacy, and safety, they are considered product-related substances. When

process changes and degradation products result in heterogeneity patterns that

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

A. 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 IV), an appropriate subset of these methods

should be selected and justified.
4

2. Biological Activity (2.1.2)

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; and

• 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 that 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 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
5

• 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 IV), the choice of relevant quantitative

assay (biological and/or physicochemical) should be justified by the

manufacturer.

3. Immunochemical Properties (2.1.3)

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., enzyme linked

immunosorbent assay (ELISA), Western-blot) utilizing antibodies that 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.

4. Purity, Impurities, and Contaminants (2.1.4)

• 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 milligram 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
6

desired product, they are considered product-related substances and not impurities

(section II.A.1).

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

should be set, as appropriate.

For the purpose of lot release (section IV), 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.B). Product-related impurities (e.g.,

precursors, certain degradation products) are molecular variants arising during

manufacture and/or storage that 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 II.C).

Examples of analytical procedures that may be employed to test for the presence

of impurities are listed in appendix B. 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 IV), an appropriate subset of these methods

should be selected and justified.
7

• 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 II.C). For the special case of adventitious

viral or mycoplasma contamination, the concept of action limits is not applicable,

and the strategies proposed in ICH guidances Q5A Quality of

Biotechnological/Biological Products: Viral Safety Evaluation of Biotechnology

Products Derived from Cell Lines of Human or Animal Origin and Q5D Quality

of Biotechnological/Biological Products: Derivation and Characterization of Cell

Substrates Used for Production of Biotechnological/Biological Products should

be considered.

5. Quantity (2.1.5)

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.

B. Analytical Considerations (2.2)

1. Reference Standards and Reference Materials (2.2.1)

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 processrelated 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.
8

2. Validation of Analytical Procedures (2.2.2)

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 guidances Q2A Validation of Analytical Procedures:

Definitions and Terminology and Q2B Validation of Analytical Procedures:

Methodology, except where there are specific issues for unique tests used for

analyzing biotechnological and biological products.

C. Process Controls (2.3)

1. Process-Related Considerations (2.3.1)

Adequate design of a process and knowledge of its capability are part of the

strategy used to develop a manufacturing process that 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 commercialscale 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 may, therefore,

sometimes be implemented after marketing authorization, in accordance with

regional regulations.

2. In-Process Acceptance Criteria and Action Limits (2.3.2)

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 II.C.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
9

action. They should be further refined as additional manufacturing experience

and data are obtained after product approval.

3. Raw Materials and Excipient Specifications (2.3.3)

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 that 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 nonpharmacopoeial

excipients.

D. Pharmacopoeial Specifications (2.4)

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 harmonization of

the analytical procedures of the pharmacopoeias. The pharmacopoeias are committed to

developing identical or methodologically equivalent test procedures and acceptance

criteria.

E. Release Limits Versus Shelf-Life Limits (2.5)

The concept of release limits versus 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 shelflife limits.
10

F. Statistical Concepts (2.6)

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.

III. JUSTIFICATION OF THE SPECIFICATION (3.0)

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 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 guidance Q5C 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 preclinical and clinical studies.

The quality of the material made at commercial scale should be representative of the lots used in

preclinical and clinical studies.
11

• Specifications are linked to analytical procedures.

Critical quality attributes may include items such as potency, the nature and quantity of productrelated 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.

IV. SPECIFICATIONS (4.0)

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, data from

stability studies, and relevant development data.

In some cases, testing at production stages rather than testing at the finished drug substance or

drug product stages may be appropriate and acceptable. In such 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.

A. Drug Substance Specification (4.1)

Generally, the following tests and acceptance criteria are considered applicable to all

drug substances (for analytical procedures, see section II.B.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.

1. Appearance and Description (4.1.1)

A qualitative statement describing the physical state (e.g., solid, liquid) and color

of a drug substance should be provided.

