Development of Therapeutic Protein Biosimilars: Comparative Analytical Assessment and Other Quality-Related Considerations Guidance for Industry

Administrative guidance, not enacted legislation. This captured publisher edition preserves the listed document and attachments; later developments are not independently consolidated.

Development of Therapeutic Protein
Biosimilars: Comparative Analytical
Assessment and Other Quality-Related
Considerations
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)
September 2025
Biosimilars

Development of Therapeutic Protein
Biosimilars: Comparative Analytical
Assessment and Other Quality-Related
Considerations
Guidance for Industry
Additional copies are available from:
Division of Drug Information
Center for Drug Evaluation and Research
Food and Drug Administration
Phone: 855-543-3784 or 301-796-3400
Email: druginfo@fda.hhs.gov
https://www.fda.gov/drugs/guidance-compliance-regulatory-information/guidances-drugs
and/or
Office of Communication, Outreach and Development
Center for Biologics Evaluation and Research
Food and Drug Administration
Phone: 800-835-4709 or 240-402-8010
Email: industry.biologics@fda.hhs.gov.
https://www.fda.gov/vaccines-blood-biologics/guidance-compliance-regulatory-information-biologics/biologics-guidances
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)
September 2025
Biosimilars

Contains Nonbinding Recommendations
TABLE OF CONTENTS
I. 	INTRODUCTION............................................................................................................. 1
II. 	BACKGROUND ............................................................................................................... 2
III. 	SCOPE ............................................................................................................................... 5
IV. 	GENERAL PRINCIPLES................................................................................................ 6
V. 	FACTORS FOR CONSIDERATION IN PERFORMING THE COMPARATIVE
ANALYTICAL ASSESSMENT .............................................................................................. 9
A. 	Expression System ......................................................................................................................... 9
B. 	Manufacturing Process................................................................................................................ 10
C. 	Physicochemical Properties......................................................................................................... 10
D. 	Functional Activities .................................................................................................................... 11
E. 	Impurities ..................................................................................................................................... 13
F. 	Reference Product........................................................................................................................ 13
G. 	Reference Materials ..................................................................................................................... 14
H. 	Finished Drug Product ................................................................................................................ 15
I. 	Stability ......................................................................................................................................... 16
VI. 	COMPARATIVE ANALYTICAL ASSESSMENT..................................................... 16
A. 	Considerations for Reference and Biosimilar Products ........................................................... 16
1. Reference Product.......................................................................................................................... 16
2. Proposed Product .......................................................................................................................... 17
3. Reference Product and Non-U.S.-Licensed Comparator Products ............................................... 17
4. Accounting for Proposed Product, Reference Product, and Non-U.S.-Licensed Comparator
Product Lots ....................................................................................................................................... 18
B. 	Considerations for Data Analysis ............................................................................................... 19
1. Risk Assessment ............................................................................................................................. 19
2. Quantitative and Qualitative Data Analysis .................................................................................. 20
C. 	Comparative Analytical Assessment Conclusions .................................................................... 22
VII. 	CONCLUSION ............................................................................................................... 23

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Development of Therapeutic Protein Biosimilars: Comparative
Analytical Assessment and Other Quality-Related Considerations
Guidance for Industry1
This guidance represents the current thinking of the Food and Drug Administration (FDA or Agency) on
this topic. It does not establish 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 office responsible for this guidance as listed on the
title page.
I. 	INTRODUCTION
This guidance describes the Agency’s recommendations on the design and evaluation of
comparative analytical studies intended to support a demonstration that a proposed therapeutic
protein2 product is biosimilar to a reference product licensed under section 351(a) of the Public
Health Service Act (PHS Act). Additionally, this guidance is intended to provide
recommendations to sponsors on the scientific and technical information for the chemistry,
manufacturing, and controls (CMC) portion of a marketing application for a proposed product
submitted under section 351(k) of the PHS Act.
Section 351(k) of the PHS Act (42 U.S.C. 262(k)) provides an abbreviated licensure pathway for
biological products shown to be biosimilar to or interchangeable with an FDA-licensed reference
product and sets forth the requirements for an application for a proposed biosimilar product and
1 This guidance has been prepared by the Center for Drug Evaluation and Research (CDER) and the Center for
Biologics Evaluation and Research (CBER) at the Food and Drug Administration.
2 21CFR 600.3(h)(6): A protein is any alpha amino acid polymer with a specific, defined sequence that is greater
than 40 amino acids in size. When two or more amino acid chains in an amino acid polymer are associated with each
other in a manner that occurs in nature, the size of the amino acid polymer for purposes of this paragraph (h)(6) will
be based on the total number of amino acids in those chains, and will not be limited to the number of amino acids in
a contiguous sequence.

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an application for a proposed interchangeable biosimilar product.3,4 Although the 351(k)
pathway applies generally to biological products, this guidance focuses on therapeutic protein
products and provides an overview of recommendations for the comparative analytical
assessment and other important scientific considerations to support a demonstration of
biosimilarity between a proposed therapeutic protein product (referred to as a proposed
biosimilar or proposed biosimilar product) and the reference product.
In general, FDA’s guidance documents do not establish legally enforceable responsibilities.
Instead, guidances describe the Agency’s 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. 	BACKGROUND
In the 1980s, FDA began to receive marketing applications for biotechnology-derived protein
products, mostly for recombinant DNA-derived versions of naturally sourced products.
Consequently, FDA established a regulatory approach for the approval of recombinant DNA-
derived protein products, which was announced in the Federal Register (51 FR 23302, June 26,
1986) in conjunction with a 1985 document titled Points to Consider in the Production and
Testing of New Drugs and Biologicals Produced by Recombinant DNA Technology.5 This
approach addresses the submission of an investigational new drug application (IND) to FDA for
evaluation before initiation of clinical investigations in human subjects and submission and
potential approval of a new drug application (NDA) or biologics license application (BLA)
before marketing products made with recombinant DNA technology, even if the active
ingredient in the product is thought to be identical to a naturally occurring substance or a
previously approved product.6 The policy set forth in those documents was developed in part
because of the challenges in evaluating protein products solely by physicochemical and
3 A BLA submitted under section 351(k) (a “351(k) BLA”) must contain, among other things, information
demonstrating that the biological product is biosimilar to a reference product based upon data derived from
analytical studies, an assessment of toxicity, and a clinical study or studies, unless FDA determines, in its discretion,
that certain studies are unnecessary in a 351(k) BLA (see section 351(k)(2) of the PHS Act).
4 In this guidance, the following terms are used to describe biological products licensed under section 351(k) of the
PHS Act: (1) biosimilar or biosimilar product refers to a product that FDA has determined to be biosimilar to the
reference product (see sections 351(i)(2) and 351(k)(2) of the PHS Act) and (2) interchangeable biosimilar or
interchangeable biosimilar product refers to a biosimilar product that FDA has determined to be interchangeable
with the reference product (see sections 351(i)(3) and 351(k)(4) of the PHS Act).
5 For more information, this document is available on FDA’s Other Recommendations for Biologics Manufacturers
web page at https://www.fda.gov/vaccines-blood-biologics/guidance-compliance-regulatory-information-
biologics/other-recommendations-biologics-manufacturers.
6 Biological products that were approved under the Federal Food, Drug, and Cosmetic Act on or before March 23,
2020, were deemed to be licensed under section 351 of the PHS Act on March 23, 2020 (section 7002(e)(2) through
(e)(4) of the Biologics Price Competition and Innovation Act of 2009).

