177 
 
Guidance for Industry  
 
SPECIFICATIONS: TEST PROCEDURES AND ACCEPTANCE 
CRITERIA FOR NEW BIOTECHNOLOGICAL/BIOLOGICAL 
VETERINARY MEDICINAL PRODUCTS 
VICH GL40 
 
FINAL GUIDANCE 
 
Comments and suggestions regarding the document should be submitted to Division of Dockets 
Management  (HFA-305),  Food  and  Drug  Administration,  5630  Fishers  Lane,  Rm.  1061, 
Rockville,  MD    20852.    Submit  electronic  comments  to  http://www.regulations.gov/.    All 
comments should be identified with the Docket No. 2005D-0200.   
For questions regarding this document, contact Dennis Bensley, Center for Veterinary Medicine, 
(HFV-143), Food and Drug Administration, 7500 Standish Place, Rockville, MD 20855,240-276-
8268, e-mail:  dennis.bensley@fda.hhs.gov.  
 
Additional copies of this final guidance may be requested from the Communications Staff (HFV-
12), Center for Veterinary Medicine, Food and Drug Administration, 7519 Standish Place, 
Rockville,  MD    20855  and  may  be  viewed  on  the  Internet  at 
http://www.fda.gov/AnimalVeterinary/GuidanceComplianceEnforcement/GuidanceforIndustry/ucm042450.htm 
 
U.S. Department of Health and Human Services 
Food and Drug Administration 
Center for Veterinary Medicine 
June 14, 2006 
 
  Contains Non-Binding Recommendations 
 
VICH GL40 (Quality) 
November 2005 
For implementation at Step 7 
 
 
 
 
 
 
S : T P
PECIFICATIONS   EST  ROCEDURES AND 
A C
CCEPTANCE  RITERIA FOR NEW 
B /B
IOTECHNOLOGICAL IOLOGICAL 
V M P  
ETERINARY  EDICINAL  RODUCTS
 
 
 
Recommended for Adoption 
at Step 7 of the VICH Process 
in November 2005  
by the VICH Steering Committee 
for implementation in November 2006 
 
 
 
 
 
 
This Guidance has been developed by the appropriate VICH Expert Working Group and is subject to 
consultation by the parties, in accordance with the VICH Process.  At Step 7 of the Process the final 
draft will be recommended for adoption to the regulatory bodies of the European Union, Japan and 
USA. 
2 of 21  Contains Non-Binding Recommendations 
 
 
SPECIFICATIONS: TEST PROCEDURES AND ACCEPTANCE CRITERIA FOR NEW 
BIOTECHNOLOGICAL/BIOLOGICAL VETERINARY MEDICINAL PRODUCTS 
VICH Harmonised Tripartite Guidance 
TABLE OF CONTENTS 
1.   INTRODUCTION.................................................................................................... 5 
  1.1  Objective  ...................................................................................................... 5 
  1.2  Background...................................................................................................... 5 
  1.3 Scope  ...................................................................................................... 6 
2.   PRINCIPLES FOR CONSIDERATION IN SETTING  
SPECIFICATIONS.................................................................................................. 6 
  2.1  Characterization............................................................................................... 6 
     2.1.1  Physicochemical properties.......................................................... 6 
     2.1.2  Biological activity........................................................................... 7 
     2.1.3  Immunochemical properties.......................................................... 8 
     2.1.4  Purity, impurities and contaminants.............................................. 8 
     2.1.5  Quantity......................................................................................... 9 
  2.2 Analytical Considerations.............................................................................. 10 
     2.2.1  Reference standards and reference materials............................ 10 
     2.2.2  Validation of analytical procedures............................................. 10 
  2.3 Process Controls........................................................................................... 10 
     2.3.1  Process-related considerations................................................... 10 
     2.3.2  In-process acceptance criteria and action limits......................... 11 
     2.3.3  Raw materials and excipient specifications................................. 11 
  2.4 Pharmacopoeial Specifications...................................................................... 11 
  2.5 Release Limits vs. Shelf-life Limits................................................................ 11 
  2.6 Statistical Concepts....................................................................................... 12 
3.   JUSTIFICATION OF THE SPECIFICATION........................................................ 12 
4.   SPECIFICATIONS................................................................................................ 13 
  4.1 Drug Substance Specification........................................................................ 13 
3 of 21  Contains Non-Binding Recommendations 
 
