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
5600 Fishers Lane
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 INDUSTRY
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.
1 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.
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.
2 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.
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.
3 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
4   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
5 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.
6   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 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.
7 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 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. 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
8 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 shelf-
life limits.
9 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.
10   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.
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.
11 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, well-
controlled 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.
12 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.
13 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.
14 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
15 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.
16 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
17 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.
18 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.
19 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 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 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.
20 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 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).
21