Scientific Considerations in 
Demonstrating Biosimilarity 
to a Reference Product 
 
Guidance for Industry 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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) 
 
 
 April 2015 
Biosimilarity 
   
Scientific Considerations in 
Demonstrating Biosimilarity 
to a Reference Product 
 
Guidance for Industry 
 
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Food and Drug Administration 
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Email: ocod@fda.hhs.gov 
http://www.fda.gov/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformation/Guidances/default.htm    
 
 
 
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) 
 
April 2015 
Biosimilarity 
 
  Contains Nonbinding Recommendations 
 
TABLE OF CONTENTS 
 
 
 
I.  INTRODUCTION............................................................................................................. 1 
II.  SCOPE ............................................................................................................................... 2 
III.  BACKGROUND ............................................................................................................... 3 
IV.  COMPLEXITIES OF PROTEIN PRODUCTS ............................................................. 4 
A.  Nature of Protein Products and Related Scientific Considerations .......................................... 5 
B.  Manufacturing Process Considerations ....................................................................................... 5 
V.  U.S.-LICENSED REFERENCE PRODUCT AND OTHER COMPARATORS ....... 6 
VI.  APPROACHES TO DEVELOPING AND ASSESSING EVIDENCE TO 
DEMONSTRATE BIOSIMILARITY ........................................................................................ 7 
A.  Using a Stepwise Approach to Demonstrate Biosimilarity ........................................................ 7 
B.  Using a Totality-of-the-Evidence Approach to Assess a Demonstration of Biosimilarity ........ 8 
VII.  DEMONSTRATING BIOSIMILARITY ....................................................................... 9 
A.  Structural Analyses ........................................................................................................................ 9 
B.  Functional Assays ........................................................................................................................ 10 
C.  Animal Data .................................................................................................................................. 11 
1. Animal Toxicity Studies.................................................................................................................. 11 
2. Inclusion of Animal PK and PD Measures .................................................................................... 13 
3. Interpreting Animal Immunogenicity Results ................................................................................ 13 
D.  Clinical Studies  General Considerations ................................................................................ 13 
1. Human Pharmacology Data .......................................................................................................... 14 
2. Clinical Immunogenicity Assessment ............................................................................................. 16 
3. Comparative Clinical Studies ........................................................................................................ 18 
4. Extrapolation of Clinical Data Across Indications ........................................................................ 21 
VIII.  POSTMARKETING SAFETY MONITORING CONSIDERATIONS .................... 22 
IX.  CONSULTATION WITH FDA..................................................................................... 22 
GLOSSARY................................................................................................................................. 24 
i  Contains Nonbinding Recommendations 
 
Scientific Considerations in Demonstrating Biosimilarity to a 
Reference Product 
1
Guidance for Industry  
 
 
This guidance represents the current thinking of the Food and Drug Administration (FDA or Agency) on 
this topic.  It does not create any rights for any person and is not binding on FDA or the public.  You can 
use an alternative approach if it satisfies the requirements of the applicable statutes and regulations.  To 
discuss an alternative approach, contact the FDA staff responsible for this guidance as listed on the title 
page.  
 
 
 
 
I.  INTRODUCTION  
 
This guidance is intended to assist sponsors in demonstrating that a proposed therapeutic protein 
product (hereinafter proposed product2) is biosimilar to a reference product for purposes of the 
submission of a marketing application under section 351(k) of the Public Health Service Act 
(PHS Act).3  The Biologics Price Competition and Innovation Act of 2009 (BPCI Act) amends 
the PHS Act and other statutes to create an abbreviated licensure pathway in section 351(k) of 
the PHS Act for biological products shown to be biosimilar to or interchangeable with an FDA-
licensed biological reference product (see sections 7001 through 7003 of the Patient Protection 
and Affordable Care Act (Affordable Care Act) (Public Law 111-148).  Although the 351(k) 
pathway applies generally to biological products, this guidance focuses on therapeutic protein 
products and gives an overview of important scientific considerations for demonstrating 
biosimilarity.  The scientific principles described in this guidance may also apply to other types 
of proposed biosimilar biological products. 
 
This guidance is one in a series of guidances that FDA is developing to implement the BPCI Act.  
These guidances address a broad range of issues, including: 
 
  Quality Considerations in Demonstrating Biosimilarity of a Therapeutic Protein 
Product to a Reference Product 
                                                 
1 This guidance has been prepared by the Office of Medical Policy in the Center for Drug Evaluation and Research 
(CDER) in cooperation with the Center for Biologics Evaluation and Research (CBER) at the Food and Drug 
Administration. 
  
2 In Section II (Scope) of this document, the term proposed product is also used to describe a product that is the 
subject of a new drug application (NDA) submitted through the pathway described by section 505(b)(2) of the 
Federal Food, Drug, and Cosmetic Act (FD&C Act). 
 
3 The statutory definition of biosimilar and definitions of selected other terms used in this guidance are provided in 
the glossary at the end of the document. 
  1  Contains Nonbinding Recommendations 
 
  Scientific Considerations in Demonstrating Biosimilarity to a Reference Product 
  Biosimilars:  Questions and Answers Regarding Implementation of the Biologics 
Price Competition and Innovation Act of 2009 
  Formal Meetings Between the FDA and Biosimilar Biological Product Sponsors or 
Applicants 
  Clinical Pharmacology Data to Support a Demonstration of Biosimilarity to a 
Reference Product 
 
When applicable, references to information in these guidances are included in this guidance.  
 
In general, FDAs guidance documents 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.  
 
II.  SCOPE 
 
This guidance gives an overview of FDAs approach to determining biosimilarity and discusses 
important scientific considerations in demonstrating biosimilarity, including: 
 
  A stepwise approach to demonstrating biosimilarity, which can include a 
comparison of the proposed product and the reference product with respect to 
structure, function, animal toxicity, human pharmacokinetics (PK) and 
pharmacodynamics (PD), clinical immunogenicity, and clinical safety and 
effectiveness    
  The totality-of-the-evidence approach that FDA will use to review applications for 
biosimilar products, consistent with a longstanding Agency approach to evaluation 
of scientific evidence4 
  General scientific principles in conducting comparative structural analyses, 
functional assays, animal testing, human PK and PD studies, clinical 
immunogenicity assessments, and comparative clinical studies (including clinical 
study design issues) 
 
                                                 
4 The guidance for industry Providing Clinical Evidence of Effectiveness for Human Drug and Biological Products 
provides insight into the concept of the totality-of-the-evidence approach in a different context (i.e., considerations 
of both the quantity and quality of the evidence to support effectiveness for drugs and biological products).  Some of 
the principles discussed in that guidance may also be relevant in the design of a development program to support a 
demonstration of biosimilarity. 
 
We update guidances periodically.  For the most recent version of a guidance, check the FDA guidance Web page at 
http://www.fda.gov/RegulatoryInformation/Guidances/default.htm. 
  2  Contains Nonbinding Recommendations 
 
Additional topics discussed include the following: 
 
  Considerations of the complexities of therapeutic protein products when designing a 
biosimilar development program, including manufacturing process considerations 
  Use of data derived from studies comparing a proposed product with a non-U.S.-
licensed comparator product 
  Postmarketing safety monitoring considerations 
 
This guidance applies to applications submitted under section 351(k) of the PHS Act.  However, 
some scientific principles described in this guidance may be informative for the development of 
certain biological products under section 505(b)(2) of the FD&C Act.5  Section 505(b)(2) of the 
FD&C Act and section 351(k) of the PHS Act are two separate statutory schemes.  This guidance 
is not intended to describe any relationship between the standards for approval under these 
schemes. 
 