     4.1.1  Appearance and description....................................................... 13 
     4.1.2  Identity......................................................................................... 13 
     4.1.3  Purity and impurities.................................................................... 13 
     4.1.4  Potency....................................................................................... 14 
     4.1.5  Quantity....................................................................................... 14 
  4.2 Medicinal Product Specification..................................................................... 14 
     4.2.1  Appearance and description....................................................... 14 
     4.2.2  Identity......................................................................................... 15 
     4.2.3  Purity and impurities.................................................................... 15 
     4.2.4  Potency....................................................................................... 15 
     4.2.5  Quantity....................................................................................... 15 
     4.2.6  General tests............................................................................... 15 
     4.2.7  Additional testing for unique dosage forms................................. 15 
5.   GLOSSARY  .................................................................................................... 16 
6.   APPENDICES   .....................................................................................18 
  6.1 Appendix for Physicochemical Characterization............................................ 18 
     6.1.1  Structural characterization and confirmation............................... 18 
     6.1.2  Physicochemical properties.........................................................19 
  6.2 Appendix for Impurities...................................................................................20 
     6.2.1  Process-related impurities and contaminants..............................20 
     6.2.2  Product-related impurities including degradation products......... 20 
 
 
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SPECIFICATIONS: TEST PROCEDURES AND ACCEPTANCE CRITERIA FOR NEW 
BIOTECHNOLOGICAL/BIOLOGICAL VETERINARY MEDICINAL PRODUCTS 
 
This guidance represents the Food and Drug Administrations (FDAs) 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.  
You can use an alternative approach if the approach satisfies the requirements of the applicable statutes 
and regulations.  If you want to discuss an alternative approach, contact the FDA staff responsible for 
implementing this guidance.  If you cannot identify the appropriate FDA staff, call the appropriate number 
listed on the title page of this guidance.  
 
1.    INTRODUCTION 
1.1   Objective 
This guidance document provides general principles on 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. 
FDAs  guidance  documents,  including  this  guidance,  do  not  establish  legally 
enforceable  responsibilities.    Instead,  guidances  describe  the  Agencys  current 
thinking on a topic and should be viewed only as recommendations, unless specific 
regulatory  or  statutory  requirements  are  cited.    The  use  of the word should in 
Agency  guidances  means  that  something  is  suggested  or  recommended,  but  not 
required. 
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 criteria to which a drug substance, medicinal 
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 medicinal 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 (GMPs), 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 medicinal 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. 
 
 
 
Page 5 of 21  Contains Non-Binding Recommendations 
1.3   Scope 
 
The principles adopted and explained in this document apply to products composed of well-
characterized  proteins  and  polypeptides,  and  their  derivatives  which  are  isolated  from 
tissues, body fluids, cell cultures, or produced using recombinant deoxyribonucleic acid  
(r-DNA)  technology.  Thus,  the  document  covers  the  generation  and  submission  of 
specifications for products such as cytokines, growth hormones and growth factors, insulins, 
and monoclonal antibodies.  This document does not cover antibiotics, heparins, vitamins, 
cell metabolites, DNA products, allergenic extracts, vaccines, cells, whole blood, and cellular 
blood components. 
 