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functional testing and because the biological system in which such a protein product is produced
can have a significant effect on the structure and function of the product itself.
Improvements in manufacturing processes, process controls, control of materials, and product
testing, as well as the availability of additional characterization tests and studies, have led to a
gradual evolution in the regulation of protein products. For example, in 1996, FDA provided
recommendations in the FDA Guidance Concerning Demonstration of Comparability of Human
Biological Products, Including Therapeutic Biotechnology-derived Products, which explains
how a sponsor may demonstrate, through a combination of analytical testing, functional assays
(in vitro and/or in vivo), assessment of pharmacokinetics (PK) and/or pharmacodynamics (PD)
and toxicity in animals, and clinical testing (clinical pharmacology, safety, and/or efficacy) that a
manufacturing change does not adversely affect the safety, identity, purity, or potency of its
FDA-approved product.
Since 1996, FDA has approved many manufacturing process changes for licensed biological
products based on a demonstration of product comparability before and after the process change,
as supported by quality criteria and analytical testing and without the need for additional
nonclinical and clinical data. In some cases, uncertainty about the effect of the change and/or the
results of the biochemical/functional comparability studies has necessitated collection and
assessment of additional data, including nonclinical and/or clinical testing, to demonstrate
product comparability. These concepts were further developed in the International Conference
on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human
Use (ICH) and resulted in the ICH guidance for industry Q5E Comparability of
Biotechnological/Biological Products Subject to Changes in Their Manufacturing Process (June
2005) (ICH Q5E).
Although the scope of ICH Q5E is limited to an assessment of the comparability of a biological
product before and after a manufacturing process change made by the same manufacturer, certain
general scientific principles described in ICH Q5E are applicable to an assessment of
biosimilarity between a proposed product and its reference product. However, demonstrating
that a proposed product is biosimilar to an FDA-licensed reference product manufactured by a
different manufacturer typically will be more complex and will likely require more extensive and
comprehensive data than assessing the comparability of a product before and after a
manufacturing process change made by the product’s sponsor. 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 biosimilar will have no direct knowledge of the manufacturing
process for the reference product and will have its own manufacturing process (e.g., different cell
line, raw materials, equipment, processes, process controls, acceptance criteria).
Therefore, comprehensive comparative analytical data are necessary to build the foundation for a
development program for a proposed biosimilar product intended for submission under section
351(k) of the PHS Act.

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Section 351(k) of the PHS Act
Section 351(k) of the PHS Act (42 U.S.C. 262(k)) provides an abbreviated licensure pathway for
biological products shown to be biosimilar to or interchangeable with an FDA-licensed reference
product and sets forth the requirements for an application for a proposed biosimilar product and
an application for a proposed interchangeable biosimilar.
The term biosimilar or biosimilarity is defined in the PHS Act “in reference to a biological
product that is the subject of an application under [section 351(k)]” 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” (section 351(i)(2) of the PHS Act). The term reference product is defined in the PHS
Act as 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 (section 351(i)(4) of the PHS Act).7 An
application submitted under section 351(k) must contain, among other things, information
demonstrating that “the biological product is biosimilar to a reference product” based upon data
derived from:
• 	Analytical studies that demonstrate that the biological product is highly similar to the
reference product notwithstanding minor differences in clinically inactive components;
• 	An assessment of toxicity (which may rely on, or consist of, a study or studies described
in section 351(k)(2)(A)(i)(I)(aa) or (cc)) of the PHS Act; and
• 	A clinical study or studies (including the assessment of immunogenicity and PK or PD)
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.8
FDA has the discretion to determine that an element above is unnecessary in a 351(k)
application.9
Interchangeable biosimilar products may be substituted for the reference product without the
intervention of the prescribing health care provider.10 To meet the standard for
7 In this guidance, the terms reference product and FDA-licensed reference product both refer to the single
biological product licensed under subsection (a) against which a biological product is evaluated in an application
submitted under subsection (k). See section 351(i)(4) of the PHS Act.
8 Section 351(k)(2)(A)(i)(I) of the PHS Act.
9 Section 351(k)(2)(A)(ii) of the PHS Act.
10 See section 351(i)(3) of the PHS Act defining “interchangeable or interchangeability” in reference to a biological
product that is shown to meet the standards described in section 351(k)(4) of the PHS Act to mean that “the

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interchangeability, an applicant must provide sufficient information to demonstrate biosimilarity
and also to demonstrate that the biological product can be expected to produce the same clinical
result as the reference product in any given patient and, if the biological product is administered
more than once to an individual, the risk in terms of safety or diminished efficacy of alternating
or switching between the use of the biological product and the reference product is not greater
than the risk of using the reference product without such alternation or switch.11 With respect to
the comparative analytical assessment, the same comparative analytical data that supports a
demonstration of biosimilarity can support a demonstration of interchangeability.
III. 	SCOPE
This document provides guidance on the use of comparative analytical studies that are relevant to
assessing whether the proposed product is biosimilar to the reference product for purposes of
submission of a marketing application under section 351(k) of the PHS Act. This document is
not intended to provide an overview of FDA’s approach to determining interchangeability, which
is addressed in separate guidance documents.12 Although this guidance applies specifically to
recombinant therapeutic protein products (including monoclonal antibodies for prophylactic use),
the general scientific principles may be informative for the development of proposed biosimilars
to other protein products (e.g., naturally derived protein products). If the reference product
cannot be adequately characterized for the purpose of demonstrating that a proposed product is
biosimilar to the reference product as recommended in this guidance, the application may not be
appropriate for submission under section 351(k) of the PHS Act.
This guidance also describes considerations for CMC information that is relevant to assessing
whether the proposed product is biosimilar to the reference product. It is critical that all product
applications contain a complete and thorough CMC section that provides the necessary and
appropriate information (e.g., characterization, adventitious agent safety, process controls,
process validation and specifications) to support that the manufacturing process consistently
delivers a product with the intended quality characteristics. This guidance should be used as a
companion to other guidances available from FDA that describe the CMC information
appropriate for evaluation of protein products.13 We encourage early interaction with FDA to
discuss specific CMC issues that may arise for a sponsor’s proposed product.
biological product may be substituted for the reference product without the intervention of the health care provider
who prescribed the reference product.”
11 See section 351(k)(4) of the PHS Act
12 See FDA’s guidance for industry Considerations in Demonstrating Interchangeability With a Reference Product
(May 2019). See also FDA’s draft guidance for industry Considerations in Demonstrating Interchangeability With a
Reference Product: Update (June 2024). When final, that guidance will represent FDA’s current thinking on this
topic.
13 For CMC requirements and recommendations for submission of a marketing application, sponsors should consult
current regulations and see the guidance for industry For the Submission on Chemistry, Manufacturing, and
Controls Information for a Therapeutic Recombinant DNA-Derived Product or a Monoclonal Antibody Product for
In-vivo Use (August 1996), as well as other applicable FDA guidance documents.

