Development of Therapeutic Protein 
Biosimilars:  Comparative Analytical 
Assessment and Other Quality-Related 
Considerations  
 
Guidance for Industry 
 
 
DRAFT GUIDANCE
 
 
  This guidance document is being distributed for comment purposes only. 
 
Comments and suggestions regarding this draft document should be submitted within 60 days of 
publication in the Federal Register of the notice announcing the availability of the draft 
guidance.  Submit electronic comments to https://www.regulations.gov.  Submit written 
comments to the Dockets Management Staff (HFA-305), Food and Drug Administration, 5630 
Fishers Lane, Rm. 1061, Rockville, MD  20852.  All comments should be identified with the 
docket number listed in the notice of availability that publishes in the Federal Register. 
 
For questions regarding this draft document, contact (CDER) Sandra Benton, 301-796-1042, or 
(CBER) Office of Communication, Outreach and Development, 800-835-4709 or 240-402-8010.  
 
 
 
 
 
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) 
 
 
 
 
May 2019 
Biosimilars 
 
 
   
Development of Therapeutic Protein 
Biosimilars:  Comparative Analytical 
Assessment and Other Quality-Related 
Considerations   
 
Guidance for Industry 
 
 
Additional copies are available from: 
 
Office of Communications, Division of Drug Information 
Center for Drug Evaluation and Research 
Food and Drug Administration 
10001 New Hampshire Ave., Hillandale Bldg., 4th Floor  
Silver Spring, MD 20993-0002  
Phone:  855-543-3784 or 301-796-3400; Fax:  301-431-6353 
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 
10903 New Hampshire Ave., Bldg. 71, Room 3128 
Silver Spring, MD 20993-0002 
Phone:  800-835-4709 or 240-402-7800 
Email:  ocod@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) 
  
 
May 2019 
Biosimilars 
 
  Contains Nonbinding Recommendations 
Draft  Not for Implementation 
 
TABLE OF CONTENTS 
 
 
I.  INTRODUCTION............................................................................................................. 1 
II.  BACKGROUND ............................................................................................................... 3 
III.  SCOPE ............................................................................................................................... 6 
IV.  GENERAL PRINCIPLES ............................................................................................... 6 
V.  FACTORS FOR CONSIDERATION IN PERFORMING THE COMPARATIVE 
ANALYTICAL ASSESSMENT ................................................................................................ 10 
A.  Expression System ....................................................................................................................... 11 
B.  Manufacturing Process ................................................................................................................ 11 
C.  Physicochemical Properties ......................................................................................................... 12 
D.  Functional Activities .................................................................................................................... 13 
E.  Target Binding ............................................................................................................................. 14 
F.  Impurities ..................................................................................................................................... 14 
G.   Reference Product and Reference Standards ............................................................................ 15 
H.  Finished Drug Product ................................................................................................................ 17 
I.   Stability ......................................................................................................................................... 18 
VI.   COMPARATIVE ANALYTICAL ASSESSMENT..................................................... 18 
A.  Considerations for Reference and Biosimilar Products ........................................................... 19 
1. Reference Product .......................................................................................................................... 19 
2. Proposed Product .......................................................................................................................... 19 
3. Accounting for Reference Product and Proposed Product Lots .................................................... 20 
4. Reference Product and Non-U.S.-Licensed Comparator Products ............................................... 20 
B.  Considerations for Data Analysis ............................................................................................... 21 
1. Risk Assessment ............................................................................................................................. 21 
2. Quantitative and Qualitative Data Analysis .................................................................................. 22 
C.  Comparative Analytical Assessment Conclusions .................................................................... 24 
VII.  CONCLUSION ............................................................................................................... 24 
VIII.  RELEVANT GUIDANCES ........................................................................................... 25 
GLOSSARY................................................................................................................................. 28 
i  Contains Nonbinding Recommendations 
Draft  Not for Implementation 
 
 
Development of Therapeutic Protein Biosimilars:  Comparative 
1 
Analytical Assessment and Other Quality-Related Considerations   
2 
 
3 
Guidance for Industry1 
4 
5   
6   
7  This draft guidance, when finalized, will represent the current thinking of the Food and Drug 
8  Administration (FDA or Agency) on this topic.  It does not establish any rights for any person and is not 
9  binding on FDA or the public.  You can use an alternative approach if it satisfies the requirements of the 
10  applicable statutes and regulations.  To discuss an alternative approach, contact the FDA staff responsible 
11  for this guidance as listed on the title page.   
12   
13   
14   
15   
16  I.  INTRODUCTION  
17   
18  This guidance describes the Agencys recommendations on the design and evaluation of 
19  comparative analytical studies intended to support a demonstration that a proposed therapeutic 
20  protein product is biosimilar to a reference product licensed under section 351(a) of the Public 
21  Health Service Act (PHS Act).  Additionally, this guidance is intended to provide 
22  recommendations to sponsors on the scientific and technical information for the chemistry, 
23  manufacturing, and controls (CMC) portion of a marketing application for a proposed product 
24  submitted under section 351(k) of the PHS Act. 
25   
26  The Biologics Price Competition and Innovation Act of 2009 (BPCI Act) amends the PHS Act 
27  and other statutes to create an abbreviated licensure pathway in section 351(k) of the PHS Act 
28  for biological products shown to be biosimilar to, or interchangeable with, an FDA-licensed 
29  reference product (see sections 7001 through 7003 of the Patient Protection and Affordable Care 
30  Act (ACA) (Public Law 111-148).  Although the 351(k) pathway applies generally to biological 
31  products, this guidance focuses on therapeutic protein products and provides an overview of 
32  recommendations for the comparative analytical assessment and other important scientific 
33  considerations to support a demonstration of biosimilarity between a proposed therapeutic 
                                                 
