[Federal Register: August 18, 1999 (Volume 64, Number 159)]
[Notices]               
[Page 44928-44935]
From the Federal Register Online via GPO Access [wais.access.gpo.gov]
[DOCID:fr18au99-73]                         

=======================================================================
-----------------------------------------------------------------------

DEPARTMENT OF HEALTH AND HUMAN SERVICES

Food and Drug Administration
[Docket No. 98D-0374]

 
International Conference on Harmonisation; Guidance on 
Specifications: Test Procedures and Acceptance Criteria for 
Biotechnological/Biological Products

AGENCY: Food and Drug Administration, HHS.

ACTION: Notice.

-----------------------------------------------------------------------

SUMMARY: The Food and Drug Administration (FDA) is publishing a 
guidance entitled ``Q6B Specifications: Test Procedures and Acceptance 
Criteria for Biotechnological/Biological Products.'' The guidance was 
prepared under the auspices of the International Conference on 
Harmonisation of Technical Requirements for Registration of 
Pharmaceuticals for Human Use (ICH). The guidance provides guidance on 
general principles for the selection of test procedures and the setting 
and justification of acceptance criteria for biotechnological and 
biological products. The guidance is intended to assist in the 
establishment of a uniform set of international specifications for 
biotechnological and biological products to support new marketing 
applications.

DATES: Effective August 18, 1999. Submit written comments at any time.

ADDRESSES: Submit written comments on the guidance to the Dockets 
Management Branch (HFA-305), Food and Drug Administration, 5630 Fishers 
Lane, rm. 1061, Rockville, MD 20852. Copies of the guidance are 
available from the Drug Information Branch (HFD-210), Center for Drug 
Evaluation and Research, Food and Drug Administration, 5600 Fishers 
Lane, Rockville, MD 20857, 301-827-4573. Single copies of the guidance 
may be obtained by mail from the Office of Communication, Training and 
Manufacturers Assistance (HFM-40), Center for Biologics Evaluation and 
Research (CBER), 1401 Rockville Pike, Rockville, MD 20852, or by 
calling the CBER Voice Information System at 1-800-835-4709 or 301-827-
1800. Copies may be obtained from CBER's FAX Information System at 1-
888-CBER-FAX or 301-827-3844.

FOR FURTHER INFORMATION CONTACT: 
    Neil D. Goldman, Center for Biologics Evaluation and Research (HFM-
20), Food and Drug Administration, 1401 Rockville Pike, Rockville, MD 
20852, 301-827-0377.

[[Page 44929]]

    Regarding ICH: Janet J. Showalter, Office of Health Affairs (HFY-
20), Food and Drug Administration, 5600 Fishers Lane, Rockville, MD 
20857, 301-827-0864.