III.  BACKGROUND  
 
The BPCI Act was enacted as part of the Affordable Care Act on March 23, 2010.  The BPCI 
Act creates an abbreviated licensure pathway for biological products demonstrated to be 
biosimilar to or interchangeable with a reference product.  Section 351(k) of the PHS Act (42 
U.S.C. 262(k)), added by the BPCI Act, sets forth the requirements for an application for a 
proposed biosimilar product and an application or a supplement for a proposed interchangeable 
product.6  Section 351(i) of the PHS Act defines biosimilarity to mean that the biological 
product is highly similar to the reference product notwithstanding minor differences in clinically 
inactive components and that there are no clinically meaningful differences between the 
biological product and the reference product in terms of the safety, purity, and potency of the 
product.7  The BPCI Act also amended the definition of biological product to include protein 
(except any chemically synthesized polypeptide).8 
 
Under section 351(k) of the PHS Act, a proposed biological product that is demonstrated to be 
biosimilar to a reference product can rely on certain existing scientific knowledge about the 
                                                 
5 A 505(b)(2) application is an NDA that contains full reports of investigations of safety and effectiveness, where at 
least some of the information required for approval comes from studies not conducted by or for the applicant and for 
which the applicant has not obtained a right of reference or use (e.g., the Agencys finding of safety and/or 
effectiveness for a listed drug or published literature).  A 505(b)(2) application that seeks to rely on a listed drug 
(i.e., the reference product) must contain adequate data and information to demonstrate that the proposed product is 
sufficiently similar to the listed drug to justify reliance, in part, on FDAs finding of safety and/or effectiveness for 
the listed drug.  Any aspects of the proposed product that differ from the listed drug must be supported by adequate 
data and information to support the safety and effectiveness of the proposed product. 
 
6 General scientific issues relating to the demonstration of interchangeability will be addressed separately. 
 
7 Section 7002(b)(3) of the Affordable Care Act, adding section 351(i)(2) of the PHS Act. 
 
8 Section 7002(b)(2) of the Affordable Care Act, amending section 351(i) of the PHS Act. 
 
  3  Contains Nonbinding Recommendations 
 
safety, purity, and potency9 of the reference product to support licensure.  FDA will license a 
proposed biological product submitted under section 351(k) of the PHS Act if FDA determines 
that the information submitted in the application . . . is sufficient to show that the biological 
product is biosimilar to the reference product . . . and the 351(k) applicant (or other appropriate 
person) consents to an inspection of the facility that is the subject of the application (i.e., a 
facility in which the proposed biological product is manufactured, processed, packed, or held).10   
 
An application submitted under section 351(k) of the PHS Act must contain, among other things, 
information demonstrating that the biological product is biosimilar to a reference product 
based upon data derived from:11 
 
  Analytical studies that demonstrate that the biological product is highly similar to the 
reference product notwithstanding minor differences in clinically inactive components; 
  Animal studies (including the assessment of toxicity); and 
  A clinical study or studies (including the assessment of immunogenicity and 
pharmacokinetics or pharmacodynamics) that are sufficient to demonstrate safety, 
purity, and potency in one or more appropriate conditions of use for which the reference 
product is licensed and intended to be used and for which licensure is sought for the 
biological product. 
 
The Agency has the discretion to determine that an element described above is unnecessary in a 
351(k) application.12  FDA advises sponsors intending to develop biosimilar products to meet 
with FDA to present their product development plans and establish a schedule of milestones that 
will serve as landmarks for future discussions with the Agency.  FDA anticipates that early 
discussions with FDA about product development plans and about approaches to providing 
adequate scientific justifications will facilitate biosimilar development.13 
 
IV.  COMPLEXITIES OF PROTEIN PRODUCTS 
 
A sponsor should consider the complexities of protein products and related scientific issues when 
designing a development program to support a demonstration of biosimilarity.   
 
                                                 
9 The standard for licensure of a biological product as potent under section 351(a) of the PHS Act has long been 
interpreted to include effectiveness (see 21 CFR 600.3(s) and the guidance for industry on Providing Clinical 
Evidence of Effectiveness for Human Drug and Biological Products).  In this guidance, we use the terms safety and 
effectiveness and safety, purity, and potency interchangeably in the discussions pertaining to biosimilar products. 
 
10 Section 7002(a)(2) of the Affordable Care Act, adding section 351(k)(3) of the PHS Act. 
 
11 Section 7002(a)(2) of the Affordable Care Act, adding section 351(k)(2)(A)(i)(I) of the PHS Act. 
 
12 Section 7002(a)(2) of the Affordable Care Act, adding section 351(k)(2)(A)(ii) of the PHS Act. 
 
13 See the draft guidance for industry Formal Meetings Between the FDA and Biosimilar Biological Product 
Sponsors or Applicants for a detailed discussion.  When final, this guidance will represent FDAs current thinking 
on this topic. 
  4  Contains Nonbinding Recommendations 
 
A.  Nature of Protein Products and Related Scientific Considerations 
 
Unlike small molecule drugs, whose structure can usually be completely defined and entirely 
reproduced, proteins are typically more complex and are unlikely to be shown to be structurally 
identical to a reference product.  Many potential differences in protein structure can arise.  
Because even minor structural differences (including certain changes in glycosylation patterns) 
can significantly affect a proteins safety and/or effectiveness, it is important to evaluate these 
differences.  
 
In general, proteins can differ in at least three ways: (1) primary amino acid sequence; 
(2) modification to amino acids, such as sugar moieties (glycosylation) or other side chains; and 
(3) higher order structure (protein folding and protein-protein interactions).  Modifications to 
amino acids may lead to heterogeneity and can be difficult to control.  Protein modifications and 
higher order structure can be affected by formulation and environmental conditions, including 
light, temperature, moisture, packaging materials, container closure systems, and delivery device 
materials.  Additionally, process- as well as product-related impurities may increase the 
likelihood and/or the severity of an immune response to a protein product, and certain excipients 
may limit the ability to characterize the protein product. 
 
Advances in analytical sciences enable some protein products to be extensively characterized 
with respect to their physicochemical and biological properties, such as higher order structures 
and functional characteristics.  These analytical methodologies have increasingly improved the 
ability to identify and characterize not only the drug substance of a protein product but also 
excipients and product- and process-related impurities. 
 
Despite such significant improvements in analytical techniques, however, current analytical 
methodology may not be able to detect all relevant structural and functional differences between 
two protein products.  In addition, there may be incomplete understanding of the relationship 
between a products structural attributes and its clinical performance.  Thus, as set forth in the 
PHS Act, data derived from analytical studies, animal studies, and a clinical study or studies are 
required to demonstrate biosimilarity unless FDA determines an element unnecessary.14 
 
B.  Manufacturing Process Considerations 
 
Different manufacturing processes may alter a protein product in a way that could affect the 
safety or effectiveness of the product.  For example, differences in biological systems used to 
manufacture a protein product may cause different posttranslational modifications, which in turn 
may affect the safety and/or effectiveness of the product.  Thus, when the manufacturing process 
for a marketed protein product is changed, the application holder must assess the effects of the 
change and demonstratethrough appropriate analytical testing, functional assays, and/or in 
some cases animal and/or clinical studiesthat the change does not have an adverse effect on 
the identity, strength, quality, purity, or potency of the product as they relate to the safety or 
                                                 
14 Section 7002(a)(2) of the Affordable Care Act, adding section 351(k)(2)(A)(i)(I) of the PHS Act. 
 
  5  Contains Nonbinding Recommendations 
 
effectiveness of the product.15  The International Conference on Harmonisation (ICH) guidance 
for industry Q5E Comparability of Biotechnological/Biological Products Subject to Changes in 
Their Manufacturing Process (ICH Q5E) describes scientific principles in the comparability 
assessment for manufacturing changes. 
 