1 This draft 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. 
 
 
 
 
 
  1 Contains Nonbinding Recommendations 
Draft  Not for Implementation 
 
 
34  protein product (referred to as a proposed biosimilar2 or proposed biosimilar product) and the 
35  reference product. 3   
36   
37  This guidance is one in a series of guidances that FDA is developing to facilitate implementation 
38  of the BPCI Act.   
39   
40  Relevant final guidance documents4 issued to date address a broad range of issues, including:   
41   
42    Scientific Considerations in Demonstrating Biosimilarity to a Reference Product 
43  (April 2015) 
44    Questions and Answers on Biosimilar Development and the BPCI Act (December 
45  2018) 
46    Clinical Pharmacology Data to Support a Demonstration of Biosimilarity to a 
47  Reference Product (December 2016) 
48    Labeling for Biosimilar Products (July 2018) 
49    Considerations in Demonstrating Interchangeability With a Reference Product 
50  (May 2019) 
51   
52  In addition, FDA has published draft guidance documents related to the BPCI Act, which, when 
53  finalized, will represent FDAs current thinking.  These draft guidance documents include: 
54   
55    Formal Meetings Between the FDA and Sponsors or Applicants of BsUFA 
56  Products (June 2018) 
57    Reference Product Exclusivity for Biological Products Filed Under Section 
58  351(a) of the PHS Act (August 2014) 
59    New and Revised Draft Q&As on Biosimilar Development and the BPCI Act 
60  (Revision 2) (December 2018) 
61   
                                                 
2 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 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).   
 
3 A 351(k) application for a proposed biosimilar product must 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.  Section 
351(k)(2)(A)(i)(I)(aa) of the PHS Act. 
 
4 We update guidances periodically.  For the most recent version of a guidance, check the FDA guidance web page 
at https://www.fda.gov/regulatory-information/search-fda-guidance-documents.   
  2 Contains Nonbinding Recommendations 
Draft  Not for Implementation 
 
 
62  When applicable, references to information in these final and draft guidances are included in this 
63  guidance. 
64   
65  In general, FDAs guidance documents do not establish legally enforceable responsibilities.  
66  Instead, guidances describe the Agencys current thinking on a topic and should be viewed only 
67  as recommendations, unless specific regulatory or statutory requirements are cited.  The use of 
68  the word should in Agency guidances means that something is suggested or recommended, but 
69  not required.  
70   
71   
72  II.  BACKGROUND 
73   
74  In the 1980s, FDA began to receive marketing applications for biotechnology-derived protein 
75  products, mostly for recombinant DNA-derived versions of naturally sourced products.  
76  Consequently, FDA established a regulatory approach for the approval of recombinant DNA-
77  derived protein products, which was announced in the Federal Register (51 FR 23302, June 26, 
78  1986), in conjunction with a 1985 document titled Points to Consider in the Production and 
79  Testing of New Drugs and Biologicals Produced by Recombinant DNA Technology.5  This 
80  approach addresses the submission of an investigational new drug application (IND) to FDA for 
81  evaluation before initiation of clinical investigations in human subjects and submission and 
82  potential approval of a new drug application (NDA) or biologics license application (BLA) 
83  before marketing products made with recombinant DNA technology, even if the active 
84  ingredient in the product is thought to be identical to a naturally occurring substance or a 
85  previously approved product.  The policy set forth in those documents was developed in part 
86  because of the challenges in evaluating protein products solely by physicochemical and 
87  functional testing and because the biological system in which such a protein product is produced 
88  can have a significant effect on the structure and function of the product itself. 
89   
90  Improvements in manufacturing processes, process controls, materials, and product testing, as 
91  well as characterization tests and studies, have led to a gradual evolution in the regulation of 
92  protein products.  For example, in 1996, FDA provided recommendations in the FDA Guidance 
93  Concerning Demonstration of Comparability of Human Biological Products, Including 
94  Therapeutic Biotechnology-derived Products, which explains how a sponsor may demonstrate, 
95  through a combination of analytical testing, functional assays (in vitro and/or in vivo), 
96  assessment of pharmacokinetics (PK) and/or pharmacodynamics (PD) and toxicity in animals, 
97  and clinical testing (clinical pharmacology, safety, and/or efficacy), that a manufacturing change 
98  does not adversely affect the safety, identity, purity, or potency of its FDA-approved product. 
99   
                                                 
5 For more information, this document is available on FDAs Other Recommendations for Biologics Manufacturers 
web page at https://www.fda.gov/vaccines-blood-biologics/guidance-compliance-regulatory-information-
biologics/other-recommendations-biologics-manufacturers.  
 