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IV. 	GENERAL PRINCIPLES
Advances in analytical sciences enable many protein products to be characterized extensively in
terms of their physicochemical and biological properties. These analytical procedures have
improved the ability to identify and characterize not only the desired product but also product-
related substances and product- and process-related impurities.14 Advances in manufacturing
science and production methods may enhance the likelihood that a proposed product can be
demonstrated to be highly similar to a reference product by better targeting the reference
product’s physiochemical and functional properties. In addition, advances in analytical sciences
may enable detection and characterization of differences between the protein products. These
differences should be further assessed to understand the impact on the biosimilar product clinical
performance relative to the reference product.
Despite improvements in analytical techniques, current analytical methodology may not be able
to detect or characterize all relevant structural and functional differences between the two protein
products. A thorough understanding of each analytical method’s limitations will be critical to a
sponsor’s successful identification of residual uncertainties and, in turn, to the design of
subsequent testing. In addition, there may be incomplete understanding of the relationship
between a product’s structural attributes and its clinical performance. Sponsors should use
appropriate analytical methodologies, including available state-of-the art technologies, that have
adequate sensitivity and specificity to detect and characterize differences between the proposed
product and the reference product.
As part of a complete CMC data submission, an application submitted under section 351(k) of
the PHS Act is required to include analytical studies that demonstrate that the biological product
is highly similar to the reference product.15 The rationale for the approach to the comparative
analytical assessment should be clearly described, with consideration of the characteristics,
known mechanism or mechanisms of action, and function of the reference product.
Comparative analytical data provide the foundation for the development of a proposed product
for submission in an application under section 351(k) of the PHS Act and can influence decisions
about the type and amount of clinical data needed to support a demonstration of biosimilarity.
Such analytical data should be available early in product development and will permit more
detailed discussion with the Agency because known quality attributes can be used to shape
biosimilar development and justify certain development decisions. FDA encourages sponsors to
submit comprehensive comparative analytical data early in the development process: at the pre-
IND stage; with the original IND submission; or with the submission of data from the initial
clinical studies, such as PK and, if applicable, PD studies. FDA will best be able to provide
meaningful input on the extent and scope of additional clinical studies for a proposed biosimilar
development program once the Agency has considered the comparative analytical data.
14 The use of the terms product-related substances and product- and process-related impurities is consistent with
their use and meaning in the ICH guidance for industry Q6B Specifications: Test Procedures and Acceptance
Criteria for Biotechnological/Biological Products (August 1999).
15 See section 351(k)(2)(A)(i)(I)(aa) of the PHS Act.

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Comprehensive, robust comparative physicochemical and functional studies (these may include
biological assays, binding assays, and enzyme kinetics assays) should be performed to evaluate
the proposed product and the reference product. A meaningful comparative analytical
assessment depends on, among other things, the capabilities of available state-of-the-art
analytical assays to assess, for example, the molecular weight of the protein, complexity of the
protein (higher order structure and posttranslational modifications), degree of heterogeneity,
functional properties, impurity profiles, and degradation profiles denoting stability. The
capability of the methods used in these analytical assessments, as well as their limitations, should
be described by the sponsor. Physicochemical and functional characterization studies should be
sufficient to establish relevant quality attributes, including those that define a product’s identity,
quantity, safety, purity, and potency. The product-related impurities and product-related
substances should be identified, characterized as appropriate, quantified, and compared using
multiple lots of the proposed product and multiple lots of the reference product, to the extent
feasible and relevant. Their potential impact on the safety, purity, and potency of the product
should be assessed as a part of the evaluation.
Because therapeutic proteins are made in living systems, heterogeneity is expected in certain
quality attributes of protein products. Heterogeneity in therapeutic proteins may arise in a
number of ways and may affect the expected clinical performance of a product. Replication
errors in the DNA encoding the protein sequence and amino acid misincorporation may occur
during translation, although the level of these errors is typically low. In addition, most protein
products undergo posttranslational modifications that can alter the functions of the protein, for
example: addition of other chemical groups such as phosphate and various lipids and
carbohydrates, proteolytic cleavage following translation, modification of the chemical nature of
an amino acid (e.g., formylation). Such modifications can result from intracellular activities
during cell culture or by deliberate modification of the protein (e.g., PEGylation). Other
posttranslational modifications can be a consequence of manufacturing process operations; for
example, glycation may occur with exposure of the product to reducing sugars. Also, certain
storage conditions may be more or less permissive for certain degradation pathways such as
oxidation, deamidation, or aggregation. All of these product-related variants may alter the
biological properties of the expressed recombinant protein. Therefore, identification and
determination of the relative levels of relevant product-related variants should be included in the
comparative analytical characterization studies.
The conformation of a protein is an important factor in its biological function. Proteins generally
exhibit complex conformations (tertiary structure and, in some cases, quaternary structure)
because of their large size and the rotational characteristics of protein alpha carbons, among
other things. The resulting flexibility enables dynamic, but subtle, changes in protein
conformation, some of which may be required for functional activity. These motions are often
dependent on low-energy interactions, such as hydrogen bonds and van der Waals forces, which
may be very sensitive to environmental conditions. Current analytical technology is capable of
evaluating the three-dimensional structure of many proteins. Using multiple, relevant, state-of-
the-art methods can help describe tertiary protein structure and, to a varying extent, quaternary
structure, and can add to the body of information supporting biosimilarity. Any differences in
higher order structure between a proposed product and a reference product should be evaluated

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in terms of a potential effect on protein function and stability. Thus, functional assays are also
critical tools for evaluating the integrity of the higher order structures.
A scientifically sound characterization that provides a comprehensive understanding of the
physicochemical and biological characteristics of the proposed product is essential to the design
of the manufacturing process and to the conduct of development studies for all biological
products. The body of knowledge that emerges will serve to support a demonstration of product
quality and the effectiveness of a suitable control system during development and support
approval of the product.
Proposed biosimilar product manufacturers should perform in-depth comparative
physicochemical and biological analyses of an appropriate number of lots of the proposed
product and the reference product and, where available and appropriate, a comparison with a
reference standard for suitable attributes (e.g., potency). Evaluation of multiple lots of a
reference product and multiple lots of a proposed product enables estimation of product
variability across lots. The number of lots needed to understand the lot-to-lot variability of both
the reference and the proposed products may differ on a case-by-case basis and should be
scientifically justified by the sponsor.
FDA encourages sponsors to consult with the Agency to ensure that an appropriate number of
lots are evaluated. Identification of specific lots of a reference product used in comparative
analytical studies, together with information on expiration dates and time frames for when the
lots were analyzed and used in other types of studies (nonclinical or clinical studies), should be
provided. This information will be useful in justifying acceptance criteria for the comparative
analytical assessment of the proposed product and the reference product. However, acceptance
criteria should be based on the totality of the analytical data and knowledge of the attribute
impact to the patient and not simply on the observed range of product attributes of the reference
product. This is because some product attributes act in combination to affect a product’s safety,
purity, and potency profile; therefore, their potential interaction should be considered when
conducting the comparative analytical assessment and setting specifications. For example, for
some glycoproteins, the content and distribution of tetra-antennary and N-acetyllactosamine
repeats can affect in vivo potency and should not be evaluated independently of each other.
An extensive analytical characterization may reveal differences between the reference product
and the proposed product, especially when using analytical techniques capable of discriminating
qualitative or quantitative differences in product attributes. Emphasis should be placed on
developing orthogonal quantitative methods to definitively identify any differences in product
attributes. The results of analytical studies assessing functional and physicochemical
characteristics, including, for example, higher order structure, posttranslational modifications,
and impurity and degradation profiles may be an appropriate scientific basis for a selective and
targeted approach to clinical studies to support a demonstration of biosimilarity. It may be useful
to compare differences in the quality attributes of the proposed product with those of the
reference product using a meaningful analysis algorithm16 that covers a large number of
16 See, for example, Kozlowski S, J Woodcock, K Midthun, and RB Sherman, 2011, Developing the Nation’s
Biosimilars Program, N Engl J Med; 365:385–388.