Demonstrating that a proposed product is biosimilar to a reference product typically will be more 
complex than assessing the comparability of a product before and after manufacturing changes 
made by the same manufacturer.  This is because a manufacturer that modifies its own 
manufacturing process has extensive knowledge and information about the product and the 
existing process, including established controls and acceptance parameters.  By contrast, the 
manufacturer of a proposed product is likely to have a different manufacturing process (e.g., 
different cell line, raw materials, equipment, processes, process controls, and acceptance criteria) 
from that of the reference product and no direct knowledge of the manufacturing process for the 
reference product.  Therefore, even though some of the scientific principles described in ICH 
Q5E may also apply in the demonstration of biosimilarity, in general, FDA anticipates that more 
data and information will be needed to establish biosimilarity than would be needed to establish 
that a manufacturers post-manufacturing change product is comparable to the pre-manufacturing 
change product. 
 
V.  U.S.-LICENSED REFERENCE PRODUCT AND OTHER COMPARATORS 
 
To obtain licensure of a proposed product under section 351(k) of the PHS Act, a sponsor must 
demonstrate that the proposed product is biosimilar to a single reference product that previously 
has been licensed by FDA.16  In general, a sponsor needs to provide information to demonstrate 
biosimilarity based on data directly comparing the proposed product with the reference product.  
As a scientific matter, analytical studies and at least one clinical PK study and, if appropriate, at 
least one PD study, intended to support a demonstration of biosimilarity for purposes of section 
351(k) of the PHS Act must include an adequate comparison of the proposed biosimilar product 
directly with the U.S.-licensed reference product unless it can be scientifically justified that such 
a study is not needed.  However, a sponsor may seek to use data derived from animal or clinical 
studies comparing a proposed product with a non-U.S.-licensed comparator product to address, 
in part, the requirements under section 351(k)(2)(A) of the PHS Act.  In such a case, the sponsor 
should provide adequate data or information to scientifically justify the relevance of these 
comparative data to an assessment of biosimilarity and establish an acceptable bridge to the U.S.-
licensed reference product.17  Sponsors are encouraged to discuss with FDA during the 
development program their plans to provide an adequate scientific justification and bridge to the 
U.S.-licensed reference product.  A final decision about the adequacy of such justification and 
bridge will be made by FDA during review of the 351(k) application. 
 
                                                 
15 See 21 CFR 601.12 and 21 CFR 314.70 for regulatory requirements for changes (including manufacturing 
changes) made to a licensed biologics license application (BLA) and an approved NDA, respectively. 
  
16 Sections 7002(a)(2) and (b)(3) of the Affordable Care Act, adding sections 351(k), 351(i)(2), and 351(i)(4) of the 
PHS Act. 
 
17 For examples of issues that a sponsor may need to address, see the guidance for industry Biosimilars:  Questions 
and Answers Regarding Implementation of the Biologics Price Competition and Innovation Act of 2009. 
  6  Contains Nonbinding Recommendations 
 
 
VI.  APPROACHES TO DEVELOPING AND ASSESSING EVIDENCE TO 
DEMONSTRATE BIOSIMILARITY 
 
FDA recommends that sponsors use a stepwise approach to develop the evidence needed to 
demonstrate biosimilarity.  FDA intends to consider the totality of the evidence provided by a 
sponsor when the Agency evaluates the sponsors demonstration of biosimilarity, consistent with 
a longstanding Agency approach to evaluating scientific evidence.18 
 
A.  Using a Stepwise Approach to Demonstrate Biosimilarity 
 
The purpose of a biosimilar development program is to support a demonstration of biosimilarity 
between a proposed product and a reference product, including an assessment of the effects of 
any observed differences between the products, but not to independently establish the safety and 
effectiveness of the proposed product.  FDA recommends that sponsors use a stepwise approach 
to developing the data and information needed to support a demonstration of biosimilarity.  At 
each step, the sponsor should evaluate the extent to which there is residual uncertainty about the 
biosimilarity of the proposed product and identify next steps to try to address that uncertainty.  
Where possible, studies conducted should be designed to maximize their contribution to 
demonstrating biosimilarity.  For example, a clinical immunogenicity study may also provide 
other useful information about the safety profile of the proposed product. 
 
The stepwise approach should start with extensive structural and functional characterization of 
both the proposed product and the reference product, which serves as the foundation of a 
biosimilar development program (sections VII.A and VII.B).  The more comprehensive and 
robust the comparative structural and functional characterizationthe extent to which these 
studies are able to identify (qualitatively or quantitatively) differences in relevant product 
attributes between the proposed product and the reference product (including the drug substance, 
excipients, and impurities)the more useful such characterization will be in determining what 
additional studies may be needed.  For example, rigorous structural and functional comparisons 
that show minimal or no difference between the proposed product and the reference product will 
strengthen the scientific justification for a selective and targeted approach to animal and/or 
clinical testing to support a demonstration of biosimilarity.  It may be useful to further quantify 
the similarity or differences between the two products using a meaningful fingerprint-like 
analyses algorithm that covers a large number of additional product attributes and their 
combinations with high sensitivity using orthogonal methods.  Such a strategy may further 
reduce the possibility of undetected structural differences between the products and lead to a 
more selective and targeted approach to animal and/or clinical testing.  A sufficient 
understanding of the mechanism of action (MOA) of the drug substance and clinical relevance of 
any observed structural differences, clinical knowledge of the reference product and its class that 
indicates low overall safety risks, and the availability of a relevant PD measure(s) may provide 
further scientific justification for a selective and targeted approach to animal and/or clinical 
studies. 
 
                                                 
18 See footnote 4. 
  7  Contains Nonbinding Recommendations 
 
The sponsor should then consider the role of animal data in assessing toxicity and, in some cases, 
in providing additional support for demonstrating biosimilarity and in contributing to the 
immunogenicity assessment (section VII.C).  The sponsor should then conduct comparative 
human PK and PD studies (if there is a relevant PD measure(s)) (section VII.D.1) and compare 
the clinical immunogenicity of the two products in an appropriate study population (section 
VII.D.2).  If there is residual uncertainty about biosimilarity after conducting structural analyses, 
functional assays, animal testing, human PK and PD studies, and the clinical immunogenicity 
assessment, the sponsor should then consider what additional clinical data may be needed to 
adequately address that uncertainty (section VII.D.3).  FDA encourages sponsors to consult 
extensively with the Agency after completion of comparative structural and functional analyses 
(before finalizing the clinical program) and throughout development as needed. 
 
FDA recognizes that some of the aforementioned investigations could be performed in parallel; 
however, the Agency recommends that sponsors use a stepwise approach to better address 
residual uncertainty about biosimilarity that might remain at each step and incorporate FDAs 
advice provided after FDA review of data and information collected at certain milestones. 
 
B.  Using a Totality-of-the-Evidence Approach to Assess a Demonstration of 
Biosimilarity 
 
In evaluating a sponsors demonstration of biosimilarity, FDA will consider the totality of the 
data and information submitted in the application, including structural and functional 
characterization, nonclinical evaluation, human PK and PD data, clinical immunogenicity data, 
and comparative clinical study(ies) data.  FDA intends to use a risk-based approach to evaluate 
all available data and information submitted in support of the biosimilarity of the proposed 
product.   
 
Thus, a sponsor may be able to demonstrate biosimilarity even though there are formulation or 
minor structural differences, provided that the sponsor provides sufficient data and information 
demonstrating that the differences are not clinically meaningful and the proposed product 
otherwise meets the statutory criteria for biosimilarity.  For example, differences in certain 
posttranslational modifications or differences in certain excipients (e.g., human serum albumin) 
might not preclude a finding of biosimilarity if data and information provided by the sponsor 
show that the proposed product is highly similar to the reference product notwithstanding minor 
differences in clinically inactive components and that there are no clinically meaningful 
differences between the products in terms of safety, purity, and potency.19  Clinically meaningful 
differences could include a difference in the expected range of safety, purity, or potency of the 
proposed product and the reference product.  By contrast, slight differences in rates of 
occurrence of certain adverse events between the two products ordinarily would not be 
considered clinically meaningful differences. 
 