  3 Contains Nonbinding Recommendations 
Draft  Not for Implementation 
 
 
100  Since 1996, FDA has approved many manufacturing process changes for licensed biological 
101  products based on a demonstration of product comparability before and after the process change, 
102  as supported by quality criteria and analytical testing and without the need for additional 
103  nonclinical data and clinical safety and/or efficacy studies.  In some cases, uncertainty about the 
104  effect of the change and/or the results of the biochemical/functional comparability studies has 
105  necessitated collection and assessment of additional data, including nonclinical and/or clinical 
106  testing, to demonstrate product comparability.  These concepts were further developed in the 
107  International Conference on Harmonisation of Technical Requirements for Registration of 
108  Pharmaceuticals for Human Use (ICH) and resulted in the ICH guidance for industry Q5E 
109  Comparability of Biotechnological/Biological Products Subject to Changes in Their 
110  Manufacturing Process (June 2005).   
111   
112  Although the scope of ICH Q5E is limited to an assessment of the comparability of a biological 
113  product before and after a manufacturing process change made by the same manufacturer, certain 
114  general scientific principles described in ICH Q5E are applicable to an assessment of 
115  biosimilarity between a proposed product and its reference product.  However, demonstrating 
116  that a proposed product is biosimilar to an FDA-licensed reference product manufactured by a 
117  different manufacturer typically will be more complex and will likely require more extensive and 
118  comprehensive data than assessing the comparability of a product before and after a 
119  manufacturing process change made by the products sponsor.  A manufacturer that modifies its 
120  own manufacturing process has extensive knowledge and information about the product and the 
121  existing process, including established controls and acceptance parameters.  By contrast, the 
122  manufacturer of a proposed biosimilar will have no direct knowledge of the manufacturing 
123  process for the reference product and will have its own manufacturing process (e.g., different cell 
124  line, raw materials, equipment, processes, process controls, acceptance criteria).  
125   
126  Therefore, comprehensive comparative analytical data are necessary to build the foundation for a 
127  development program for a proposed biosimilar product intended for submission under section 
128  351(k) of the PHS Act. 
129   
130  The BPCI Act 
131   
132  The BPCI Act, enacted as part of the (ACA) on March 23, 2010, amends the PHS Act and other 
133  statutes to create an abbreviated licensure pathway for biological products shown to be 
134  biosimilar to, or interchangeable with, an FDA-licensed biological reference product (see 
135  sections 7001 through 7003 of the ACA).  Section 351(k) of the PHS Act (42 U.S.C. 262(k)), 
136  added by the BPCI Act, sets forth the requirements for an application for a proposed biosimilar 
137  product or a proposed interchangeable product.  An application submitted under section 351(k) 
  4 Contains Nonbinding Recommendations 
Draft  Not for Implementation 
 
 
138  must contain, among other things, information demonstrating that the biological product is 
139  biosimilar to a reference product based upon data derived from:  
140   
141    Analytical studies that demonstrate that the biological product is highly similar to the 
142  reference product notwithstanding minor differences in clinically inactive components; 
143    Animal studies (including the assessment of toxicity); and 
144    A clinical study or studies (including the assessment of immunogenicity and PK or PD) 
145  that are sufficient to demonstrate safety, purity, and potency in one or more appropriate 
146  conditions of use for which the reference product is licensed and intended to be used and 
147  for which licensure is sought for the biological product.6 
148   
149  FDA has the discretion to determine that an element above is unnecessary in a 351(k) 
150  application.7 
151   
152  The term biosimilar or biosimilarity is defined in the PHS Act in reference to a biological 
153  product that is the subject of an application under [section 351(k)] to mean that the biological 
154  product is highly similar to the reference product notwithstanding minor differences in clinically 
155  inactive components and that there are no clinically meaningful differences between the 
156  biological product and the reference product in terms of the safety, purity, and potency of the 
157  product (section 351(i)(2) of the PHS Act).  The term reference product is defined in the PHS 
158  Act as the single biological product licensed under section 351(a) of the PHS Act against which a 
159  biological product is evaluated in a 351(k) application (section 351(i)(4) of the PHS Act). 
160   
161  Section 351(k)(4) of the PHS Act provides that upon review of an application submitted under 
162  section 351(k) or any supplement to such application, FDA will determine the biological product 
163  to be interchangeable with the reference product if FDA determines that the information 
164  submitted in the application (or a supplement to such application) is sufficient to show that the 
165  biological product is biosimilar to the reference product and can be expected to produce the 
166  same clinical result as the reference product in any given patient8 and that for a biological 
167  product that is administered more than once to an individual, the risk in terms of safety or 
168  diminished efficacy of alternating or switching between use of the biological product and the 
169  reference product is not greater than the risk of using the reference product without such 
170  alternation or switch.9  
171   
172  The term interchangeable or interchangeability is defined in the PHS Act, in reference to a 
173  biological product that is shown to meet the standards described in section 351(k)(4) of the PHS 
                                                 
6 Section 351(k)(2)(A)(i)(I) of the PHS Act.  
 
7 Section 351(k)(2)(A)(ii) of the PHS Act. 
 
8 Section 351(k)(4)(A) of the PHS Act. 
 
9 Section 351(k)(4)(B) of the PHS Act. 
 
  5 Contains Nonbinding Recommendations 
Draft  Not for Implementation 
 
 
174  Act, to mean that the biological product may be substituted for the reference product without 
175  the intervention of the health care provider who prescribed the reference product (section 
176  351(i)(3) of the PHS Act). 
177   
178   
179  III.  SCOPE 
180   
181  This document provides guidance on the use of comparative analytical studies that are relevant to 
182  assessing whether the proposed product is biosimilar to a reference product for purposes of 
183  submission of a marketing application under section 351(k) of the PHS Act.  This document is 
184  not intended to provide an overview of FDAs approach to determining interchangeability, which 
185  is addressed in a separate guidance document.10  Although this guidance applies specifically to 
186  therapeutic protein products, the general scientific principles may be informative for the 
187  development of proposed biosimilars to other protein products, such as in vivo protein diagnostic 
188  products.  If the reference product cannot be adequately characterized for the purpose of 
189  demonstrating that a proposed product is biosimilar to the reference product as recommended in 
190  this guidance, the application may not be appropriate for submission under section 351(k) of the 
191  PHS Act.  
192   
193  This guidance also describes considerations for CMC information that is relevant to assessing 
194  whether the proposed product is biosimilar to the reference product.  It is critical that all product 
195  applications contain a complete and thorough CMC section that provides the necessary and 
196  appropriate information (e.g., characterization, adventitious agent safety, process controls, and 
197  specifications) to support that the manufacturing process consistently delivers a product with the 
198  intended quality characteristics.  This guidance should be used as a companion to other 
199  guidances available from FDA that describe the CMC information appropriate for evaluation of 
200  protein products.11  We encourage early interaction with FDA to discuss specific CMC issues 
201  that may arise for a sponsors proposed product. 
202   
203   
204  IV.  GENERAL PRINCIPLES 
205   
206  Advances in analytical sciences (both physicochemical and biological) enable some protein 
207  products to be characterized extensively in terms of their physicochemical and biological 
208  properties.  These analytical procedures have improved the ability to identify and characterize 
                                                 
10 See FDAs guidance for industry, Considerations in Demonstrating Interchangeability With a Reference Product 
(May 2019).   
 