A separate VICH Guidance (GL39), "Specifications:  Test Procedures and Acceptance for 
New Veterinary Drug Substances and Medicinal 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.    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  important  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 and data from stability studies, and relevant development data. 
Extensive characterization is performed in the development phase and, where important, 
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 
Page 6 of 21  Contains Non-Binding Recommendations 
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 important. 
Heterogeneity  can  also  be  produced  during  manufacture  and/or  storage  of  the  drug 
substance or medicinal product.  Since the heterogeneity of these products defines their 
quality, the degree and profile of this heterogeneity should be characterized, to assure 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  which  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: 
  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; 
  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 which 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 important.  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 
Page 7 of 21  Contains Non-Binding Recommendations 
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 
  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. 
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 medicinal products, the protein molecule may need to be 
examined using immunochemical procedures (e.g., ELISA, Western-blot) utilizing antibodies 
which 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 
  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 mg of product) which is also highly method-dependent.  Consequently, the purity of the 
drug  substance  and  medicinal  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 
Page 8 of 21  Contains Non-Binding Recommendations 
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. 
  Impurities 
In addition to evaluating the purity of the drug substance and medicinal 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 necessary and, where appropriate, 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, which 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 important (section 2.3). 
Examples of analytical procedures which 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. 
  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 medicinal 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  Harmonised  Tripartite  Guidances    Quality  of 
Biotechnological/Biological  Products:  Viral  Safety  Evaluation  of  Biotechnology  Derived 
Products  Derived  from  Cell  Lines  of  Human  or  Animal  Origin  and    Quality  of 
Biotechnological/Biological  Products:  Derivation  and  Characterization  of  Cell  Substrates 
Used for Production of Biotechnological/Biological Products may be considered. 
Page 9 of 21  Contains Non-Binding Recommendations 
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 important.  Also, distinct reference materials for product-
related  substances,  product-related  impurities  and  process-related  impurities,  may  be 
important  to  establish.    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 VICH 
Harmonised  Tripartite  Guidances    Validation  of  Analytical  Procedures:  Definitions  and 
Terminology and 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  which  is  controlled  and  reproducible,  yielding  a  drug 
substance or medicinal 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 medicinal product may not 
be important 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. 
Page 10 of 21  Contains Non-Binding Recommendations 
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 medicinal 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 medicinal 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 medicinal 
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 which 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  medicinal  product.    Appropriate  information  pertaining  to  the 
antibody should be made available. 
The quality of the excipients used in the medicinal 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 non-pharmacopoeial 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  medicinal  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  harmonisation  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 vs. Shelf-life Limits 
The concept of different acceptance criteria for release vs. shelf-life specifications applies to 
medicinal products only; it pertains to the establishment of more restrictive criteria for the 
release of a medicinal product than are applied to the shelf-life. Examples where this may be 
applicable include assay and impurity (degradation product) levels.  In some regions, the 
Page 11 of 21  Contains Non-Binding Recommendations 
concept of release limits may only be applicable to in-house limits and not to the regulatory 
shelf-life limits.   Thus, in these regions, the regulatory acceptance criteria are the same from 
release throughout shelf-life; however, an applicant may choose to have tighter in-house 
limits at the time of release to provide increased assurance to the applicant that the product 
will  remain  within  the  regulatory  acceptance  criterion  throughout  its  shelf-life.    In  the 
European Union there is a regulatory requirement for distinct specifications for release and 
for shelf-life where different. 
2.6   Statistical Concepts 
Appropriate  statistical  analysis  should  be  applied,  when  important,  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.    JUSTIFICATION OF THE SPECIFICATION 
The setting of specifications for drug substance and medicinal 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 medicinal product. 
Degradation of drug substance and medicinal 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 VICH Harmonised 
Tripartite Guidance: 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 pre-clinical and clinical 
studies.  The quality of the material made at commercial scale should be representative of 
the lots used in preclinical and clinical studies. 
Page 12 of 21  Contains Non-Binding Recommendations 
  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.     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, and data from stability studies, and relevant development data.  
In some cases, testing at production stages rather than at the drug substance or medicinal 
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 medicinal 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 important. 
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 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: 
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  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. 
  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  important 
(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  medicinal  product.    When  an 
appropriate potency assay is used for the medicinal 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, an alternate determination of quantity may not be important. 
4.2   Medicinal product Specification 
Generally,  the  following  tests  and  acceptance  criteria  are  considered  applicable  to  all 
medicinal products.  Each section (4.2.1 - 4.2.5) is cross-referenced to respective sections 
(4.1.1 - 4.1.5) under Drug Substance.  Pharmacopoeial requirements apply to the relevant 
dosage forms (see 21 U.S.C.   351(b)).  Typical tests found in the pharmacopoeia include, 
but  are  not  limited  to  sterility,  microbial  limits,  volume  in  container,  particulate  matter, 
uniformity  of  dosage  units,  and  moisture  content  for  lyophilized  medicinal  products.    If 
appropriate, testing for uniformity of dosage units may be performed as in-process controls 
and corresponding acceptance criteria set. 
4.2.1  Appearance and description 
A qualitative statement describing the physical state (e.g., solid, liquid), color, and clarity of 
the medicinal product should be provided. 
4.2.2  Identity 
The identity test(s) should be highly specific for the medicinal product and should be based 
on unique aspects of its molecular structure and for 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 
important to establish identity for some products.  Some of the methods typically used for 
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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 
medicinal 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  medicinal 
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 important.  If impurities are known to be introduced or formed during the production 
and/or storage of the medicinal 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 medicinal product, to measure changes in the drug substance 
during the manufacture and/or storage of the medicinal 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  medicinal  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 medicinal 
product. However, the rationale for such a choice should be provided. 
4.2.5  Quantity 
The quantity of the drug substance in the medicinal product, usually based on protein content 
(mass),  should  be  determined  using  an  appropriate  assay.    In  cases  where  product 
manufacture is based upon potency, an alternate determination of quantity may not be 
important. 
4.2.6  General tests 
Physical description and the measurement of other quality attributes is often important for the 
evaluation of the medicinal 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 (see 21 U.S.C.  360b(b)(1)(D)). 
 