 
                                                 
19 In this example, because some excipients may affect the ability to characterize products, a sponsor should provide 
evidence that the excipients used in the reference product will not affect the ability to characterize and compare the 
products. 
  8  Contains Nonbinding Recommendations 
 
VII.  DEMONSTRATING BIOSIMILARITY 
 
This section discusses scientific considerations in the stepwise approach to developing data and 
information needed to support a demonstration of biosimilarity.  To demonstrate biosimilarity, a 
sponsor must provide sufficient data and information to show that the proposed product and the 
reference product are highly similar notwithstanding minor differences in clinically inactive 
components and that there are no clinically meaningful differences between the two products in 
terms of safety, purity, and potency.20  The type and amount of analyses and testing that will be 
sufficient to demonstrate biosimilarity will be determined on a product-specific basis. 
 
A.  Structural Analyses 
 
The PHS Act requires that a 351(k) application include information demonstrating biosimilarity 
based on data derived from, among other things, analytical studies that demonstrate that the 
biological product is highly similar to the reference product notwithstanding minor differences in 
clinically inactive components, unless FDA determines that an element is unnecessary in a 
351(k) application.21  FDA expects that first, a sponsor will extensively characterize the 
proposed product and the reference product with state-of-the-art technology, because extensive 
characterization of both products serves as the foundation for a demonstration of biosimilarity.  It 
is expected that the expression construct for a proposed product will encode the same primary 
amino acid sequence as its reference product.  However, minor modifications such as N- or C-
terminal truncations that are not expected to change the product performance may be justified 
and should be explained by the sponsor.  Additionally, sponsors should consider all relevant 
characteristics of the protein product (e.g., the primary, secondary, tertiary, and quaternary 
structure; posttranslational modifications; and biological activities) to demonstrate that the 
proposed product is highly similar to the reference product notwithstanding minor differences in 
clinically inactive components.  The more comprehensive and robust the comparative structural 
and functional characterization is, the stronger the scientific justification for a selective and 
targeted approach to animal and/or clinical testing.   
 
Sponsors should use appropriate analytical methodologies with adequate sensitivity and 
specificity for structural characterization of the proteins.  Generally, such tests include the 
following comparisons of the proposed product and the reference product: 
 
  Primary structures, such as amino acid sequence 
  Higher order structures, including secondary, tertiary, and quaternary structure 
(including aggregation)  
  Enzymatic posttranslational modifications, such as glycosylation and 
phosphorylation 
                                                 
20 Section 7002(b)(3) of the Affordable Care Act, adding section 351(i)(2) of the PHS Act. 
 
21 Section 7002(a)(2) of the Affordable Care Act, adding sections 351(k)(2)(A)(i)(I)(aa) and 351(k)(2)(A)(ii) of the 
PHS Act. 
  9  Contains Nonbinding Recommendations 
 
  Other potential variations, such as protein deamidation and oxidation 
  Intentional chemical modifications, such as PEGylation sites and characteristics 
 
Sponsors should conduct extensive structural characterization of both the proposed product and 
the reference product in multiple representative lots to understand the lot-to-lot variability of 
both products in the manufacturing processes.  Lots used for the analyses should support the 
biosimilarity of both the clinical material used in the clinical study(ies) intended to support a 
demonstration of biosimilarity, and the to-be-marketed proposed product, to the reference 
product.  Characterization of lots manufactured during process development for the proposed 
product may also be useful.  Sponsors should justify the selection of the representative lots, 
including the number of lots. 
 
In addition, FDA recommends that sponsors analyze the finished dosage form of multiple lots of 
the proposed product and the reference product, assessing excipients and any formulation effect 
on purity, product- and process-related impurities, and stability.22  Differences in formulation 
between the proposed product and the reference product are among the factors that may affect 
the extent and nature of subsequent animal or clinical testing.23  A sponsor considering 
manufacturing changes after completing the initial analytical similarity assessment or after 
completing clinical testing intended to support a 351(k) application should perform an additional 
analytical similarity assessment with lots manufactured by the new process and the reference 
product and establish comparability of the proposed product manufactured by the old and new 
manufacturing processes.  The nature and extent of the changes may determine the extent of the 
analytical similarity and comparability studies and any necessary additional studies. 
 
If the reference product or the proposed product cannot be adequately characterized with state-
of-the-art technology, the application for the proposed product may not be appropriate for 
submission under section 351(k) of the PHS Act; and the sponsor should consult FDA for 
guidance on the appropriate submission pathway. 
 
B.  Functional Assays 
 
The pharmacologic activity of protein products should be evaluated by in vitro and/or in vivo 
functional assays.  In vitro assays may include, but are not limited to, biological assays, binding 
assays, and enzyme kinetics.  In vivo assays may include the use of animal models of disease 
(e.g., models that exhibit a disease state or symptom) to evaluate functional effects on 
pharmacodynamic markers or efficacy measures.  A functional evaluation comparing a proposed 
product to the reference product using these types of assays is also an important part of the 
foundation that supports a demonstration of biosimilarity and may be used to scientifically 
justify a selective and targeted approach to animal and/or clinical testing. 
 
                                                 
22 See also the guidance for industry Quality Considerations in Demonstrating Biosimilarity of a Therapeutic 
Protein Product to a Reference Product. 
 
23 See also the guidance for industry Quality Considerations in Demonstrating Biosimilarity of a Therapeutic 
Protein Product to a Reference Product. 
  10  Contains Nonbinding Recommendations 
 
Sponsors can use functional assays to provide additional evidence that the biologic activity and 
potency of the proposed product are highly similar to those of the reference product and/or to 
support a conclusion that there are no clinically meaningful differences between the proposed 
product and the reference product.  Such assays also may be used to provide additional evidence 
that the MOA of the two products is the same to the extent the MOA of the reference product is 
known.  Functional assays can be used to provide additional data to support results from 
structural analyses, investigate the consequences of observed structural differences, and explore 
structure-activity relationships.24  These assays are expected to be comparative so they can 
provide evidence of similarity or reveal differences in the performance of the proposed product 
compared to the reference product, especially differences resulting from variations in structure 
that cannot be detected using current analytical methods.  FDA also recommends that sponsors 
discuss limitations of the assays they used when interpreting results in their submissions to FDA.  
Such discussions would be useful for the evaluation of analytical data and may guide whether 
additional analytical testing would be necessary to support a demonstration of biosimilarity. 
 
Functional assays can also provide information that complements the animal and clinical data in 
assessing the potential clinical effects of minor differences in structure between the proposed 
product and the reference product.  For example, cell-based bioactivity assays may be used to 
detect the potential for inducing cytokine release syndrome in vivo.  The available information 
about these assays, including sensitivity, specificity, and extent of validation, can affect the 
amount and type of additional animal or clinical data that may be needed to establish 
biosimilarity.  As is the case for the structural evaluation, sponsors should justify the selection of 
the representative lots, including the number of lots. 
 
C.  Animal Data 
 
The PHS Act also requires that a 351(k) application include information demonstrating 
biosimilarity based on data derived from animal studies (including the assessment of toxicity), 
unless FDA determines that such studies are not necessary in a 351(k) application.25  Results 
from animal studies may be used to support the safety evaluation of the proposed product and 
more generally to support the demonstration of biosimilarity between the proposed product and 
the reference product.  
 
1.  Animal Toxicity Studies 
 
As a scientific matter, animal toxicity data are considered useful when, based on the 
results of extensive structural and functional characterization, uncertainties remain about 
the safety of the proposed product that need to be addressed before initiation of clinical 
studies in humans (assuming results from animal studies can meaningfully address the 
remaining uncertainties).  
 