11 For CMC requirements for submission of a marketing application, sponsors should consult current regulations and 
see the guidance for industry 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.  
 
 
  6 Contains Nonbinding Recommendations 
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209  not only the desired product but also product-related substances and product- and process-related 
210  impurities.12  Advances in manufacturing science and production methods may enhance the 
211  likelihood that a proposed product can be demonstrated to be highly similar to a reference 
212  product by better targeting the reference products physiochemical and functional properties.  In 
213  addition, advances in analytical sciences may enable detection and characterization of 
214  differences between the protein products.  These differences should be further assessed to 
215  understand the impact on the biosimilar product clinical performance relative to the reference 
216  product.   
217   
218  Despite improvements in analytical techniques, current analytical methodology may not be able 
219  to detect or characterize all relevant structural and functional differences between the two protein 
220  products.  A thorough understanding of each analytical method's limitations will be critical to a 
221  sponsor's successful identification of residual uncertainties and, in turn, to the design of 
222  subsequent testing.  In addition, there may be incomplete understanding of the relationship 
223  between a product's structural attributes and its clinical performance.  FDA encourages the use of 
224  available state-of-the-art technology.  Sponsors should use appropriate analytical methodologies 
225  that have adequate sensitivity and specificity to detect and characterize differences between the 
226  proposed product and the reference product.   
227   
228  As part of a complete CMC data submission, an application submitted under section 351(k) of 
229  the PHS Act is required to include analytical studies that demonstrate that the biological product 
230  is highly similar to the reference product.13  The rationale for the approach to the comparative 
231  analytical assessment should be clearly described, with consideration of the characteristics, 
232  known mechanism of action(s), and function of the reference product. 
233   
234  Comparative analytical data provide the foundation for the development of a proposed product 
235  for submission in an application under section 351(k) of the PHS Act and can influence decisions 
236  about the type and amount of animal and clinical data needed to support a demonstration of 
237  biosimilarity.  Such analytical data should be available early in product development and will 
238  permit more detailed discussion with the Agency because known quality attributes can be used to 
239  shape biosimilar development and justify certain development decisions.  Thus, in addition to the 
240  preliminary comparative analytical data that should be submitted to support an initial advisory 
241  meeting,14 FDA encourages sponsors to submit comprehensive comparative analytical data early 
                                                 
12 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). 
 