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additional product attributes and their combinations with high sensitivity using orthogonal
methods. Enhanced approaches in manufacturing science, as discussed in the ICH guidance for
industry Q8(R2) Pharmaceutical Development (November 2009), may facilitate production
processes that can better match a reference product’s quality profile.17 Such a strategy could
further quantify the overall similarity between two molecules and may lead to additional bases
for a more selective and targeted approach to subsequent clinical studies.
The type, nature, and extent of any differences between the proposed product and the reference
product, introduced by design or observed from comprehensive analytical characterization of
multiple lots, should be clearly described and discussed (see section VI.A of this guidance). The
discussion should include identification and comparison of relevant quality attributes from
product characterization. The potential clinical effects of observed structural and functional
differences between the two products should be assessed and appropriately justified.
V. 	FACTORS FOR CONSIDERATION IN PERFORMING THE COMPARATIVE
ANALYTICAL ASSESSMENT
When performing the comparative analytical assessment to support a demonstration of
biosimilarity, manufacturers should consider a number of factors, including the following:
A. 	Expression System
Therapeutic protein products can be produced in microbial cells (prokaryotic or eukaryotic), cell
lines (e.g., mammalian, avian, insect, plant), or tissues derived from animals or plants. The
expression construct in a cell line for a proposed product should encode the same primary amino
acid sequence as its reference product. However, minor modifications, such as N- or C-terminal
truncations (e.g., the heterogeneity of C-terminal lysine of a monoclonal antibody) that are not
expected to change the product performance, may be justified, and should be explained by the
sponsor. Possible differences between the chosen expression system (e.g., host cell and the
expression construct) of the proposed product and that of the reference product should be
carefully considered because the type of expression system will affect the types of process- and
product-related substances, impurities, and contaminants (including potential adventitious
agents) that may be present in the protein product. For example, the expression system can have
a significant effect on the types and extent of translational and posttranslational modifications
that are imparted to the proposed product, which may introduce additional uncertainty into the
demonstration that the proposed product is biosimilar to the reference product.
Minimizing differences between the proposed product and reference product expression systems
to the extent possible can enhance the likelihood of producing a biosimilar protein product. Use
of different expression systems will be evaluated on a case-by-case basis.
17 See the ICH guidances for industry Q8(R2), Q9(R1) Quality Risk Management (May 2023), Q10 Pharmaceutical
Quality System (April 2009), and Q11 Development and Manufacture of Drug Substances (November 2012) for
guidance on enhanced approaches in manufacturing science.

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B. 	Manufacturing Process
A comprehensive understanding of all steps in the manufacturing process for the proposed
product should be established during product development. As a scientific matter,
characterization tests, process controls, and specifications that will emerge from information
gained during process development must be specific for the proposed product and manufacturing
process. The use of enhanced approaches to pharmaceutical development, along with quality
risk management and effective quality systems,18 will facilitate the consistent manufacturing of a
high-quality product. As with biological products originally licensed under section 351(a) of the
PHS Act, an application for a biological product submitted for licensure under section 351(k) of
the PHS Act may not incorporate by reference drug substance, drug substance intermediate, or
drug product information contained in a master file (MF).19 Other types of contract
manufacturing arrangements can be considered if the sponsor does not intend to manufacture the
product for licensure.20
A sponsor considering manufacturing changes after completing the initial comparative analytical
assessment or after completing clinical studies intended to support a 351(k) application, before
the initial BLA approval, should demonstrate comparability between the pre- and post-change
proposed product and may need to conduct additional studies. The nature and extent of the
changes may determine the extent of these additional studies. The comparative analytical studies
should include a sufficient number of lots of the proposed biosimilar product used in clinical
studies as well as from the proposed commercial process including lots used to demonstrate
process consistency (e.g., process performance qualification (PPQ)).
C. 	Physicochemical Properties
Physicochemical assessment of the proposed product and the reference product should consider
all relevant characteristics of the protein product (e.g., the primary, secondary, tertiary, and
quaternary structure; posttranslational modifications; and functional activity or activities). The
objective of this assessment is to detect potential differences in quality attributes between the
proposed product and the reference product.
18 See ICH Q8(R2), ICH Q9(R1), ICH Q10, and ICH Q11 for guidance on enhanced approaches in manufacturing
science.
19 21 CFR 601.2(g). Exceptions are described in 21 CFR 601.2(g)(2) and (3). An MF for drug substance, drug
substance intermediate, or drug product information for a biological product may be referenced to support an IND
for a proposed biosimilar product § 601.2(g)(5). Assurance of product quality should be provided on each lot of
material produced by the MF holder. Procedures should also be in place to ensure that the IND sponsor is notified
by the MF holder of significant changes to the MF potentially affecting product quality. The IND sponsor is then
expected to provide notification to the Agency that changes were made to the MF in order to initiate a reevaluation
of the MF.
20 See the guidance for industry Cooperative Manufacturing Arrangements for Licensed Biologics (November
2008).

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The sponsor should address the concept of the desired product (and its variants) as discussed
in ICH Q6B21 when designing and conducting the characterization studies. Thus, it will be
important to understand the heterogeneity of the proposed product and the reference product
(e.g., the nature, location, and levels of glycosylation) and the ranges of variability of different
isoforms, including those that result from posttranslational modifications.
Analytical methodologies can be used to assess specific physicochemical characteristics of
proteins. These methodologies are described in published documents, including scientific
literature, regulatory guidelines, and pharmacopeial methods. Some techniques provide
information on multiple characteristics. 	Appropriate analytical test methods should be selected
based on the nature of the protein being characterized and knowledge regarding the structure and
heterogeneity of the reference product and the proposed product, as well as characteristics
critical to product performance.
To address the full range of physicochemical properties or biological activities adequately, it is
often necessary to apply more than one analytical procedure to evaluate the same quality
attribute. Methods that use different physicochemical or biological principles to assess the same
attribute are especially valuable because they provide independent data to support the quality of
that attribute (e.g., orthogonal methods to assess aggregation). In addition, the use of
complementary analytical techniques in series, such as peptide mapping or capillary
electrophoresis combined with mass spectrometry of the separated molecules, should provide a
meaningful and sensitive method for comparing products.
Unlike routine quality control assays, methods used to characterize the product do not
necessarily need to be validated; however, the methods used to characterize the product should
be scientifically sound, fit for their intended use, and provide results that are reproducible and
reliable. In selecting these methods, it is important to consider the characteristics of the protein
product, including known and potential impurities. Information regarding the ability of a method
to discern relevant differences between a proposed product and a reference product should be
submitted by the applicant as part of the comparison. The methods should be demonstrated to be
of appropriate sensitivity and specificity to provide meaningful information as to whether the
proposed product and the reference product are highly similar.
D. 	Functional Activities
Functional assays serve multiple purposes in the characterization of protein products. These tests
complement physicochemical analyses and are a quantitative measure of the function of the
protein product.
Depending on the structural complexity of the protein and available analytical technology, the
physicochemical analysis may be unable to confirm the integrity of the higher order structures.
Instead, the integrity of such structures can usually be inferred from the product’s biological
activity. If the clinically relevant mechanism or mechanisms of action are known for the
reference product or can reasonably be determined, the functional assays should reflect such
21 See ICH Q6B.