                                                 
24 See also the guidance for industry Quality Considerations in Demonstrating Biosimilarity of a Therapeutic 
Protein Product to a Reference Product. 
 
25 Section 7002(a)(2) of the Affordable Care Act, adding sections 351(k)(2)(A)(i)(I)(bb) and 351(k)(2)(A)(ii) of the 
PHS Act. 
  11  Contains Nonbinding Recommendations 
 
The scope and extent of any animal toxicity studies will depend on information about the 
reference product, information about the proposed product, and the extent of known 
similarities or differences between the two.  As described further in section IX, FDA 
encourages sponsors to initiate early discussions with the Agency with regard to their 
biosimilar development plans, including identifying appropriate scientific justifications 
for not conducting an animal toxicity study or for the scope and extent of such a study. 
 
If comparative structural and functional data using the proposed product provide strong 
support for analytical similarity to a reference product, then limited animal toxicity data 
may be sufficient to support initial clinical use of the proposed product.  Such a study 
may be non-sacrificial and include endpoints that measure in-life parameters, PD, and PK 
(with an assessment of immunogenicity). 
 
If the structural and functional data are limited in scope or there are concerns about the 
proposed product quality, a general toxicology study may be needed that includes full 
animal pathology, histopathology, PD, PK, and immunogenicity assessments.  When 
animal toxicology studies are conducted, it will be useful to perform a comparative study 
with the proposed product and the reference product (i.e., comparative bridging 
toxicology studies).  The selection of dose, regimen, duration, and test species for these 
studies should provide a meaningful toxicological comparison between the two products.  
It is important to understand the limitations of such animal studies (e.g., small sample 
size, intra-species variations) when interpreting results comparing the proposed product 
and the reference product.  For a detailed discussion on the design of animal toxicology 
studies relevant to biological products, see the ICH guidance for industry S6(R1) 
Preclinical Safety Evaluation of Biotechnology-Derived Pharmaceuticals (ICH S6(R1)). 
 
Safety data derived from animal toxicity studies generally are not expected if clinical data 
(e.g., from studies or marketing experience outside the United States) using the proposed 
product are available (with the same proposed route of administration and formulation) 
that provide sufficient evidence for its safe use, unless animal toxicity studies are 
otherwise needed to address a specific product quality concern.   
 
Animal toxicity studies are generally not useful if there is no animal species that can 
provide pharmacologically relevant data for the product (i.e., no species in which the 
biologic activity of the product mimics the human response).  For a detailed discussion 
about demonstrating species relevance, see the criteria described in ICH S6(R1).  
However, there may be some instances when animal data from a pharmacologically 
nonresponsive species (including rodents) may be useful to support clinical studies with a 
proposed product that has not been previously tested in human subjects, for example, 
comparative PK and systemic tolerability studies.  If animal toxicity studies are not 
warranted based on an acceptable scientific justification, additional comparative in vitro 
testing (using human cells or tissues when appropriate) is encouraged.  Data derived 
using human cells can provide important comparative information between the proposed 
product and the reference product regarding potential clinical effects (section VII.B), 
particularly in situations where there are no animal species available for safety testing. 
 
  12  Contains Nonbinding Recommendations 
 
In general, nonclinical safety pharmacology, reproductive and developmental toxicity, 
and carcinogenicity studies are not warranted when the proposed product and the 
reference product have been demonstrated to be highly similar through extensive 
structural and functional characterization and animal toxicity studies (if such studies were 
conducted). 
 
2.  Inclusion of Animal PK and PD Measures 
 
Under certain circumstances, a single-dose study in animals comparing the proposed 
product and the reference product using PK and PD measures may contribute to the 
totality of evidence that supports a demonstration of biosimilarity.  Specifically, sponsors 
can use results from animal studies to support the degree of similarity based on the PK 
and PD profiles of the proposed product and the reference product.  PK and PD measures 
also can be incorporated into a single animal toxicity study, where appropriate.  Animal 
PK and PD assessment will not negate the need for human PK and PD studies. 
 
3.  Interpreting Animal Immunogenicity Results 
 
Animal immunogenicity assessments are conducted to assist in the interpretation of the 
animal study results and generally do not predict potential immune responses to protein 
products in humans.  However, when differences in manufacturing (e.g., impurities or 
excipients) between the proposed product and the reference product may result in 
differences in immunogenicity, measurement of anti-therapeutic protein antibody 
responses in animals may provide useful information.  Additionally, differences observed 
in animal immunogenicity assessments may reflect potential structural or functional 
differences between the two products not captured by other analytical methods. 
 
D.  Clinical Studies  General Considerations 
 
The sponsor of a proposed product must include in its submission to FDA information 
demonstrating that there are no clinically meaningful differences between the biological product 
and the reference product in terms of the safety, purity, and potency of the product.26 
 
The nature and scope of the clinical study or studies will depend on the nature and extent of 
residual uncertainty about biosimilarity after conducting structural and functional 
characterization and, where relevant, animal studies.  The frequency and severity of safety risks 
                                                 
26 Section 7002(b)(3) of the Affordable Care Act, adding section 351(i)(2)(B) of the PHS Act.  To support a 
demonstration of biosimilarity, the statute also requires a clinical study or studies (including the assessment of 
immunogenicity and PK or PD) sufficient to demonstrate safety, purity, and potency in one or more appropriate 
conditions of use for which the reference product is licensed and intended to be used and for which licensure is 
sought for the biological product, unless FDA determines an element unnecessary (section 7002(a)(2) of the 
Affordable Care Act, adding section 351(k)(2)(A)(i)(I)(cc) of the PHS Act).  As a general matter, FDA anticipates 
that the recommendations described in this guidance designed to demonstrate that the proposed product is highly 
similar to its reference product notwithstanding minor differences in clinically inactive components and to 
demonstrate that no clinically meaningful differences exist between the two products will provide data sufficient to 
demonstrate the safety, purity, and potency of the proposed product.  FDA recommends that sponsors identify which 
study or studies will provide data regarding no clinically meaningful differences prior to starting clinical studies. 
  13  Contains Nonbinding Recommendations 
 
and other safety and effectiveness considerations (e.g., poor relationship between pharmacologic 
effects and effectiveness) for the reference product may also affect the design of the clinical 
program.  The scope of the clinical program and the type of clinical studies (i.e., comparative 
human PK, PD, clinical immunogenicity, or clinical safety and effectiveness) should be 
scientifically justified by the sponsor. 
 
As a scientific matter, FDA expects a sponsor to conduct comparative human PK and PD studies 
(if there is a relevant PD measure(s))27 and a clinical immunogenicity assessment.  In certain 
cases, the results of these studies may provide adequate clinical data to support a conclusion that 
there are no clinically meaningful differences between the proposed biosimilar product and the 
reference product.  However, if residual uncertainty about biosimilarity remains after conducting 
these studies, an additional comparative clinical study or studies would be needed to further 
evaluate whether there are clinically meaningful differences between the two products.   
 
1.  Human Pharmacology Data28 
 
Human PK and PD profiles of a protein product often cannot be adequately predicted 
from functional assays and/or animal studies alone.  Therefore, human PK and PD studies 
comparing a proposed product to the reference product generally are fundamental 
components in supporting a demonstration of biosimilarity.  Both PK and PD studies 
(where there is a relevant PD measure(s)) generally will be expected to establish 
biosimilarity, unless a sponsor can scientifically justify that such a study is not needed.29  
Even if relevant PD measures are not available, sensitive PD endpoints may be assessed 
if such assessment may help reduce residual uncertainty about biosimilarity. 
 