13 See section 351(k)(2)(A)(i)(I)(aa) of the PHS Act. 
 
14 See the draft guidance for industry Formal Meetings Between the FDA and Sponsors or Applicants of BsUFA 
Products (June 2018), which provides recommendations to industry on all formal meetings between the FDA and 
sponsors or applicants for proposed biosimilar products or proposed interchangeable products intended to be 
submitted under 351(k) of the PHS Act.  When final, this guidance will represent FDAs current thinking on this 
topic. 
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242  in the development process:  at the pre-IND stage; with the original IND submission; or with the 
243  submission of data from the initial clinical studies, such as PK and PD studies.  FDA will best be 
244  able to provide meaningful input on the extent and scope of animal and additional clinical studies 
245  for a proposed biosimilar development program once the Agency has considered the comparative 
246  analytical data.  
247   
248  Comprehensive, robust comparative physicochemical and functional studies (these may include 
249  biological assays, binding assays, and enzyme kinetics) should be performed to evaluate the 
250  proposed product and the reference product.  A meaningful comparative analytical assessment 
251  depends on, among other things, the capabilities of available state-of-the-art analytical assays to 
252  assess, for example, the molecular weight of the protein, complexity of the protein (higher order 
253  structure and posttranslational modifications), degree of heterogeneity, functional properties, 
254  impurity profiles, and degradation profiles denoting stability.  The capability of the methods used 
255  in these analytical assessments, as well as their limitations, should be described by the sponsor.  
256  Physicochemical and functional characterization studies should be sufficient to establish relevant 
257  quality attributes, including those that define a products identity, quantity, safety, purity, and 
258  potency.  The product-related impurities and product-related substances should be identified, 
259  characterized as appropriate, quantified, and compared using multiple lots of the proposed 
260  product and multiple lots of the reference product, to the extent feasible and relevant, as part of 
261  an assessment of the potential impact on the safety, purity, and potency of the product.   
262   
263  Because therapeutic proteins are made in living systems, there may be heterogeneity in certain 
264  quality attributes of these products.  Heterogeneity in therapeutic proteins may arise in a number 
265  of ways and may affect the expected clinical performance of a protein product.  Replication 
266  errors in the DNA encoding the protein sequence and amino acid misincorporation may occur 
267  during translation, although the level of these errors is typically low.  In addition, most protein 
268  products undergo posttranslational modifications that can alter the functions of the protein by 
269  attaching other biochemical groups such as phosphate and various lipids and carbohydrates; by 
270  proteolytic cleavage following translation; by changing the chemical nature of an amino acid 
271  (e.g., formylation); or by many other mechanisms.  Such modifications can result from 
272  intracellular activities during cell culture or by deliberate modification of the protein (e.g., 
273  PEGylation).  Other posttranslational modifications can be a consequence of manufacturing 
274  process operations; for example, glycation may occur with exposure of the product to reducing 
275  sugars.  Also, certain storage conditions may be more or less permissive for certain degradation 
276  pathways such as oxidation, deamidation, or aggregation.  All of these product-related variants 
277  may alter the biological properties of the expressed recombinant protein.  Therefore, 
278  identification and determination of the relative levels of these variants should be included in the 
279  comparative analytical characterization studies.   
280   
281  The three-dimensional conformation of a protein is an important factor in its biological function.  
282  Proteins generally exhibit complex three-dimensional conformations (tertiary structure and, in 
283  some cases, quaternary structure) because of their large size and the rotational characteristics of 
284  protein alpha carbons, among other things.  The resulting flexibility enables dynamic, but subtle, 
285  changes in protein conformation over time, some of which may be required for functional 
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286  activity.  These rotations are often dependent on low-energy interactions, such as hydrogen 
287  bonds and van der Waals forces, which may be very sensitive to environmental conditions.  
288  Current analytical technology is capable of evaluating the three-dimensional structure of many 
289  proteins.  Using multiple, relevant, state-of-the-art methods can help define tertiary protein 
290  structure and, to varying extent, quaternary structure, and can add to the body of information 
291  supporting biosimilarity.  At the same time, a proteins three-dimensional conformation can often 
292  be difficult to define precisely using current physicochemical analytical technology.  Any 
293  differences in higher order structure between a proposed product and a reference product should 
294  be evaluated in terms of a potential effect on protein function and stability.  Thus, functional 
295  assays are also critical tools for evaluating the integrity of the higher order structures. 
296   
297  A scientifically sound characterization that provides a comprehensive understanding of the 
298  chemical, physical, and biological characteristics of the proposed product is essential to the 
299  design of the manufacturing process and to the conduct of development studies for all biological 
300  products.  The body of knowledge that emerges will serve to support a demonstration of product 
301  quality and the effectiveness of a suitable control system during development, and support 
302  approval of the product.   
303   
304  Proposed biosimilar product, manufacturers should perform in-depth chemical, physical, and 
305  bioactivity comparisons with side-by-side analyses of an appropriate number of lots of the 
306  proposed product and the reference product and, where available and appropriate, a comparison 
307  with a reference standard for suitable attributes (e.g., potency).  For a discussion of reference 
308  standards, see section V.G of this guidance.  Evaluation of multiple lots of a reference product 
309  and multiple lots of a proposed product enables estimation of product variability across lots.  The 
310  number of lots needed to understand the lot-to-lot variability of both the reference and proposed 
311  products may differ on a case-by-case basis and should be scientifically justified by the sponsor.   
312   
313  FDA encourages sponsors to consult with the Agency to ensure that an appropriate number of 
314  lots are evaluated.  Identification of specific lots of a reference product used in comparative 
315  analytical studies, together with expiration dates and time frames and when the lots were 
316  analyzed and used in other types of studies (nonclinical or clinical studies), should be provided.  
317  This information will be useful in justifying acceptance criteria to ensure product consistency, as 
318  well as to support the comparative analytical assessment of the proposed product and the 
319  reference product.  However, acceptance criteria should be based on the totality of the analytical 
320  data and not simply on the observed range of product attributes of the reference product.  This is 
321  because some product attributes act in combination to affect a products safety, purity, and 
322  potency profile; therefore, their potential interaction should be considered when conducting the 
323  comparative analytical assessment and setting specifications.  For example, for some 
324  glycoproteins, the content and distribution of tetra-antennary and N-acetyllactosamine repeats 
325  can affect in vivo potency and should not be evaluated independently of each other.   
326   
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327  Additionally, data obtained for lots used in nonclinical and clinical studies and relevant 
328  information on the relationship between an attribute and the performance of the drug product 
329  (see ICH Q8(R2))15 can also be used to help establish acceptance criteria. 
330   
331  An extensive analytical characterization may reveal differences between the reference product 
332  and the proposed product, especially when using analytical techniques capable of discriminating 
333  qualitative or quantitative differences in product attributes.  Emphasis should be placed on 
334  developing orthogonal quantitative methods to definitively identify any differences in product 
335  attributes.  Based on the results of analytical studies assessing functional and physicochemical 
336  characteristics, including, for example, higher order structure posttranslational modifications, 
, 
337  and impurity and degradation profiles, the sponsor may have an appropriate scientific basis for a 
338  selective and targeted approach to subsequent animal and/or clinical studies to support a 
339  demonstration of biosimilarity.  It may be useful to compare differences in the quality attributes 
340  of the proposed product with those of the reference product using a meaningful fingerprint-like 
341  analysis algorithm16 that covers a large number of additional product attributes and their 
342  combinations with high sensitivity using orthogonal methods.  Enhanced approaches in 
343  manufacturing science, as discussed in ICH Q8(R2), may facilitate production processes that can 
344  better match a reference products fingerprint.17  Such a strategy could further quantify the 
345  overall similarity between two molecules and may lead to additional bases for a more selective 
346  and targeted approach to subsequent animal and/or clinical studies. 
347   
348  The type, nature, and extent of any differences between the proposed product and the reference 
349  product, introduced by design or observed from comprehensive analytical characterization of 
350  multiple manufacturing lots, should be clearly described and discussed.  The discussion should 
351  include identification and comparison of relevant quality attributes from product 
352  characterization.  The potential clinical effects of observed structural and functional differences 
353  between the two products should be assessed and supported by animal or clinical studies, if 
354  necessary. 
355   
356   
357  V.  FACTORS FOR CONSIDERATION IN PERFORMING THE COMPARATIVE 
358  ANALYTICAL ASSESSMENT 
359   
360  When performing the comparative analytical assessment to support a demonstration of 
361  biosimilarity, manufacturers should consider a number of factors, including the following:  
                                                 
15 See the ICH guidance for industry Q8(R2) Pharmaceutical Development (November 2009).  
 
16 For more information on fingerprint-like analysis, refer to Kozlowski S, J Woodcock, K Midthun, RB Sherman, 
2011, Developing the Nation's Biosimilars Program, N Engl J Med; 365:385-388. 
 