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mechanism or mechanisms of action to the extent possible.22 Multiple functional assays should,
in general, be performed as part of the comparative analytical assessments. The assessment of
functional activity is also useful in providing an estimate of the specific activity of a product as
an indicator of manufacturing process consistency, as well as product purity, potency, and
stability.
If the intended reference product exhibits multiple functional activities, sponsors should perform
a set of appropriate assays designed to evaluate the range of relevant activities for that product.
For example, for proteins that possess multiple functional domains expressing enzymatic and
receptor-mediated activities, or monoclonal antibodies that have both target and effector
functions, sponsors should evaluate each activity. For products for which functional activity can
be measured by more than one parameter (e.g., enzyme kinetics or interactions with blood
clotting factors), the comparative characterization of each parameter between products should be
assessed.
The sponsor should recognize the potential limitations of some types of functional assays, such
as high variability, that might preclude detection of small but significant differences between the
proposed product and the reference product. Because an assay that lacks precision or accuracy
may not provide a meaningful assessment to inform whether the proposed product is highly
similar to the reference product, sponsors are encouraged to develop functional assays that are
less variable and are sensitive to potential changes in the functional activities of the proposed
product.
In addition, in vitro functional assays should be considered in the context of other product
information such as glycosylation, post-translational modification and whether or not there may
be subtle differences in these attributes. Finally, functional assays may aid in designing
neutralizing antibody assays to be used in clinical immunogenicity assessments.
Target binding assays provide additional critical information about certain aspects of the
mechanism of action and the integrity of the higher order structures of the proposed product.
These analytical tests should be performed to characterize the proposed product in terms of its
specific binding properties, as appropriate. For example, if binding to a receptor is inherent to
protein function, this property should be measured and evaluated in the comparative analytical
studies (see ICH Q6B for additional details). Various methods, such as surface plasmon
resonance, enzyme-linked immunosorbent assay, microcalorimetry, or classical Scatchard
analysis, can provide information on the kinetics and thermodynamics of binding. Such
information can be related to the functional activity and characterization of the proposed
product’s higher order structure and should be proposed in early discussions with the Agency.
22 Generally, to the extent the mechanism or mechanisms of action are known for the reference product, such
information can be found in publicly available information regarding the Secretary’s previous determination that the
reference product is safe, pure, and potent. See section 351(k)(2)(A)(iii)(I) of the PHS Act; see also Q.I.13 of the
guidance for industry Questions and Answers on Biosimilar Development and the BPCI Act (September 2021)
(explaining that “publicly-available information” in this context generally includes the current FDA-approved
labeling for the reference product and the types of information found in the “action package” for a BLA). In
addition, sponsors may also consider submitting any additional information in support of the application, including
publicly-available information about known or putative mechanisms of action for the reference product to
scientifically justify the scope of functional assays to be included in the comparative analytical assessment.

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E. 	Impurities
The sponsor should characterize, identify, and quantify product-related impurities in the
proposed product and the reference product, to the extent feasible.23 If a comparative
physicochemical analysis reveals comparable product-related impurities at similar levels for both
products or at lower levels for the proposed product, pharmacological/toxicological studies to
characterize potential biological effects of specific impurities may not be necessary. However, if
the manufacturing process used to produce the proposed product introduces different impurities
or higher levels of impurities than those present in the reference product, additional
pharmacological/toxicological or other studies may be necessary. As discussed in the ICH
guidance for industry S6(R1) Preclinical Safety Evaluation of Biotechnology-Derived
Pharmaceuticals (May 2012), “[i]t is preferable to rely on purification processes to remove
impurities … rather than to establish a preclinical testing program for their qualification.”
Process-related impurities arising from cell substrates (e.g., host cell DNA, host cell proteins),
cell culture components (e.g., antibiotics, media components), and downstream processing steps
(e.g., reagents, residual solvents, leachables) should be evaluated. The process-related impurities
in the proposed product are not expected to match those observed in the reference product and
are generally not evaluated as part of the comparative analytical assessment.24 The chosen
analytical procedures should be adequate to detect, identify, and accurately quantify impurities.
25 In particular, results of immunological methods used to detect host cell proteins depend on the
assay reagents and the cell substrate used. Such assays should be validated using the proposed
product cell substrate and orthogonal methodologies to ensure accuracy and sensitivity.
As with any biological product, the safety of the proposed product with regard to adventitious
agents or endogenous viral contamination should be ensured by screening critical raw materials
and process intermediates and confirmation of robust virus removal and inactivation achieved by
the manufacturing process.26
F. 	Reference Product
A thorough physicochemical and biological assessment of the reference product should provide a
base of information from which to develop the proposed product and justify reliance on e.g.,
FDA’s findings of safety, purity, and potency as described in FDA-approved labeling for the
reference product.
23 The use of the terms product- and process-related impurities is consistent with their use and meaning in ICH
Q6B.
24 Process-related impurities are expected to be evaluated as part of process development and validation.
25 See the ICH guidance for industry Q2B Validation of Analytical Procedures: Methodology (May 1997).
26 See the ICH guidance for industry Q5A Viral Safety Evaluation of Biotechnology Products Derived From Cell
Lines of Human or Animal Origin (September 1998).

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If the protein has been extracted from the reference product to conduct analytical studies, the
sponsor should describe the extraction procedure and demonstrate that the procedure itself does
not alter relevant product quality attributes. This undertaking would include consideration of
alteration or loss of the desired products and impurities and relevant product-related substances,
and it should include appropriate controls to ensure that relevant characteristics of the protein are
not significantly altered by the extraction procedure.
G. 	Reference Materials
An in-house reference material should always be qualified and used for control of the
manufacturing process and product.27
An in-house reference material is typically developed from early development lots or lots used in
a clinical study or studies. Additional reference materials may be qualified later in development
and for a BLA submission. A sponsor should establish and properly qualify primary and
working reference materials that are representative of proposed product lots used in clinical
studies that support the application.
For the development of a proposed product, a reference product lot or pool of several reference
product lots, or a lot of a non-U.S.-licensed comparator product (see section VI. A. 4 of this
guidance) may be qualified as an initial reference material. The sponsor may also qualify a
development lot of the proposed product as an initial reference material by demonstrating that
the development lot is within the reference product’s ranges. Once clinical lots, or lots that have
been demonstrated to be representative of the clinical lots, have been manufactured, one of these
lots should be properly qualified (including bridging to previous reference materials) for use as a
reference material for release and stability, as well as comparative analytical testing. If possible,
once an in-house reference material is properly qualified, there should be sufficient quantities to
use throughout the development of the proposed product. All lots of reference materials used
during the development of a proposed product should be properly qualified. As a scientific
matter, as part of qualification, each new reference material needs to be bridged to previous
reference materials. In addition to release testing methods, the qualification protocol for
reference materials should include all relevant analytical methods to fully characterize the
reference material.
For all methods for which the result is reported relative to the reference material, the
qualification of the reference material should be appropriate considering, among other things, the
nature of the result and the criticality of the quality attribute. For example, the assignment of a
potency of 100% to a reference material should include a narrow acceptable potency range and
27 See ICH Q6B. Where an international or national standard is available and appropriate, reference materials
should be calibrated against it. If there is a suitable, publicly available, and well-established international reference
standard for the protein, a physicochemical and/or functional comparison of certain quality attributes of the
proposed product with this standard may also provide useful information. For example, if an international reference
standard for calibration of potency is available, a comparison of the relative potency of the proposed product with
this potency standard may be useful. Although comparison with an international or national reference standard may
be useful, that alone is not sufficient to satisfy the PHS Act’s requirement to demonstrate the biosimilarity of the
proposed product to the U.S.-licensed reference product.