Sponsors should provide a scientific justification for the selection of the human PK and 
PD study population (e.g., patients versus healthy subjects) and parameters, taking into 
consideration the relevance and sensitivity of such population and parameters, the 
population and parameters studied for the licensure for the reference product, as well as 
the current knowledge of the intra-subject and inter-subject variability of human PK and 
PD for the reference product.  For example, comparative human PK and PD studies 
should use a population, dose(s), and route of administration that are adequately sensitive 
to allow for the detection of differences in PK and PD profiles.  FDA recommends that, 
to the extent possible, the sponsor select PD measures that (1) are relevant to clinical 
                                                 
27 A PD study may also incorporate PK measures (i.e., a combined PK/PD study). 
 
28 See the draft guidance for industry Clinical Pharmacology Data to Support a Demonstration of Biosimilarity to a 
Reference Product for a more detailed discussion on the design and use of clinical pharmacology studies to support 
a demonstration of biosimilarity.  When final, this guidance will represent FDAs current thinking on this topic. 
 
29 PK and PD studies provide quite different types of information.  In simple terms, a PK study measures how the 
body acts on a drug (how the drug is absorbed, distributed, metabolized, and eliminated), and a PD study measures 
how the drug acts on the body (typically assessing a measure(s) related to the drugs biochemical and physiologic 
effects on the body).  Therefore, one type of study does not duplicate or substitute for the information provided by 
the other.  Both PK studies and PD studies provide important information for assessing biosimilarity; and therefore, 
as a scientific matter, comparative human PK studies and PD studies (where there is a relevant PD measure(s)) 
generally will be expected. 
  14  Contains Nonbinding Recommendations 
 
outcomes (e.g., on mechanistic path of MOA or disease process related to effectiveness 
or safety); (2) are measurable for a sufficient period of time after dosing to ascertain the 
full PD response and with appropriate precision; and (3) have the sensitivity to detect 
clinically meaningful differences between the proposed product and the reference 
product.  Use of multiple PD measures that assess different domains of activities may 
also be of value. 
 
When there are established dose-response or systemic exposure-response relationships 
(response may be PD measures or clinical endpoints), it is important to select, whenever 
possible, a dose(s) for study on the steep part of the dose-response curve for the proposed 
product.  Studying doses that are on the plateau of the dose-response curve is unlikely to 
detect clinically meaningful differences between the two products.  Sponsors should 
predefine and justify the criteria for PK and PD parameters for studies included in the 
application to demonstrate biosimilarity. 
 
A human PK study that demonstrates similar exposure (e.g., serum concentration over 
time) for the proposed product and the reference product may provide support for a 
demonstration of biosimilarity.  A human PK study may be particularly useful when the 
exposure correlates with clinical safety and effectiveness.  A human PD study that 
demonstrates a similar effect on a relevant PD measure(s) related to effectiveness or 
specific safety concerns (except for immunogenicity, which is evaluated separately) 
represents even stronger support for a biosimilarity determination.   
 
In certain cases, establishing a similar clinical PK, PD, and immunogenicity profile may 
provide sufficient clinical data to support a conclusion that there are no clinically 
meaningful differences between the two products.  PK and PD parameters are generally 
more sensitive than clinical efficacy endpoints in assessing the similarity of two products.    
For example, an effect on thyroid stimulating hormone (TSH) levels would provide a 
more sensitive comparison of two thyroxine products than an effect on clinical symptoms 
of euthyroidism.   
 
In cases where there is a meaningful correlation between PK and PD results and clinical 
effectiveness, convincing PK and PD results may make a comparative efficacy study 
unnecessary.  For example, similar dose-response curves of the proposed product and the 
reference product on a relevant PD measure, combined with a similar human PK profile 
and clinical immunogenicity profile, could provide sufficient evidence to support a 
conclusion of no clinically meaningful differences.  Even if there is still residual 
uncertainty about biosimilarity based on PK and PD results, establishing a similar human 
PK and PD profile may provide a scientific basis for a selective and targeted approach to 
subsequent clinical testing. 
 
For PD studies using products with a short half-life (e.g., shorter than 5 days), a rapid PD 
response, and a low incidence of immunogenicity, a crossover design is appropriate, 
when feasible.  For products with a longer half-life (e.g., more than 5 days), a parallel 
design will usually be needed.  Sponsors should provide a scientific justification for the 
selection of study dose (e.g., one dose or multiple doses) and route of administration.  
  15  Contains Nonbinding Recommendations 
 
FDA recommends that sponsors consider the duration of time it takes for a PD measure 
to change and the possibility of nonlinear PK.  FDA also encourages consideration of the 
role of modeling and simulation in designing comparative human PK and PD studies.   
 
2.  Clinical Immunogenicity Assessment 
 
The goal of the clinical immunogenicity assessment is to evaluate potential differences 
between the proposed product and the reference product in the incidence and severity of 
human immune responses.  Immune responses may affect both the safety and 
effectiveness of the product by, for example, altering PK, inducing anaphylaxis, or 
promoting development of neutralizing antibodies that neutralize the product as well as 
its endogenous protein counterpart.  Thus, establishing that there are no clinically 
meaningful differences in immune response between a proposed product and the 
reference product is a key element in the demonstration of biosimilarity.  Structural, 
functional, and animal data30 are generally not adequate to predict immunogenicity in 
humans.  Therefore, at least one clinical study that includes a comparison of the 
immunogenicity of the proposed product to that of the reference product will be expected.  
FDA encourages that, where feasible, sponsors collect immunogenicity data in any 
clinical study, including human PK or PD studies. 
 
The extent and timing of the clinical immunogenicity assessment will vary depending on 
a range of factors, including the extent of analytical similarity between the proposed 
product and the reference product, and the incidence and clinical consequences of 
immune responses for the reference product.  For example, if the clinical consequence is 
severe (e.g., when the reference product is a therapeutic counterpart of an endogenous 
protein with a critical, nonredundant biological function or is known to provoke 
anaphylaxis), a more extensive immunogenicity assessment will likely be needed to 
support a demonstration of biosimilarity.  If the immune response to the reference 
product is rare, a premarketing evaluation to assess apparent differences in immune 
responses between the two products may be adequate to support biosimilarity.  In 
addition, in some cases certain safety risks may need to be evaluated through 
postmarketing surveillance or studies. 
 
The overall immunogenicity assessment should consider the nature of the immune 
response (e.g., anaphylaxis, neutralizing antibody), the clinical relevance and severity of 
consequences (e.g., loss of efficacy of life-saving therapeutic and other adverse effects), 
the incidence of immune responses, and the population being studied.  FDA recommends 
use of a comparative parallel design (i.e., a head-to-head study) in treatment-naive 
patients as the most sensitive design for a premarketing study to assess potential 
differences in the risk of immunogenicity.  However, depending on the clinical 
experience of the reference and proposed products (taking into consideration the 
conditions of use and patient population), a sponsor may need to evaluate a subset of 
patients to provide a substantive descriptive assessment of whether a single cross-over 
                                                 
30 Section VII.C.3 of this guidance contains a discussion concerning the interpretation of animal immunogenicity 
results. 
 
  16  Contains Nonbinding Recommendations 
 
from the reference product to the proposed biosimilar would result in a major risk in 
terms of hypersensitivity, immunogenicity, or other reactions.  The design of any study to 
assess immunogenicity and acceptable differences in the incidence and other parameters 
of immune response should be discussed with FDA before initiating the study.  
Differences in immune responses between a proposed product and the reference product 
in the absence of observed clinical sequelae may be of concern and may warrant further 
evaluation (e.g., extended period of follow-up evaluation). 
 