17 See the ICH guidances for industry Q8(R2) Pharmaceutical Development (November 2009), Q9 Quality Risk 
Management (June 2006), 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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362   
363  A.  Expression System 
364   
365  Therapeutic protein products can be produced in microbial cells (prokaryotic or eukaryotic), cell 
366  lines (e.g., mammalian, avian, insect, plant), or tissues derived from animals or plants.  It is 
367  expected that the expression construct for a proposed product will encode the same primary 
368  amino acid sequence as its reference product.  However, minor modifications, such as N- or C-
369  terminal truncations (e.g., the heterogeneity of C-terminal lysine of a monoclonal antibody) that 
370  are not expected to change the product performance, may be justified and should be explained by 
371  the sponsor.  Possible differences between the chosen expression system (i.e., host cell and the 
372  expression construct) of the proposed product and that of the reference product should be 
373  carefully considered because the type of expression system will affect the types of process- and 
374  product-related substances, impurities, and contaminants (including potential adventitious 
375  agents) that may be present in the protein product.  For example, the expression system can have 
376  a significant effect on the types and extent of translational and posttranslational modifications 
377  that are imparted to the proposed product, which may introduce additional uncertainty into the 
378  demonstration that the proposed product is biosimilar to the reference product.   
379   
380  Minimizing differences between the proposed product and reference product expression systems 
381  to the extent possible can enhance the likelihood of producing a biosimilar protein product.  Use 
382  of different expression systems will be evaluated on a case-by-case basis.   
383   
384  B.  Manufacturing Process 
385   
386  A comprehensive understanding of all steps in the manufacturing process for the proposed 
387  product should be established during product development.  As a scientific matter, 
388  characterization tests, process controls, and specifications that will emerge from information 
389  gained during process development must be specific for the proposed product and manufacturing 
390  process.  The use of enhanced approaches18 to pharmaceutical development, along with quality 
391  risk management and effective quality systems, will facilitate the consistent manufacturing of a 
392  high-quality product.  As a scientific matter, as with biological products originally licensed under 
393  section 351(a) of the PHS Act, an application for a biological product submitted for licensure 
394  under section 351(k) of the PHS Act may not incorporate by reference drug substance, drug 
395  substance intermediate, or drug product information contained in a Master File (MF) because a 
396  license holder is generally expected to have knowledge of and control over the manufacturing 
397  process for the biological product for which it has a license.19  Other types of contract 
                                                 
18 See the ICH guidances for industry Q8(R2) Pharmaceutical Development (November 2009), Q9 Quality Risk 
Management (June 2006), Q10 Pharmaceutical Quality System (April 2009), and Q11 Development and 
Manufacture of Drug Substances (November 2012) for guidance on enhanced approaches in manufacturing science. 
 
19 A MF for drug substance, drug substance intermediate, or drug product information for a biological product may 
be referenced to support an investigational new drug application (IND) for a proposed biosimilar product.  
Assurance of product quality should be provided on each lot of material produced by the MF holder.  Procedures 
 
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398  manufacturing arrangements can be considered if the sponsor does not intend to manufacture the 
399  product for licensure.20 
400   
401  A sponsor considering manufacturing changes after completing the initial comparative analytical 
402  assessment or after completing clinical studies intended to support a 351(k) application will need 
403  to demonstrate comparability between the pre- and post-change proposed product and may need 
404  to conduct additional studies.  The nature and extent of the changes may determine the extent of 
405  these additional studies.  The comparative analytical studies should include a sufficient number 
406  of lots of the proposed biosimilar product used in clinical studies as well as from the proposed 
407  commercial process if the process used to produce the material used in the clinical studies is 
408  different.   
409   
410  C.  Physicochemical Properties  
411   
412  Physicochemical assessment of the proposed product and the reference product should consider 
413  all relevant characteristics of the protein product (e.g., the primary, secondary, tertiary, and 
414  quaternary structure; posttranslational modifications; and functional activity(ies)).  The objective 
415  of this assessment is to maximize the potential for detecting differences in quality attributes 
416  between the proposed product and the reference product.   
417   
418  The sponsor should address the concept of the desired product (and its variants) as discussed in 
419  ICH Q6B21 when designing and conducting the characterization studies.  Thus, it will be 
420  important to understand the heterogeneity of the proposed product and the reference product 
421  (e.g., the nature, location, and levels of glycosylation) and the ranges of variability of different 
422  isoforms, including those that result from posttranslational modifications.   
423   
424  Particular analytical methodologies can be used to assess specific physicochemical 
425  characteristics of proteins.  These methodologies are described in published documents, 
426  including scientific literature, regulatory guidelines, and pharmacopeial compendia.  Some 
427  techniques provide information on multiple characteristics.  It is expected that appropriate 
428  analytical test methods will be selected based on the nature of the protein being characterized 
429  and knowledge regarding the structure and heterogeneity of the reference product and the 
430  proposed product, as well as characteristics critical to product performance.   
431   
                                                 
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 sponsor is expected to provide notification to the Agency of any relevant 
change in the IND in order to initiate a reevaluation of the MF. 
 