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ensure control over product drift. For example, a sponsor could consider the use of a
predetermined two-sided confidence interval (CI) of the mean of the replicates, where the mean
relative potency and the 95% CI are included within a sufficiently narrow range (e.g., 90% to
110%). There should be an evaluation across the qualification history of multiple reference
materials to address potential drift.
A sponsor generally should not use a correction factor to account for any differences in, for
example, potency or biological activity between reference materials. Under certain situations,
the use of a small correction factor or factors may be considered if proposed and scientifically
justified by the sponsor. 	If a sponsor intends to propose the use of a correction factor,
discussion with the Agency during product development is recommended.
Use of reference materials inadequately qualified and/or bridged for analytical methods that
report results relative to the reference material is likely to raise concerns regarding the
comparative analytical assessment. Before submission of a 351(k) application, the prospective
applicant may choose to retest the proposed product, reference product, and, if applicable, non-
U.S.-licensed comparator product lots using the same reference materials for those methods that
report the result relative to the reference material for the final comparative analytical assessment.
H. 	Finished Drug Product
Product characterization studies of a proposed product should be performed on the most
downstream intermediate best suited for the analytical procedures used. The attributes evaluated
should be stable through any further processing steps. For these reasons, characterization studies
are often performed on the drug substance. However, if a drug substance is reformulated and/or
exposed to new materials in the finished dosage form, the impact of these changes should be
considered. Whenever possible, if the finished drug product is best suited for a particular
analysis, the sponsors should analyze the finished drug product. If an analytical method more
sensitively detects specific attributes in the drug substance but the attributes it measures are
critical and/or may change during manufacture of the finished drug product, comparative
characterization on both the drug substance and the finished drug product is recommended.
Proteins are very sensitive to their environment. Therefore, differences in excipients or primary
packaging may affect product stability and/or clinical performance. Differences in formulation
and primary packaging28 between the proposed product and the reference product are among the
factors that may affect whether or how subsequent clinical studies may take a selective and
targeted approach.29 Sponsors should clearly identify excipients used in the proposed product
that differ from those in the reference product. The acceptability of the type, nature, and extent
of any differences between the finished proposed product and the finished reference product
should be evaluated and supported by appropriate data and rationale. Additionally, different
excipients in the proposed product should be supported by existing toxicology data for the
28 See ICH Q8(R2).
29 For more discussion on selective and targeted approaches, please refer to the guidance for industry Scientific
Considerations in Demonstrating Biosimilarity to a Reference Product.

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excipient or by additional toxicity studies with the formulation of the proposed product if the
excipient safety profile is not well known. Excipient interactions as well as direct toxicities
should be considered.
I. 	Stability
As part of an appropriate physicochemical and functional comparison of the stability profile of
the proposed product with that of the reference product, accelerated and stress stability studies,
as well as forced degradation studies, should be used to establish degradation profiles and to
provide a direct stability comparison of the proposed product with the reference product. These
comparative studies should be conducted under forced degradation conditions (e.g., high
temperature, freeze thaw, light exposure, and agitation) that can cause incremental product
degradation over a defined time period. Results of these studies may reveal product differences
that warrant additional evaluation and also identify conditions under which additional controls
should be employed in manufacturing and storage.30 	As differences in formulation could affect
the stability profile, any profile differences should be scientifically justified. Sufficient real-time,
real-condition stability data from the proposed product should be provided to support the
proposed shelf life.
VI. 	COMPARATIVE ANALYTICAL ASSESSMENT
A thorough understanding of the reference product is critical for a successful biosimilar
development program. The Agency recommends that sponsors approach the comparative
analytical assessment by first understanding the physicochemical and biological characteristics
of the reference product. A full characterization of the reference product, in addition to
consideration of publicly available information, will form the basis of product understanding. As
described previously, protein products are complex molecules that generally are manufactured in
living cells and purified using a variety of technologies; therefore, they have a certain degree of
inherent lot-to-lot variability in terms of quality characteristics. The observed lot-to-lot
variability may derive from manufacturing conditions and from analytical assay variability.
Factors that contribute to lot-to-lot variability in the manufacture of a protein product include the
source of certain raw materials (e.g., growth medium, resins, or separation materials) and
different manufacturing sites. Therefore, in the comparative analytical assessment, it is
important to adequately characterize the lot-to-lot variability of the reference product and the
proposed biosimilar product.
A. 	Considerations for Reference and Biosimilar Products
1. 	Reference Product
To ensure that the full range of product variability is accurately captured, sponsors should
acquire multiple reference product lots throughout the development program of a proposed
30 See ICH guidances for industry Q5C Quality of Biotechnological Products: Stability Testing of
Biotechnological/Biological Products (July 1996) and Q1A(R2) Stability Testing of New Drug Substances and
Products (November 2003).

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biosimilar in sufficient quantity to conduct multiple physiochemical and functional assays.
Considering the inherent heterogeneity present in protein products and the expected lot-to-lot
variability stemming from manufacturing processes, the Agency recommends that a sponsor
include at least 10 reference product lots (acquired over a time frame that spans expiration dates
of several years) in the analytical assessment to ensure that the variability of the reference
product is captured adequately. Lots used in the comparative PK, PD (if applicable) and/or
clinical studies should be included in the comparative analytical assessment. The final number
of lots should be sufficient to provide adequate information regarding the variability of the
reference product. In cases where limited numbers of reference product lots are available (e.g.,
for certain orphan drugs), alternate flexible comparative analytical assessment plans should be
proposed and discussed with the Agency.
2. 	Proposed Product
The Agency recommends that a sponsor include at least 6 to 10 lots of the proposed product in
the comparative analytical assessment to ensure 1) adequate characterization of the proposed
product and understanding of manufacturing variability and 2) adequate comparison to the
reference product. These should include lots used in the clinical studies, lots manufactured with
commercial-scale processes, and validation lots, and may include additional lots manufactured at
different scales, including engineering lots. These lots should be representative of the intended
commercial manufacturing process. If there is a manufacturing process change during
development and comparability of the product was demonstrated, it may be possible, with
adequate scientific justification, to use data generated from lots manufactured by both processes
in the comparative analytical assessment. These data should be provided in the 351(k) BLA to
support comparability of drug substance and drug product manufactured with the different
processes and/or scales. 	The extent of process development design (as described in the
guidance documents ICH Q8(R2) Pharmaceutical Development and ICH Q11 Development and
Manufacture of Drug Substances) and process understanding should be used in support of the
number of proposed biosimilar product lots proposed for inclusion in the comparative analytical
assessment in the 351(k) application.
To the extent possible, proposed biosimilar lots included in the comparative analytical
assessment described in section VI. B, Considerations for Data Analysis, should be derived from
independent drug substance batches to adequately represent the variability of attributes inherent
to the drug substance manufacturing process. Drug product lots derived from the same drug
substance batch or batches are not considered sufficiently representative of such variability,
except for use in testing certain drug product attributes for which variability is mostly dependent
on the drug product manufacturing process (e.g., protein concentration). Although it is
preferable to compare the proposed drug product lots to the reference product lots, it may be
acceptable to also include independent drug substance batches (if the drug substance was not
used to make drug product), if needed, to attain a sufficient number of lots for the comparative
analytical assessment.
3. 	Reference Product and Non-U.S.-Licensed Comparator Products