The study population used to compare immunogenicity should be justified by the sponsor 
and agreed to by the Agency.  If a sponsor is seeking to extrapolate immunogenicity 
findings for one condition of use to other conditions of use, the sponsor should consider 
using a study population and treatment regimen that are adequately sensitive for 
predicting a difference in immune responses between the proposed product and the 
reference product across the conditions of use.  Usually, this will be the population and 
regimen for the reference product for which development of immune responses with 
adverse outcomes is most likely to occur (e.g., patients on background 
immunosuppressants would be less likely to develop immune responses than patients 
who are not immunosuppressed). 
 
The selection of clinical immunogenicity endpoints or PD measures associated with 
immune responses to therapeutic protein products (e.g., antibody formation and cytokine 
levels) should take into consideration the immunogenicity issues that have emerged 
during the use of the reference product.  Sponsors should prospectively define the clinical 
immune response criteria (e.g., definitions of significant clinical events such as 
anaphylaxis), using established criteria where available, for each type of potential 
immune response and should obtain agreement from FDA on these criteria before 
initiating the study. 
 
The duration of follow-up evaluation should be determined based on (1) the time course 
for the generation of immune responses (such as the development of neutralizing 
antibodies, cell-mediated immune responses) and expected clinical sequelae (informed by 
experience with the reference product), (2) the time course of disappearance of the 
immune responses and clinical sequelae following cessation of therapy, and (3) the length 
of administration of the product.  For example, for chronically administered agents, the 
follow-up period is recommended to be 1 year unless a shorter duration can be 
scientifically justified based on the totality of the evidence to support biosimilarity. 
 
As a scientific matter, a sponsor should evaluate the following antibody parameters in the 
clinical immunogenicity assessment: 
 
  Titer, specificity, relevant isotype distribution, time course of development, 
persistence, disappearance, impact on PK, and association with clinical sequelae 
  Neutralization of product activity: neutralizing capacity to all relevant functions 
(e.g., uptake and catalytic activity, neutralization for replacement enzyme 
therapeutics) 
  17  Contains Nonbinding Recommendations 
 
 
The sponsor should develop assays capable of sensitively detecting immune responses, 
even in the presence of the circulating drug product (proposed product and reference 
product).31  The proposed product and the reference product should be assessed in the 
same assay with the same patient sera whenever possible.  FDA recommends that 
immunogenicity assays be developed and validated early in development, and the 
validation should consider both the proposed product and the reference product.  
Sponsors should consult with FDA on the sufficiency of assays before initiating any 
clinical immunogenicity assessment.  
 
3.  Comparative Clinical Studies 
 
As a scientific matter, a comparative clinical study will be necessary to support a 
demonstration of biosimilarity if there is residual uncertainty about whether there are 
clinically meaningful differences between the proposed product and the reference product 
based on structural and functional characterization, animal testing, human PK and PD 
data, and clinical immunogenicity assessment.  A sponsor should provide a scientific 
justification if it believes that a comparative clinical study is not necessary. 
 
The following are examples of factors that may influence the type and extent of the 
comparative clinical study data needed: 
 
a.  The nature and complexity of the reference product, the extensiveness of 
structural and functional characterization, and the findings and limitations of 
comparative structural, functional, and nonclinical testing, including the extent of 
observed differences 
b.  The extent to which differences in structure, function, and nonclinical 
pharmacology and toxicology predict differences in clinical outcomes, in 
conjunction with the degree of understanding of the MOA of the reference 
product and disease pathology 
c.  The extent to which human PK or PD is known to predict clinical outcomes (e.g., 
PD measures known to be relevant to effectiveness or safety) 
d.  The extent of clinical experience with the reference product and its therapeutic 
class, including the safety and risk-benefit profile (e.g., whether there is a low 
potential for off-target adverse events), and appropriate endpoints and biomarkers 
for safety and effectiveness (e.g., availability of established, sensitive clinical 
endpoints) 
e.  The extent of any other clinical experience with the proposed product (e.g., if the 
proposed product has been marketed outside the United States) 
                                                 
31 See the draft guidance for industry Assay Development for Immunogenicity Testing of Therapeutic Proteins for a 
detailed discussion.  When final, this guidance will represent FDAs current thinking on this topic. 
  18  Contains Nonbinding Recommendations 
 
A sponsor should provide a scientific justification for how it intends to use these factors 
to determine what type(s) of clinical study(ies) are needed and the design of any 
necessary study(ies).  For example, if a comparative clinical study is needed, a sponsor 
should explain how these factors were considered in determining the design of such a 
study, including the endpoint(s), population, similarity margin, and statistical analyses. 
 
Additionally, specific safety or effectiveness concerns regarding the reference product 
and its class (including history of manufacturing- or source-related adverse events) may 
warrant more comparative clinical data.  Alternatively, if there is information regarding 
other biological products that could support a biosimilarity determination (with marketing 
histories that demonstrate no apparent differences in clinical safety and effectiveness 
profiles), such information may be an additional factor supporting a selective and 
targeted approach to the clinical program. 
 
Endpoints 
 
A sponsor should use endpoints that can assess clinically meaningful differences between 
the proposed product and the reference product in a comparative clinical study.  The 
endpoints may be different from those used as primary endpoints in the reference 
products clinical studies if they are scientifically supported.  As discussed in 
section VII.D.1, certain endpoints (such as PD measures) are more sensitive than clinical 
endpoints and, therefore, may enable more precise comparisons of relevant therapeutic 
effects.  There may be situations when the assessment of multiple PD measures in a 
comparative clinical study will enhance the sensitivity of the study.  The adequacy of the 
endpoints depends on the extent to which PD measures correlate with clinical outcome, 
the extent of structural and functional data support for biosimilarity, the understanding of 
MOA, and the nature or seriousness of outcome affected. 
 
Study Population 
 
The choice of study population should allow for an assessment of clinically meaningful 
differences between the proposed product and the reference product.  Often the study 
population will have characteristics consistent with those of the population studied for the 
licensure of the reference product for the same indication.  However, there are cases 
where a study population could be different from that in the clinical studies that 
supported the licensure of the reference product.  For example, if a genetic predictor of 
response was developed following licensure of the reference product, it may be possible 
to use patients with the response marker as the study population. 
 
Sample Size and Duration of Study 
 
The sample size for and duration of the comparative clinical study should be adequate to 
allow for the detection of clinically meaningful differences between the two products.  As 
discussed in section VII.D.1, certain endpoints, such as PD measures, may be more 
sensitive than clinical endpoints and facilitate the conduct of a smaller study of limited 
duration.  In such cases where the size and duration of the comparative clinical study may 
  19  Contains Nonbinding Recommendations 
 
not be adequate for the detection of relevant safety signals, a separate assessment of 
safety and immunogenicity may be needed. 
 
Study Design and Analyses 
 
A comparative clinical study for a biosimilar development program should be designed to 
investigate whether there are clinically meaningful differences between the proposed 
product and the reference product.  The design should take into consideration the nature 
and extent of residual uncertainty that remains about biosimilarity based on data 
generated from comparative structural and functional characterization, animal testing, 
human PK and PD studies, and clinical immunogenicity assessment. 
 
Generally, FDA expects a clinical study or studies designed to establish statistical 
evidence that the proposed product is neither inferior to the reference product by more 
than a specified margin nor superior to the reference product by more than a (possibly 
different) specified margin.  Typically, an equivalence design with symmetric inferiority 
and superiority margins would be used.  Symmetric margins would be reasonable when, 
for example, there are dose-related toxicities. 
 
In some cases, it would be appropriate to use an asymmetric interval with a larger upper 
bound to rule out superiority than lower bound to rule out inferiority.  An asymmetric 
interval could be reasonable, for example, if the dose used in the clinical study is near the 
plateau of the dose-response curve and there is little likelihood of dose-related effects 
(e.g., toxicity).  In most cases, use of an asymmetric interval would generally allow for a 
smaller sample size than would be needed with symmetric margins.  However, if there is 
a demonstration of clear superiority, then further consideration should be given as to 
whether the proposed product can be considered biosimilar to the reference product.   
 