SUPPLEMENTARY INFORMATION: In recent years, many important initiatives 
have been undertaken by regulatory authorities and industry 
associations to promote international harmonization of regulatory 
requirements. FDA has participated in many meetings designed to enhance 
harmonization and is committed to seeking scientifically based 
harmonized technical procedures for pharmaceutical development. One of 
the goals of harmonization is to identify and then reduce differences 
in technical requirements for drug development among regulatory 
agencies.
    ICH was organized to provide an opportunity for tripartite 
harmonization initiatives to be developed with input from both 
regulatory and industry representatives. FDA also seeks input from 
consumer representatives and others. ICH is concerned with 
harmonization of technical requirements for the registration of 
pharmaceutical products among three regions: The European Union, Japan, 
and the United States. The six ICH sponsors are the European 
Commission, the European Federation of Pharmaceutical Industries 
Associations, the Japanese Ministry of Health and Welfare, the Japanese 
Pharmaceutical Manufacturers Association, the Centers for Drug 
Evaluation and Research and Biologics Evaluation and Research, FDA, and 
the Pharmaceutical Research and Manufacturers of America. The ICH 
Secretariat, which coordinates the preparation of documentation, is 
provided by the International Federation of Pharmaceutical 
Manufacturers Associations (IFPMA).
    The ICH Steering Committee includes representatives from each of 
the ICH sponsors and the IFPMA, as well as observers from the World 
Health Organization, the Canadian Health Protection Branch, and the 
European Free Trade Area.
    In the Federal Register of June 9, 1998 (63 FR 31506), FDA 
published a draft tripartite guidance entitled ``Q6B Specifications: 
Test Procedures and Acceptance Criteria for Biotechnological/Biological 
Products.'' The notice gave interested persons an opportunity to submit 
comments by July 24, 1998.
    After consideration of the comments received and revisions to the 
guidance, a final draft of the guidance was submitted to the ICH 
Steering Committee and endorsed by the three participating regulatory 
agencies on March 11, 1999.
    In accordance with FDA's good guidance practices (62 FR 8961, 
February 27, 1997), this document has been designated a guidance, 
rather than a guideline.
    The guidance provides guidance on general principles for the 
selection of test procedures and the setting and justification of 
acceptance criteria for biotechnological and biological products. The 
guidance is intended to assist in the establishment of a uniform set of 
international specifications for biotechnological and biological 
products to support new marketing applications.
    This guidance represents the agency's current thinking on the 
selection of test procedures and the setting and justification of 
acceptance criteria for biotechnological and biological products. 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 statute, 
regulations, or both.
    As with all of FDA's guidances, the public is encouraged to submit 
written comments with new data or other new information pertinent to 
this guidance. The comments in the docket will be periodically 
reviewed, and, where appropriate, the guidance will be amended. The 
public will be notified of any such amendments through a notice in the 
Federal Register.
    Interested persons may, at any time, submit written comments on the 
guidance to the Dockets Management Branch (address above). Two copies 
of any comments are to be submitted, except that individuals may submit 
one copy. Comments are to be identified with the docket number found in 
brackets in the heading of this document. The guidance and received 
comments may be seen in the office above between 9 a.m. and 4 p.m., 
Monday through Friday. An electronic version of this guidance is 
available on the Internet at ``http://www.fda.gov/cder/guidance/
index.htm'' or at CBER's World Wide Web site at ``http://www.fda.gov/
cber/publications.htm''.
    The text of the guidance follows:

Q6B Specifications: Test Procedures and Acceptance Criteria for 
Biotechnological/Biological Products\1\
---------------------------------------------------------------------------

    \1\This guidance represents the agency's current thinking on the 
selection of test procedures and the setting and justification of 
acceptance criteria for biotechnological and biological products. 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 statute, regulations, or both.
---------------------------------------------------------------------------

Table of Contents

1.0 Introduction
    1.1 Objective
    1.2 Background
    1.3 Scope
2.0 Principles for Consideration in Setting Specifications
    2.1 Characterization
    2.1.1 Physicochemical Properties
    2.1.2 Biological Activity
    2.1.3 Immunochemical Properties
    2.1.4 Purity, Impurities and Contaminants
    2.1.5 Quantity
    2.2 Analytical Considerations
    2.2.1 Reference Standards and Reference Materials
    2.2.2 Validation of Analytical Procedures
    2.3 Process Controls
    2.3.1 Process-Related Considerations
    2.3.2 In-process Acceptance Criteria and Action Limits
    2.3.3 Raw Materials and Excipient Specifications
    2.4 Pharmacopoeial Specifications
    2.5 Release Limits versus Shelf-Life Limits
    2.6 Statistical Concepts
3.0 Justification of the Specification
4.0 Specifications
    4.1 Drug Substance Specification
    4.1.1 Appearance and Description
    4.1.2 Identity
    4.1.3 Purity and Impurities
    4.1.4 Potency
    4.1.5 Quantity
    4.2 Drug Product Specification
    4.2.1 Appearance and Description
    4.2.2 Identity
    4.2.3 Purity and Impurities
    4.2.4 Potency
    4.2.5 Quantity
    4.2.6 General Tests
    4.2.7 Additional Testing for Unique Dosage Forms
5.0 Glossary
6.0 Appendices
    6.1 Appendix for Physicochemical Characterization
    6.1.1 Structural Characterization and Confirmation
    6.1.2 Physicochemical Properties
    6.2 Appendix for Impurities
    6.2.1 Process-Related Impurities and Contaminants
    6.2.2 Product-Related Impurities Including Degradation Products

1.0  Introduction

1.1  Objective

    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.