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As described in other guidances,31 if a sponsor seeks to use a non-U.S.-licensed comparator in
certain studies to support a demonstration that the proposed biological product is biosimilar to
the U.S.-licensed reference product, the sponsor should provide adequate data or information to
scientifically justify the relevance of these comparative data to an assessment of biosimilarity by
establishing an acceptable bridge to the U.S.-licensed reference product. With respect to
comparative analytical bridging data, the sponsor should provide comparative analytical data and
analysis for each of the three pairwise comparisons (i.e., proposed biosimilar product versus
U.S.-licensed reference product, proposed biosimilar product versus non-U.S.-licensed
comparator product, and non-U.S.-licensed comparator product versus U.S.-licensed reference
product).
The acceptance criteria used to support a demonstration that a proposed biosimilar product is
highly similar to the reference product should be derived from data generated from a sponsor’s
analysis of the reference product. The comparative analytical assessment should be based on a
direct comparison of the proposed product to the reference product. As a scientific matter,
combining data from the reference product and non-U.S.-licensed comparator product to
determine the acceptance criteria or to perform the comparative analytical assessment for the
proposed product would not be acceptable to support a demonstration that the proposed product
is biosimilar to the reference product.
Sponsors are encouraged to discuss with FDA, as early as feasible during product development,
any plans to submit data derived from comparator products approved outside of the United States
(i.e., non-U.S.-licensed comparator products) in support of a 351(k) application.
4. 	Accounting for Proposed Product, Reference Product, and Non-U.S.-Licensed
Comparator Product Lots
Sponsors should account for all the reference product lots acquired and characterized. The
351(k) BLA should include data and information from all reference product and proposed
product lots that were evaluated during development, including the specific physicochemical,
functional, and clinical studies for which a lot was used. When a lot is specifically selected to be
included in or excluded from certain analytical studies, a justification should be provided. The
date of the analytical testing as well as the product expiration date should be provided in the
application. In general, expired reference product lots should not be included in the comparative
analytical assessment because lots analyzed beyond their expiration date could lead to results
outside the range that would normally be observed in unexpired lots, which may result in
overestimated reference product variability. Testing of lots past expiry may be acceptable if
samples are stored under adequate conditions that ensures the quality attributes of the product
remain stable (e.g., frozen at -80°C) and provided that sponsors submit data and information
demonstrating that storage does not impact the quality of the product.
31 See guidance for industry Scientific Considerations in Demonstrating Biosimilarity to a Reference Product and
question Q.I.8 and its answer in the guidance for industry Questions and Answers on Biosimilar Development and
the BPCI Act.

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The same type of information and data described above to be collected for reference product lots
should also be provided on every manufactured drug substance and drug product lot of the
proposed product and as applicable, any non-U.S.-licensed comparator product lots.
Lots of the proposed product, reference product, and/or, as applicable, non-U.S.-licensed
comparator product used in a clinical study should be included in the comparative analytical
assessment.
B. 	Considerations for Data Analysis
Sponsors should develop a comparative analytical assessment plan and discuss the approach with
the Agency as early as practicable. A final comparative analytical assessment report should be
available at the time a 351(k) BLA is submitted.
The Agency recommends development of a comparative analytical assessment plan using a
stepwise approach. The first step is a determination of the quality attributes that characterize the
reference product in terms of its structural/physicochemical and functional properties. These
quality attributes are then ranked according to their risk to potentially impact activity, PK, PD,
safety, efficacy, and immunogenicity. Finally, the attributes are evaluated using quantitative
analysis, considering the risk ranking of the quality attributes, as well as other factors. It should
be noted, however, that some attributes may be highly critical (e.g., protein sequence) but not
amenable to quantitative analysis.
1. 	Risk Assessment
FDA recommends that sponsors develop a risk assessment tool to evaluate and rank the reference
product quality attributes in terms of potential impact on the mechanism or mechanisms of action
and function of the product.32 Certain quality evaluations of the reference product (e.g., its
degradation rates, which are determined from stability or forced degradation studies) generally
should not be included in the risk ranking. However, these evaluations should still factor into the
comparative analytical assessment of the proposed biosimilar and reference product.
For the purpose of the comparative analytical assessment, development of the risk assessment
tool should be informed by relevant factors, including the following:
• 	Potential impact of an attribute on clinical performance: Specifically, FDA recommends
that sponsors consider the potential impact of an attribute on activity, PK, PD, safety,
efficacy, and immunogenicity. Sponsors should consider publicly available information
regarding FDA’s previous determination that the reference product is safe, pure, and
32 This guidance addresses development of a risk assessment tool for purposes of the comparative analytical
assessment for a proposed biosimilar product. A risk assessment tool used for the purpose of a comparative
analytical assessment may also inform the risk assessment used to design the propose biosimilar product’s
manufacturing and control strategy. Considerations for the quality risk management of pharmaceutical products,
including proposed biosimilars, are outside the scope of this guidance and can be found in ICH Q9.

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potent (e.g., approved product labeling) and other publicly-available information (e.g.,
published literature), as well as the sponsor’s own characterization of the reference
product, in determining the potential impact of an attribute on clinical performance.
• 	The degree of uncertainty surrounding a certain quality attribute: The degree of
uncertainty can impact the critical quality attribute (CQA) risk ranking. For example,
when there is limited understanding of the relationship between the degree of change in a
potentially low-risk attribute and the resulting clinical impact, FDA recommends that that
attribute be ranked higher in the CQA risk ranking because of the uncertainty raised.
FDA recommends that an attribute that is a high risk for any one of the performance categories
(i.e., activity of the molecule, PK, PD, safety, efficacy, and immunogenicity) be classified as
high risk. Ideally, the risk assessment tool should result in a list of attributes ordered by the risk
to the patient. The risk scores for attributes should, therefore, be proportional to patient risk.
The scoring criteria used in the risk assessment should be clearly defined and justified, and the
risk ranking for each attribute should be justified with appropriate citations to the literature and
data provided.
2. 	Quantitative and Qualitative Data Analysis
Appropriate analyses of the comparative analytical data are necessary to support a demonstration
that the proposed product is highly similar to the reference product notwithstanding minor
differences in clinically inactive components.33 One approach to data analysis would be the use
of descriptive quality ranges (QR) for assessing quantitative quality attributes of high and
moderate risk, and the use of raw data/graphical comparisons for quality attributes with the
lowest risk ranking or for those quality attributes that cannot be quantitatively measured (e.g.,
primary sequence). The data collected for some attributes may need both quantitative and
qualitative analyses. For example, the quantitative analysis for potency assays should be
accompanied by a qualitative comparison or dose-response curves. The acceptance criteria for
the QR method in the comparative analytical assessment should be based on the results of the
sponsor’s own analysis of the reference product for a specific quality attribute. The QR should
be defined as (𝜇𝜇̂ 𝑅𝑅 − 𝑋𝑋𝜎𝜎�𝑅𝑅, 𝜇𝜇̂ 𝑅𝑅 + 𝑋𝑋𝜎𝜎�𝑅𝑅) , where 𝜇𝜇̂ 𝑅𝑅 is the sample mean and 𝜎𝜎�𝑅𝑅 is the sample
standard deviation based on the reference product lots.
The multiplier (X) should be scientifically justified for that attribute. Generally, the application
of 3 standard deviations (SDs) has provided adequately constrained quality ranges. However, a
multiplier smaller than 3 may be necessary for certain high-risk attributes. Use of a multiplier
greater than 3 SDs should be discussed with the Agency. Based on our experience to date,
methods such as tolerance intervals are not recommended for establishing the similarity
acceptance criteria because a very large number of lots (typically 30) would be required to
establish meaningful intervals. The sponsor can propose other methods of data analysis,
including equivalence testing.
33 See section 351(i)(2)(A) of the PHS Act.