20 See the guidance for industry Cooperative Manufacturing Arrangements for Licensed Biologics (November 
2008). 
 
21 See the ICH guidance for industry Q6B Specifications:  Test Procedures and Acceptance Criteria for 
Biotechnological/Biological Products (August 1999). 
 
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432  To address the full range of physicochemical properties or biological activities adequately, it is 
433  often necessary to apply more than one analytical procedure to evaluate the same quality 
434  attribute.  Methods that use different physicochemical or biological principles to assess the same 
435  attribute are especially valuable because they provide independent data to support the quality of 
436  that attribute (e.g., orthogonal methods to assess aggregation).  In addition, the use of 
437  complementary analytical techniques in series, such as peptide mapping or capillary 
438  electrophoresis combined with mass spectrometry of the separated molecules, should provide a 
439  meaningful and sensitive method for comparing products. 
440   
441  Unlike routine quality control assays, tests used to characterize the product do not necessarily 
442  need to be validated; however, the tests used to characterize the product should be scientifically 
443  sound, fit for their intended use, and provide results that are reproducible and reliable.  In 
444  selecting these tests, it is important to consider the characteristics of the protein product, 
445  including known and potential impurities.  Information regarding the ability of a method to 
446  discern relevant differences between a proposed product and a reference product should be 
447  submitted as part of the comparison.  The methods should be demonstrated to be of appropriate 
448  sensitivity and specificity to provide meaningful information as to whether the proposed product 
449  and the reference product are highly similar.   
450   
451  D.  Functional Activities  
452   
453  Functional assays serve multiple purposes in the characterization of protein products.  These tests 
454  act to complement physicochemical analyses and are a quantitative measure of the function of 
455  the protein product.  
456   
457  Depending on the structural complexity of the protein and available analytical technology, the 
458  physicochemical analysis may be unable to confirm the integrity of the higher order structures.  
459  Instead, the integrity of such structures can usually be inferred from the products biological 
460  activity.  If the clinically relevant mechanism(s) of action are known for the reference product or 
461  can reasonably be determined, the functional assays should reflect such mechanism(s) of action 
462  to the extent possible.  Multiple functional assays should, in general, be performed as part of the 
463  comparative analytical assessments.  The assessment of functional activity is also useful in 
464  providing an estimate of the specific activity of a product as an indicator of manufacturing 
465  process consistency, as well as product purity, potency, and stability.  
466   
467  If a reference product exhibits multiple functional activities, sponsors should perform a set of 
468  appropriate assays designed to evaluate the range of relevant activities for that product.  For 
469  example, with proteins that possess multiple functional domains expressing enzymatic and 
470  receptor-mediated activities, sponsors should evaluate both activities to the extent that these 
471  activities are relevant to product performance.  For products where functional activity can be 
472  measured by more than one parameter (e.g., enzyme kinetics or interactions with blood clotting 
473  factors), the comparative characterization of each parameter between products should be 
474  assessed.  
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475   
476  The sponsor should recognize the potential limitations of some types of functional assays, such 
477  as high variability, that might preclude detection of small but significant differences between the 
478  proposed product and the reference product.  Because a highly variable assay may not provide a 
479  meaningful assessment as to whether the proposed product is highly similar to the reference 
480  product, sponsors are encouraged to develop assays that are less variable and are sensitive to 
481  changes in the functional activities of the product.  In addition, in vitro bioactivity assays may 
482  not fully reflect the clinical activity of the protein.  For example, these assays generally do not 
483  predict the bioavailability (PK and biodistribution) of the product, which can affect PD and 
484  clinical performance.  Also, bioavailability can be dramatically altered by subtle differences in 
485  glycoform distribution or other posttranslational modifications.  Thus, these limitations should be 
486  taken into account when assessing the robustness of the quality of data supporting biosimilarity 
487  and the need for additional information that may address residual uncertainties.  Finally, 
488  functional assays are important in assessing the occurrence of neutralizing antibodies in 
489  nonclinical and clinical studies.  
490    
491  E.  Target Binding   
492   
493  When binding is part of the activity attributed to the protein product, analytical tests should be 
494  performed to characterize the proposed product in terms of its specific binding properties (e.g., if 
495  binding to a receptor is inherent to protein function, this property should be measured and used 
496  in comparative studies) (see ICH Q6B for additional details).  Various methods such as surface 
497  plasmon resonance, microcalorimetry, or classical Scatchard analysis can provide information on 
498  the kinetics and thermodynamics of binding.  Such information can be related to the functional 
499  activity and characterization of the proposed products higher order structure.  
500   
501  F.  Impurities 
502   
503  The sponsor should characterize, identify, and quantify product-related impurities in the 
504  proposed product and the reference product, to the extent feasible.22  If a comparative 
505  physicochemical analysis reveals comparable product-related impurities at similar levels 
506  between the two products, pharmacological/toxicological studies to characterize potential 
507  biological effects of specific impurities may not be necessary.  However, if the manufacturing 
508  process used to produce the proposed product introduces different impurities or higher levels of 
509  impurities than those present in the reference product, additional pharmacological/toxicological 
510  or other studies may be necessary.  As discussed in the ICH guidance for industry S6(R1) 
511  Preclinical Safety Evaluation of Biotechnology-Derived Pharmaceuticals (May 2012), [i]t is 
                                                 