2. Identity (4.1.2)

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 II.A and in appendix A may be employed and/or modified as

appropriate for the purpose of establishing identity.
12

3. Purity and Impurities (4.1.3)

The absolute purity of biotechnological and biological products is difficult to

determine and the results are method dependent (section II.A.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 II.A.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, wellcontrolled manufacturing processes.

• Product-related impurities (section II.A.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 necessary (section II.C).

4. Potency (4.1.4)

A relevant, validated potency assay (section II.A.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

IV.B.4), an alternative method (physicochemical and/or biological) may suffice

for quantitative assessment at the drug substance stage. In some cases, the

measurement of specific activity may provide additional useful information.

5. Quantity (4.1.5)

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.
13

B. Drug Product Specification (4.2)

Generally, the following tests and acceptance criteria are considered applicable to all

drug products. The sections (IV.B.1 to IV.B.5) are cross-referenced to respective

sections (IV.A.1 to IV.A.5) under Drug Substance Specification. 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 are set.

1. Appearance and Description (4.2.1)

A qualitative statement describing the physical state (e.g., solid, liquid), color, and clarity

of the drug product should be provided.

2. Identity (4.2.2)

The identity test(s) should be highly specific for the drug product and should be based on

unique aspects of its molecular structure and 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 II.A and in appendix A,

may be employed and/or modified as appropriate for the purpose of establishing identity.

3. Purity and Impurities (4.2.3)

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 considered 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.

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.
14

4. Potency (4.2.4)

A relevant, validated potency assay (section II.A.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.

5. Quantity (4.2.5)

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.

6. General Tests (4.2.6)

Physical description and the measurement of other quality attributes are often

important for the evaluation of the drug product functions. Examples of such tests

include pH and osmolarity.

7. Additional Testing for Unique Dosage Forms (4.2.7)

It should be recognized that certain unique dosage forms may need additional

tests other than those mentioned above.
15

GLOSSARY (5.0)

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 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 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 that has the expected structure, or (2) the protein that 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 that 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 that is not the desired

product, a product-related substance, or an 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
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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 (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.

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: International or national standards.

Specification: 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.
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APPENDICES (6.0)

A. Appendix for Physiochemical Characterization (6.1)

This appendix provides examples of technical approaches that 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.

1. Structural Characterization and Confirmation (6.1.1)

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.

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 high pressure liquid chromatography (HPLC) or other appropriate analytical

procedures. The peptide fragments should be identified to the extent possible
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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 nonreducing 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 are analyzed, to the extent possible.

2. Physicochemical Properties (6.1.2)

a. Molecular weight or size

Molecular weight (or size) is determined using size exclusion chromatography,

sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (under reducing

and/or nonreducing 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 ultraviolet (UV)/visible

wavelength (e.g., 280 nanometers). The extinction coefficient is determined 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. If UV absorption is used to measure protein

content, the extinction coefficient for the particular product should be used.
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d. Electrophoretic patterns

Electrophoretic patterns and data on identity, homogeneity, and purity can be

obtained by polyacrylamide gel electrophoresis, isoelectric focusing, SDSpolyacrylamide 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 UV and visible absorption spectra are determined as appropriate. The higherorder structure of the product is examined using procedures such as circular

dichroism, nuclear magnetic resonance (NMR), or other suitable techniques as

appropriate.
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B. Appendix for Impurities (6.2)

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.

1. Process-Related Impurities and Contaminants (6.2.1)

These are derived from the manufacturing process (section II.A.4) and are classified into

three major categories: Cell substrate-derived, cell culture-derived and downstreamderived.

a. Cell substrate-derived impurities include, but are not limited to, proteins

derived from the host organism and 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 substratederived impurities such as nucleic acids and host cell proteins may

sometimes be used to eliminate the need for establishing acceptance

criteria for these impurities.

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, nonmetallic 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 guidance Q5A Viral Safety

Evaluation of Biotechnology Products Derived From Cell Lines of Human

or Animal Origin.
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2. Product-Related Impurities Including Degradation Products (6.2.2)

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 in significant amounts during manufacture

and/or storage 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 SDSPAGE. 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).