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The objective of the comparative analytical assessment is to verify that each attribute, as
observed in the proposed biosimilar and the reference product, has a similar distribution.
Comparative analysis of a quality attribute would generally support a finding that the proposed
product is highly similar to the reference product when a sufficient percentage of biosimilar lot
values (e.g., 90%) fall within the QR defined for that attribute.
In addition to risk ranking, other factors should be considered in determining which type of
quantitative data analysis should be applied to a particular attribute or assay. Some additional
factors that should be considered when determining the appropriate type of data evaluation and
analysis of results include the following:
• 	Nature of the attribute: Attributes that are known to be of high risk should be prioritized
over attributes with unknown but potentially high risk (i.e., attributes with a high-risk
ranking attributable to uncertainty).
• 	Distribution of the attribute: In general, the Agency recommends that sponsors develop
the manufacturing process to target the center of distribution of the quality attributes of
the reference product as closely as possible. For example, the distribution of an attribute
in the proposed biosimilar product that is biased toward one side of the reference product
distribution may raise concerns depending on the nature of the attribute and the role the
attribute plays in, for example, the mechanism of action. If such a distribution is
observed, appropriate justification should be provided, as a scientific matter, to support
the comparative analytical assessment of the proposed product with the reference product
(e.g. control strategy ensures containment within the reference product range; sufficient
product knowledge is available to exclude a potential impact on clinical performance). In
cases where an attribute in the reference product is not normally distributed or when the
mean of distribution shifts significantly over time, sponsors should consult with the
Agency.
• 	Abundance of the attribute: The abundance of the attribute should be confirmed in both
the reference product (as determined by the proposed product sponsor’s analysis of the
reference product) and the proposed product. Limit assays do not necessarily need to be
evaluated using QR; however, the selected limits regarding the amount of an attribute
should be defined and justified. The justification should also include consideration of
how the amount of the attribute changes over time.
• 	Sensitivity of assay used for assessing an attribute: Although multiple, orthogonal assays
are encouraged for assessing an attribute, not all assays assessing the attribute need to be
evaluated in the same manner. Although the most sensitive assay for detecting product
differences should be evaluated using QR, it may be appropriate to evaluate the results of
other assays for the same attribute using a graphical comparison. A justification should
be provided for the method of evaluation used for each type of assay.
• 	Types of attributes/assays: Quantitative analyses may not be applicable to some
attributes, (e.g., protein sequence or certain assays used for higher order structure
evaluation, or assays that are only qualitative). The comparative analytical assessment

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plan should clearly define specific assays where quantitative data analyses would not be
applied and the rationale for that decision.
• 	Publicly available information: Publicly available information may be relevant to the
appropriate type of data analysis and acceptance criteria in the comparative analytical
assessment. A sponsor should seek additional advice from the Agency on the inclusion
of any publicly available information in the comparative analytical assessment.
For qualitative analyses of lower risk attributes, FDA recommends side-by-side data presentation
(e.g., spectra, thermograms, graphical representation of data) to allow for a visual comparison of
the proposed product to the reference product.
The final comparative analytical assessment plan should include the risk ranking of attributes,
the type of data evaluation to be used for each attribute/assay, and the final data analysis plan.
The plan should specify the anticipated availability of both proposed biosimilar and reference
product lots for evaluation of each attribute/assay and should include a rationale for why the
proposed number of lots should be considered sufficient for the evaluation. The comparative
analytical assessment plan should be discussed with the Agency as early in the biosimilar
development program as possible so that agreement can be reached on which attributes/assays
should be evaluated. The final comparative analytical assessment plan is recommended to be
submitted to the Agency before initiating the final analytical assessments; typically, this occurs
in a meeting with the Agency.
C. 	Comparative Analytical Assessment Conclusions
In the comparative analytical assessment, risk ranking and data analysis are used to evaluate a
large number of attributes, which are often assessed using multiple orthogonal assays. FDA
evaluates the totality of the analytical data; if the results of a particular assay do not meet
prespecified criteria, this alone does not preclude a demonstration that the proposed product is
highly similar to the reference product notwithstanding minor differences in clinically inactive
components. For example, if differences between products are observed as part of the
comparative analytical assessment, the sponsor may provide additional scientific information
(risk assessment and additional data) and a justification for why these differences do not preclude
a demonstration that the products are highly similar.
In certain situations, changes to the manufacturing process of the biosimilar product may be
needed to resolve differences observed in the comparative analytical assessment. Data should be
provided demonstrating that the observed differences were resolved by any manufacturing
changes and that other quality attributes were not substantially affected. If other attributes were
affected by the manufacturing change, data should be provided to demonstrate that the impact of
the change has been evaluated and addressed. The nature and extent of the changes may
determine the extent of the data needed and may include additional comparative analytical
studies of the proposed biosimilar and reference product to support the 351(k) application.

Contains Nonbinding Recommendations
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VII. 	CONCLUSION
The foundation for an assessment and a demonstration of biosimilarity between a proposed
product and its reference product includes analytical studies that demonstrate that the proposed
product is highly similar to the reference product notwithstanding minor differences in clinically
inactive components. The demonstration that the proposed product is biosimilar to the reference
product involves robust characterization of the proposed product, including comparative
physicochemical and functional studies with the reference product. The information gained from
these studies is necessary for the development of a proposed product as a biosimilar. In addition,
a 351(k) application for a proposed product must contain, among other things, information
demonstrating biosimilarity based on data derived from an assessment of toxicity and a clinical
study or studies (including the assessment of immunogenicity and PK or PD), unless the Agency
determines that an element is unnecessary in a particular 351(k) application.34 A sponsor’s
ability to discern and understand the impact of relevant analytical differences between the
proposed product and its reference product is critical to determining whether the statutory
standard for biosimilarity can be met.
34 Section 351(k)(2)(A)(i)(I) of the PHS Act. See also section 351(k)(2)(A)(ii) of the PHS Act.