1.2  Background

    A specification is defined as a list of tests, references to 
analytical procedures, and appropriate acceptance criteria which are 
numerical limits, ranges, or other criteria for the tests described. 
It establishes the set of

[[Page 44930]]

criteria to which a drug substance, drug product, or materials at 
other stages of its manufacture should conform to be considered 
acceptable for its intended use. ``Conformance to specification'' 
means that the drug substance and drug product, when tested 
according to the listed analytical procedures, will meet the 
acceptance criteria. Specifications are critical quality standards 
that are proposed and justified by the manufacturer and approved by 
regulatory authorities as conditions of approval.
    Specifications are one part of a total control strategy designed 
to ensure product quality and consistency. Other parts of this 
strategy include thorough product characterization during 
development, upon which many of the specifications are based, 
adherence to good manufacturing practices, a validated manufacturing 
process, raw materials testing, in-process testing, stability 
testing, etc.
    Specifications are chosen to confirm the quality of the drug 
substance and drug product rather than to establish full 
characterization and should focus on those molecular and biological 
characteristics found to be useful in ensuring the safety and 
efficacy of the product.

1.3  Scope

    The principles adopted and explained in this document apply to 
proteins and polypeptides, their derivatives, and products of which 
they are components (e.g., conjugates). These proteins and 
polypeptides are produced from recombinant or 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.

2.0 Principles for Consideration in Setting Specifications

2.1 Characterization

    Characterization of a biotechnological or biological product 
(which includes the determination of physicochemical properties, 
biological activity, immunochemical properties, purity, and 
impurities) by appropriate techniques is necessary to allow relevant 
specifications to be established. Acceptance criteria should be 
established and justified based on data obtained from lots used in 
preclinical and/or clinical studies, data from lots used for 
demonstration of manufacturing consistency, data from stability 
studies, and relevant development data.
    Extensive characterization is performed in the development phase 
and, where necessary, following significant process changes. At the 
time of submission, the product should have been compared with an 
appropriate reference standard, if available. When feasible and 
relevant, it should be compared with its natural counterpart. Also, 
at the time of submission, the manufacturer should have established 
appropriately characterized in-house reference materials which will 
serve for biological and physicochemical testing of production lots. 
New analytical technology and modifications to existing technology 
are continually being developed and should be utilized when 
appropriate.

2.1.1 Physicochemical properties

    A physicochemical characterization program will generally 
include a determination of the composition, physical properties, and 
primary structure of the desired product. In some cases, information 
regarding higher-order structure of the desired product (the 
fidelity of which is generally inferred by its biological activity) 
may be obtained by appropriate physicochemical methodologies.
    An inherent degree of structural heterogeneity occurs in 
proteins due to the biosynthetic processes used by living organisms 
to produce them; therefore, the desired product can be a mixture of 
anticipated post-translationally modified forms (e.g., glycoforms). 
These forms may be active and their presence may have no deleterious 
effect on the safety and efficacy of the product (section 2.1.4). 
The manufacturer should define the pattern of heterogeneity of the 
desired product and demonstrate consistency with that of the lots 
used in preclinical and clinical studies. If a consistent pattern of 
product heterogeneity is demonstrated, an evaluation of the 
activity, efficacy, and safety (including immunogenicity) of 
individual forms may not be necessary.
    Heterogeneity can also be produced during manufacture and/or 
storage of the drug substance or drug product. Since the 
heterogeneity of these products defines their quality, the degree 
and profile of this heterogeneity should be characterized to 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 6.1. New analytical technology and modifications 
to existing technology are continually being developed and should be 
utilized when appropriate.
    For the purpose of lot release (section 4), an appropriate 
subset of these methods should be selected and justified.