In some cases, depending on the study population and endpoint(s), ruling out only 
inferiority may be adequate to establish that there are no clinically meaningful differences 
between the proposed product and the reference product.  For example, if it is well 
established that doses of a reference product pharmacodynamically saturate the target at 
the clinical dose level and it would be unethical to use lower than clinically approved 
doses, a non-inferiority (NI) design may be sufficient.32  
 
A sponsor should provide adequate scientific justification for the choice of study design, 
study population, study endpoint(s), estimated effect size for the reference product, and 
margin(s) (how much difference to rule out).  Sponsors should discuss their study 
proposal(s) and overall clinical development plan with FDA before initiating the 
comparative clinical study(ies). 
 
                                                 
32 If an NI design is considered appropriate, sponsors are encouraged to refer to the draft guidance for industry Non-
inferiority Clinical Trials.  When final, this guidance will represent FDAs current thinking on this topic. 
  20  Contains Nonbinding Recommendations 
 
4.  Extrapolation of Clinical Data Across Indications  
 
If the proposed product meets the statutory requirements for licensure as a biosimilar 
product under section 351(k) of the PHS Act based on, among other things, data derived 
from a clinical study or studies sufficient to demonstrate safety, purity, and potency in an 
appropriate condition of use, the applicant may seek licensure of the proposed product for 
one or more additional conditions of use for which the reference product is licensed.  
However, the applicant would need to provide sufficient scientific justification for 
extrapolating clinical data to support a determination of biosimilarity for each condition 
of use for which licensure is sought.   
 
Such scientific justification for extrapolation should address, for example, the following 
issues for the tested and extrapolated conditions of use:  
 
  The MOA(s) in each condition of use for which licensure is sought; this may 
include: 
  The target/receptor(s) for each relevant activity/function of the product 
  The binding, dose/concentration response, and pattern of molecular signaling 
upon engagement of target/receptor(s) 
  The relationships between product structure and target/receptor interactions 
  The location and expression of the target/receptor(s)  
  The PK and bio-distribution of the product in different patient populations 
(Relevant PD measures may also provide important information on the MOA.) 
  The immunogenicity of the product in different patient populations 
  Differences in expected toxicities in each condition of use and patient population 
(including whether expected toxicities are related to the pharmacological activity of 
the product or to off-target activities) 
  Any other factor that may affect the safety or efficacy of the product in each 
condition of use and patient population for which licensure is sought 
 
Differences between conditions of use with respect to the factors described above do not 
necessarily preclude extrapolation.  A scientific justification should address these 
differences in the context of the totality of the evidence supporting a demonstration of 
biosimilarity. 
 
In choosing which condition of use to study that would permit subsequent extrapolation 
of clinical data to other conditions of use, FDA recommends that a sponsor consider 
choosing a condition of use that would be adequately sensitive to detect clinically 
meaningful differences between the two products.   
  21  Contains Nonbinding Recommendations 
 
 
The sponsor of a proposed product may obtain licensure only for a condition of use that 
has been previously licensed for the reference product.  If a reference product has a 
condition of use that was licensed under section 506(c) of the FD&C Act and 21 CFR 
part 601, subpart E (accelerated approval), and the reference products clinical benefit in 
this condition of use has not yet been verified in postmarketing studies, the proposed 
product sponsor should consider studying another condition of use for which the 
reference product is licensed to avoid potential complications in the event that 
postmarketing studies fail to verify the clinical benefit of the reference product for the 
condition of use. 
 
VIII.  POSTMARKETING SAFETY MONITORING CONSIDERATIONS 
 
Robust postmarketing safety monitoring is an important component in ensuring the safety and 
effectiveness of biological products, including biosimilar therapeutic protein products. 
 
Postmarketing safety monitoring should first take into consideration any particular safety or 
effectiveness concerns associated with the use of the reference product and its class, the 
proposed product in its development and clinical use (if marketed outside the United States), the 
specific condition of use and patient population, and patient exposure in the biosimilar 
development program.  Postmarketing safety monitoring for a proposed product should also have 
adequate mechanisms in place to differentiate between the adverse events associated with the 
proposed product and those associated with the reference product, including the identification of 
adverse events associated with the proposed product that have not been previously associated 
with the reference product.  Rare, but potentially serious, safety risks (e.g., immunogenicity) may 
not be detected during preapproval clinical testing because the size of the population exposed 
likely will not be large enough to assess rare events.  In particular cases, such risks may need to 
be evaluated through postmarketing surveillance or studies.  In addition, as with any other 
biological product, FDA may take any appropriate action to ensure the safety and effectiveness 
of a proposed product, including, for example, requiring a postmarketing study or clinical trial to 
evaluate certain safety risks.33  
 
Because some aspects of postmarketing safety monitoring are product-specific, FDA encourages 
sponsors to consult with appropriate FDA divisions to discuss the sponsors proposed approach 
to postmarketing safety monitoring. 
 
IX.  CONSULTATION WITH FDA 
 
Many product-specific factors can influence the components of a product development program 
intended to establish that a proposed product is biosimilar to a reference product.  Therefore, 
FDA will ordinarily provide feedback on a case-by-case basis on the components of a 
development program for a proposed product.  In addition, it may not be possible to identify in 
advance all the necessary components of a development program; and the assessment of one 
element (e.g., structural analyses) at one step can influence decisions about the type and amount 
                                                 
33 See, for example, sections 505(o)(3) and 505(p)(1)(A)(ii) of the FD&C Act. 
  22  Contains Nonbinding Recommendations 
 
of subsequent data for the next step.  For these reasons, FDA recommends that sponsors use a 
stepwise approach to establish the totality of the evidence that supports a demonstration of 
biosimilarity. 
 
FDA also advises sponsors intending to develop biosimilar products to meet with FDA to present 
their product development plans and establish a schedule of milestones that will serve as 
landmarks for future discussions with the Agency.  FDA anticipates that early discussions with 
FDA about product development plans and about the approaches to providing adequate scientific 
justifications will facilitate biosimilar development. 
  23  Contains Nonbinding Recommendations 
 
GLOSSARY 
 
As used in this guidance, the following terms are defined below:  
 
  Biological product means a virus, therapeutic serum, toxin, antitoxin, vaccine, blood, blood 
component or derivative, allergenic product, protein (except any chemically synthesized 
polypeptide), or analogous product, or arsphenamine or derivative of arsphenamine (or any 
other trivalent organic arsenic compound), applicable to the prevention, treatment, or cure of 
a disease or condition of human beings.34 
  Biosimilar or biosimilarity means that the biological product is highly similar to the 
reference product notwithstanding minor differences in clinically inactive components, and 
that there are no clinically meaningful differences between the biological product and the 
reference product in terms of the safety, purity, and potency of the product.35 
  Chemically synthesized polypeptide means any alpha amino acid polymer that (a) is made 
entirely by chemical synthesis and (b) is less than 100 amino acids in size. 
  Product, when used without modifiers in this guidance, is intended to refer to the 
intermediates, drug substance, and/or drug product, as appropriate.  The use of the term 
product is consistent with the use of the term in ICH Q5E. 
  Protein means any alpha amino acid polymer with a specific defined sequence that is greater 
than 40 amino acids in size.  
  Reference product means the single biological product licensed under section 351(a) of the 
PHS Act against which a biological product is evaluated in a 351(k) application.36 
 
                                                 
34 Section 7002(b)(2) of the Affordable Care Act, amending section 351(i)(1) of the PHS Act. 
 
35 Section 7002(b)(3) of the Affordable Care Act, adding section 351(i)(2) of the PHS Act. 
 
36 Section 7002(b)(3) of the Affordable Care Act, adding section 351(i)(4) of the PHS Act. 
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