 
5.    GLOSSARY 
Acceptance Criteria 
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Numerical  limits,  ranges,  or  other  suitable  measures  for  acceptance  of  the  results  of 
analytical procedures.  
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 medicinal 
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 which has the expected structure, or (2) the protein which 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. 
Excipient 
An  ingredient  added  intentionally  to  the  drug  substance  which  should  not  have 
pharmacological properties in the quantity used. 
Impurity 
(1) Any component of the new veterinary drug substance which is not the chemical entity 
defined as the new veterinary drug substance.  
(2) Any component of the medicinal product which is not the chemical entity defined as 
the drug substance or an excipient in the medicinal product. 
 
In-house Primary Reference Material 
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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 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. 
New Veterinary Drug Substance 
The designated therapeutic moiety that has not been previously registered in a region or 
member state in a veterinary medicinal product (also referred to as a new molecular entity or 
new chemical entity). It can be a complex, simple ester, or salt of a previously approved drug 
substance. 
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 medicinal product. 
These variants possess properties comparable to the desired product and are not considered 
impurities. 
Reference Standards 
Refer to international or national standards. 
 
Specification 
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, medicinal 
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 
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substance and medicinal 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  (see  21 
U.S.C.  360b(b)(1)(D)). 
6.     APPENDICES 
6.1   Appendix for Physicochemical Characterization 
This appendix provides examples of technical approaches which might be considered for 
structural characterization and confirmation, and evaluation of physicochemical properties of 
the  desired  product,  drug  substance  and/or  medicinal  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 
important.  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 
HPLC or other appropriate analytical procedure.  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  medicinal  product  using  an  appropriately  validated  procedure  is  a 
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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  non-reducing  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 is 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, SDS-
polyacrylamide gel electrophoresis (under reducing and/or non-reducing 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 UV/visible wavelength (e.g., 280 
nm).  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, etc.  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 
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The ultraviolet 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, 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,  non  metallic  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. The 
strategy  described  in  ICH  Harmonised  Tripartite  Guidance  Viral  Safety  Evaluation  of 
Biotechnology Products Derived from Cell Lines of Human or Animal Origin should be 
considered. 
 
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 
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considerable  effort  in  isolation  and  characterization  in  order  to  identify  the  type  of 
modification(s).    Degradation  products  arising  during  manufacture  and/or  storage  in 
significant amounts 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).   
 
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