2.1.2 Biological activity

    Assessment of the biological properties constitutes an equally 
essential step in establishing a complete characterization profile. 
An important property is the biological activity that describes the 
specific ability or capacity of a product to achieve a defined 
biological effect.
    A valid biological assay to measure the biological activity 
should be provided by the manufacturer. Examples of procedures used 
to measure biological activity include:
    <bullet>  Animal-based biological assays, which measure an 
organism's biological response to the product;
    <bullet>  Cell culture-based biological assays, which measure 
biochemical or physiological response at the cellular level; and
    <bullet>  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:
    <bullet>  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
    <bullet>  There exists a well-established manufacturing history.
    Where physicochemical tests alone are used to quantitate the 
biological activity (based on appropriate correlation), results 
should be expressed in mass.
    For the purpose of lot release (section 4), the choice of 
relevant quantitative assay (biological and/or physicochemical) 
should be justified by the manufacturer.

[[Page 44931]]

2.1.3 Immunochemical properties

    When an antibody is the desired product, its immunological 
properties should be fully characterized. Binding assays of the 
antibody to purified antigens and defined regions of antigens should 
be performed, as feasible, to determine affinity, avidity and 
immunoreactivity (including cross-reactivity). In addition, the 
target molecule bearing the relevant epitope should be biochemically 
defined and the epitope itself defined, when feasible.
    For some drug substances or drug products, the protein molecule 
may need to be examined using immunochemical procedures (e.g., 
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.

2.1.4 Purity, impurities, and contaminants

    <bullet>  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 desired product, they are considered product-related 
substances and not impurities (section 2.1.1).
    Individual and/or collective acceptance criteria for product-
related substances should be set, as appropriate.
    For the purpose of lot release (section 4), an appropriate 
subset of methods should be selected and justified for determination 
of purity.
    <bullet>  Impurities
    In addition to evaluating the purity of the drug substance and 
drug product, which may be composed of the desired product and 
multiple product-related substances, the manufacturer should also 
assess impurities which may be present. Impurities may be either 
process- or product-related. They can be of known structure, 
partially characterized, or unidentified. When adequate quantities 
of impurities can be generated, these materials should be 
characterized to the extent possible and, where possible, their 
biological activities should be evaluated.
    Process-related impurities encompass those that are derived from 
the manufacturing process, i.e., cell substrates (e.g., host cell 
proteins, host cell DNA), cell culture (e.g., inducers, antibiotics, 
or media components), or downstream processing (see appendix, 
section 6.2.1). Product-related impurities (e.g., precursors, 
certain degradation products) are molecular variants arising during 
manufacture and/or storage 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 2.3).
    Examples of analytical procedures that may be employed to test 
for the presence of impurities are listed in appendix 6.2. New 
analytical technology and modifications to existing technology are 
continually being developed and should be utilized when appropriate.
    For the purpose of lot release (section 4), an appropriate 
subset of these methods should be selected and justified.
    <bullet>  Contaminants
    Contaminants in a product include all adventitiously introduced 
materials not intended to be part of the manufacturing process, such 
as chemical and biochemical materials (e.g., microbial proteases) 
and/or microbial species. Contaminants should be strictly avoided 
and/or suitably controlled with appropriate in-process acceptance 
criteria or action limits for drug substance or drug product 
specifications (section 2.3). For the special case of adventitious 
viral or mycoplasma contamination, the concept of action limits is 
not applicable, and the strategies proposed in ICH 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.

2.1.5 Quantity

    Quantity, usually measured as protein content, is critical for a 
biotechnological and biological product and should be determined 
using an appropriate assay, usually physicochemical in nature. In 
some cases, it may be demonstrated that the quantity values obtained 
may be directly related to those found using the biological assay. 
When this correlation exists, it may be appropriate to use 
measurement of quantity rather than the measurement of biological 
activity in manufacturing processes, such as filling.

2.2 Analytical Considerations

2.2.1 Reference standards and reference materials

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

2.2.2 Validation of analytical procedures

    At the time the application is submitted to the regulatory 
authorities, applicants should have validated the analytical 
procedures used in the specifications in accordance with the ICH 
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.

2.3 Process Controls

2.3.1 Process-related considerations

    Adequate design of a process and knowledge of its capability are 
part of the strategy used to develop a manufacturing process 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 
commercial-scale production.
    For certain impurities, testing of either the drug substance or 
the drug product may not be necessary and may not need to be 
included in the specifications if efficient control or removal to 
acceptable levels is demonstrated by suitable studies. This testing 
can include verification at commercial scale in accordance with 
regional regulations. It is recognized that only limited data may be 
available at the time of submission of an application. This concept 
may, therefore, sometimes be implemented after marketing 
authorization, in accordance with regional regulations.

2.3.2 In-process acceptance criteria and action limits

    In-process tests are performed at critical decision-making steps 
and at other steps where data serve to confirm consistency of the 
process during the production of either the drug substance or the 
drug product. The results of in-process testing may be recorded as 
action limits or reported as acceptance criteria. Performing such 
testing may

[[Page 44932]]

eliminate the need for testing of the drug substance or drug product 
(section 2.3.1). In-process testing for adventitious agents at the 
end of cell culture is an example of testing for which acceptance 
criteria should be established.
    The use of internal action limits by the manufacturer to assess 
the consistency of the process at less critical steps is also 
important. Data obtained during development and validation runs 
should provide the basis for provisional action limits to be set for 
the manufacturing process. These limits, which are the 
responsibility of the manufacturer, may be used to initiate 
investigation or further action. They should be further refined as 
additional manufacturing experience and data are obtained after 
product approval.

2.3.3 Raw materials and excipient specifications

    The quality of the raw materials used in the production of the 
drug substance (or drug product) should meet standards appropriate 
for their intended use. Biological raw materials or reagents may 
require careful evaluation to establish the presence or absence of 
deleterious endogenous or adventitious agents. Procedures 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.

2.4 Pharmacopoeial Specifications

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

2.5 Release Limits Versus Shelf-Life Limits

    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 shelf-life limits.

2.6 Statistical Concepts

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

3.0 Justification of the Specification

    The setting of specifications for drug substance and drug 
product is part of an overall control strategy which includes 
control of raw materials and excipients, in-process testing, process 
evaluation or validation, adherence to good manufacturing practices, 
stability testing, and testing for consistency of lots. When 
combined in total, these elements provide assurance that the 
appropriate quality of the product will be maintained. Since 
specifications are chosen to confirm the quality rather than to 
characterize the product, the 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.
    <bullet>  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.
    <bullet>  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.''
    <bullet>  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.
    <bullet>  Specifications are linked to analytical procedures.
    Critical quality attributes may include items such as potency, 
the nature and quantity of product-related substances, product-
related impurities, and process-related impurities. Such attributes 
can be assessed by multiple analytical procedures, each yielding 
different results. In the course of product development, it is not 
unusual for the analytical technology to evolve in parallel with the 
product. Therefore, it is important to confirm that data generated 
during development correlate with those generated at the time the 
marketing application is filed.

4.0 Specifications

    Selection of tests to be included in the specifications is 
product specific. The rationale used to establish the acceptable 
range of acceptance criteria should be described. Acceptance 
criteria should be established and justified based on data obtained 
from lots used in preclinical and/or clinical studies, data from 
lots used for demonstration of manufacturing consistency, 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.

4.1 Drug Substance Specification

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

4.1.1 Appearance and description

    A qualitative statement describing the physical state (e.g., 
solid, liquid) and color of a drug substance should be provided.

4.1.2 Identity

    The identity test(s) should be highly specific for the drug 
substance and should be based on unique aspects of its molecular 
structure and/or other specific properties. More than one test 
(physicochemical, biological, and/or immunochemical) may be 
necessary to establish identity. The identity test(s) can be 
qualitative in nature. Some of the methods typically used for 
characterization of the product as described in section 2.1 and in 
appendix 6.1 may be employed and/or modified as appropriate for the 
purpose of establishing identity.

4.1.3 Purity and impurities

    The absolute purity of biotechnological and biological products 
is difficult to determine and the results are method dependent 
(section 2.1.4). Consequently, the purity of the drug substance is 
usually estimated by a combination of methods. The

[[Page 44933]]

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:
    <bullet>   Process-related impurities (section 2.1.4) in the 
drug substance may include cell culture media, host cell proteins, 
DNA, monoclonal antibodies or chromatographic media used in 
purification, solvents, and buffer components. These impurities 
should be minimized by the use of appropriate, well-controlled 
manufacturing processes.
    <bullet>  Product-related impurities (section 2.1.4) in the drug 
substance are molecular variants with properties different from 
those of the desired product formed during manufacture and/or 
storage.
    For the impurities, the choice and optimization of analytical 
procedures should focus on the separation of the desired product and 
product-related substances from impurities. Individual and/or 
collective acceptance criteria for impurities should be set, as 
appropriate. Under certain circumstances, acceptance criteria for 
selected impurities may not be necessary (section 2.3).

4.1.4 Potency

    A relevant, validated potency assay (section 2.1.2) should be 
part of the specifications for a biotechnological or biological drug 
substance and/or drug product. When an appropriate potency assay is 
used for the drug product (section 4.2.4), an alternative method 
(physicochemical and/or biological) may suffice for quantitative 
assessment at the drug substance stage. In some cases, the 
measurement of specific activity may provide additional useful 
information.

4.1.5 Quantity

    The quantity of the drug substance, usually based on protein 
content (mass), should be determined using an appropriate assay. The 
quantity determination may be independent of a reference standard or 
material. In cases where product manufacture is based upon potency, 
there may be no need for an alternate determination of quantity.

4.2 Drug Product Specification

    Generally, the following tests and acceptance criteria are 
considered applicable to all drug products. Each section (4.2.1-
4.2.5) is cross-referenced to respective sections (4.1.1-4.1.5) 
under Drug Substance 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.

4.2.1 Appearance and description

    A qualitative statement describing the physical state (e.g., 
solid, liquid), color, and clarity of the drug product should be 
provided.

4.2.2 Identity

    The identity test(s) should be highly specific for the drug 
product and should be based on unique aspects of its molecular 
structure and other specific properties. The identity test(s) can be 
qualitative in nature. While it is recognized that in most cases a 
single test is adequate, more than one test (physicochemical, 
biological, and/or immunochemical) may be necessary to establish 
identity for some products. Some of the methods typically used for 
characterization of the product as described in section 2.1 and in 
appendix 6.1 may be employed and/or modified as appropriate for the 
purpose of establishing identity.

4.2.3 Purity and impurities

    Impurities may be generated or increased during manufacture and/
or storage of the drug product. These may be either the same as 
those occurring in the drug substance itself, process-related, or 
degradation products which form specifically in the drug product 
during formulation or during storage. If impurities are 
qualitatively and quantitatively (i.e., relative amounts and/or 
concentrations) the same as in the drug substance, testing is not 
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.

4.2.4 Potency

    A relevant, validated potency assay (section 2.1.2) should be 
part of the specifications for a biotechnological and biological 
drug substance and/or drug product. When an appropriate potency 
assay is used for the drug substance, an alternative method 
(physicochemical and/or biological) may suffice for quantitative 
assessment of the drug product. However, the rationale for such a 
choice should be provided.

4.2.5 Quantity

    The quantity of the drug substance in the drug product, usually 
based on protein content (mass), should be determined using an 
appropriate assay. In cases where product manufacture is based upon 
potency, there may be no need for an alternate determination of 
quantity.

4.2.6 General tests

    Physical description and the measurement of other quality 
attributes are often important for the evaluation of the drug 
product functions. Examples of such tests include pH and osmolarity.

4.2.7 Additional testing for unique dosage forms

    It should be recognized that certain unique dosage forms may 
need additional tests other than those mentioned above.

5.0 Glossary

    Acceptance criteria: Numerical limits, ranges, or other suitable 
measures for acceptance of the results of analytical procedures 
which the drug substance or drug product or materials at other 
stages of 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 
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

[[Page 44934]]

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.

6.0 Appendices

6.1 Appendix for Physiochemical Characterization

    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.

6.1.1 Structural characterization and confirmation

    (a) Amino acid sequence
    The amino acid sequence of the desired product should be 
determined to the extent possible using approaches such as those 
described in items (b) through (e) and then compared with the 
sequence of the amino acids deduced from the gene sequence of the 
desired product.
    (b) Amino acid composition
    The overall amino acid composition is determined using various 
hydrolytic and analytical procedures and compared with the amino 
acid composition deduced from the gene sequence for the desired 
product, or the natural counterpart, if considered necessary. In 
many cases, amino acid composition analysis provides some useful 
structural information for peptides and small proteins, but such 
data are generally less definitive for large proteins. Quantitative 
amino acid analysis data can also be used to determine protein 
content in many cases.
    (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 
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.

6.1.2 Physicochemical properties

    (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.
    (d) Electrophoretic patterns
    Electrophoretic patterns and data on identity, homogeneity, and 
purity can be obtained by polyacrylamide gel electrophoresis, 
isoelectric focusing, SDS-polyacrylamide gel electrophoresis, 
Western-blot, capillary electrophoresis, or other suitable 
procedures.
    (e) Liquid chromatographic patterns
    Chromatographic patterns and data on the identity, homogeneity, 
and purity can be obtained by size exclusion chromatography, 
reverse-phase liquid chromatography, ion-exchange liquid 
chromatography, affinity chromatography, or other suitable 
procedures.
    (f) Spectroscopic profiles
    The UV and visible absorption spectra are determined as 
appropriate. The higher-order structure of the product is examined 
using procedures such as circular dichroism, nuclear magnetic 
resonance (NMR), or other suitable techniques as appropriate.

6.2 Appendix for Impurities

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

6.2.1 Process-related impurities and contaminants

    These are derived from the manufacturing process (section 2.1.4) 
and are classified into three major categories: Cell substrate-
derived, cell culture-derived and downstream-derived.
    (a) Cell substrate-derived impurities include, but are not 
limited to, proteins derived from the host organism 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

[[Page 44935]]

product-coding gene, fusion partners, or other appropriate cell 
lines. The level of DNA from the host cells can be detected by 
direct analysis on the product (such as hybridization techniques). 
Clearance studies, which could include spiking experiments at the 
laboratory scale, to demonstrate the removal of cell substrate-
derived impurities such as nucleic acids and host cell proteins may 
sometimes be used to eliminate the need for establishing acceptance 
criteria for these impurities.
    (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.''

6.2.2 Product-related impurities including degradation products

    The following represents the most frequently encountered 
molecular variants of the desired product and lists relevant 
technology for their assessment. Such variants may need considerable 
effort in isolation and characterization in order to identify the 
type of modification(s). Degradation products arising 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 SDS-PAGE. Peptide mapping may be useful, depending on the 
property of the variant.
    (b) Other modified forms. Deamidated, isomerized, mismatched S-S 
linked, oxidized, or altered conjugated forms (e.g., glycosylation, 
phosphorylation) may be detected and characterized by 
chromatographic, electrophoretic, and/or other relevant analytical 
methods (e.g., HPLC, capillary electrophoresis, mass spectroscopy, 
circular dichroism).
    (c) Aggregates. The category of aggregates includes dimers and 
higher multiples of the desired product. These are generally 
resolved from the desired product and product-related substances and 
quantitated by appropriate analytical procedures (e.g., size 
exclusion chromatography, capillary electrophoresis).

    Dated: August 11, 1999.
Margaret M. Dotzel,
Acting Associate Commissioner for Policy.
[FR Doc. 99-21352 Filed 8-17-99; 8:45 am]
BILLING CODE 4160-01-F