22 The use of the terms product- and process-related impurities is consistent with their use and meaning in 
ICH Q6B. 
 
 
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512  preferable to rely on purification processes to remove impurities . . . rather than to establish a 
513  preclinical testing program for their qualification.23  
514   
515  Process-related impurities arising from cell substrates (e.g., host cell DNA, host cell proteins), 
516  cell culture components (e.g., antibiotics, media components), and downstream processing steps 
517  (e.g., reagents, residual solvents, leachables, endotoxin, bioburden) should be evaluated.  The 
518  process-related impurities in the proposed product are not expected to match those observed in 
519  the reference product and are not included in the comparative analytical assessment.  The chosen 
520  analytical procedures should be adequate to detect, identify, and accurately quantify biologically 
521  significant levels of impurities. 24  In particular, results of immunological methods used to detect 
522  host cell proteins depend on the assay reagents and the cell substrate used.  Such assays should 
523  be validated using the product cell substrate and orthogonal methodologies to ensure accuracy 
524  and sensitivity.   
525   
526  As with any biological product, the safety of the proposed product with regard to adventitious 
527  agents or endogenous viral contamination, should be ensured by screening critical raw materials 
528  and confirmation of robust virus removal and inactivation achieved by the manufacturing 
529  process. 25   
530   
531  G.   Reference Product and Reference Standards  
532   
533  A thorough physicochemical and biological assessment of the reference product should provide a 
534  base of information from which to develop the proposed product and justify reliance on certain 
535  existing scientific knowledge about the reference product.  Sufficient evidence that the proposed 
536  product is highly similar to the reference product must be provided to support a selective and 
537  targeted approach in early product development (e.g., selected animal studies and/or additional 
538  clinical studies).26 
539   
540  The comparative analytical assessment submitted with the marketing application to support the 
541  demonstration of biosimilarity of the proposed product to the reference product should include 
542  lots of the proposed product used in principal clinical study(ies), as well as the proposed 
543  commercial product.  As stated earlier in section V.B, a sponsor considering manufacturing 
544  changes after completing the initial comparative analytical assessment or after completing 
545  clinical studies intended to support a 351(k) application may need to conduct additional 
                                                 
23 See the ICH guidance for industry S6(R1) Preclinical Safety Evaluation of Biotechnology-Derived 
Pharmaceuticals (May 2012), page 2. 
 
24 See the ICH guidance for industry Q2B Validation of Analytical Procedures:  Methodology (May 1997). 
 
25 See the ICH guidance for industry Q5A Viral Safety Evaluation of Biotechnology Products Derived From Cell 
Lines of Human or Animal Origin (September 1998). 
 
26 See 21 CFR 312.23 for IND application content and format. 
 
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546  comparative analytical studies of the proposed product and the reference product.  The nature 
547  and extent of the changes may determine the extent of these additional analytical studies. 
548   
549  If the drug substance has been extracted from the reference product to conduct analytical studies, 
550  the sponsor should describe the extraction procedure and provide support that the procedure 
551  itself does not alter relevant product quality attributes.  This undertaking would include 
552  consideration of alteration or loss of the desired products and impurities and relevant product-
553  related substances, and it should include appropriate controls to ensure that relevant 
554  characteristics of the protein are not significantly altered by the extraction procedure. 
555   
556  If there is a suitable, publicly available, and well-established reference standard for the protein, a 
557  physicochemical and/or functional comparison of the proposed product with this standard may 
558  also provide useful information.27  For example, if an international standard for calibration of 
559  potency is available, a comparison of the relative potency of the proposed product with this 
560  potency standard should be performed.  As recommended in ICH Q6B, an in-house reference 
561  standard(s) should always be qualified and used for control of the manufacturing process and 
562  product.  
563   
564  An in-house reference standard is typically developed from early development lots or lots used in 
565  a clinical study(ies).  Additional reference standards may be qualified later in development and 
566  for a BLA submission.  Ideally, a sponsor will have established and properly qualified primary 
567  and working reference standards that are representative of proposed product lots used in clinical 
568  studies that support the application. 
569   
570  For the development of a proposed product, a reference product lot or a lot of a non-U.S.-
571  licensed comparator product (see section VI.A.4 of this guidance) is typically qualified as an 
572  initial reference standard.  Once clinical lots of the proposed product have been manufactured, it 
573  is expected that one of these lots will be properly qualified (including bridging to previous 
574  reference standards) for use as a reference standard for release and stability, as well as 
575  comparative analytical testing.  If possible, once an in-house reference standard is properly 
576  qualified, there should be sufficient quantities to use throughout the development of the proposed 
577  product.  All lots of reference standards used during the development of a proposed product 
578  should be properly qualified.  In addition to release testing methods, the qualification protocol 
579  for reference standards should include all analytical methods that report the result relative to the 
580  reference standard.   
581   
582  For all methods where the result is reported relative to the reference standard, the assignment of 
583  a potency of 100% should include a narrow acceptable potency range and ensure control over 
584  product drift.  For example, a sponsor should consider the use of a pre-determined two-sided 
585  confidence interval (CI) of the mean of the replicates, where the mean relative potency and the 
586  95% CI are included within a sufficiently narrow range (e.g., 90-110%).  There should be an 
                                                 
27 Although studies with such a reference standard may be useful, they are not sufficient to satisfy the BPCI Acts 
requirement to demonstrate the biosimilarity of the proposed product to the U.S.-licensed reference product.   
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587  evaluation across the history of multiple reference standard qualifications to address potential 
588  drift. 
589   
590  A sponsor generally should not use a correction factor to account for any differences in, for 
591  example, potency or biological activity between reference standards.   
592   
593  Use of reference standards inadequately qualified for analytical methods that report results 
594  relative to the reference standard is likely to raise concerns regarding the comparative analytical 
595  assessment.  One approach to address these concerns, if applicable, may be to store the reference 
596  product and non-U.S.-licensed comparator product lots under conditions that maintain stability 
597  long term, if feasible.  Prior to submission of a 351(k) application, the prospective applicant 
598  should conduct a reevaluation of all proposed product, reference product, and non-U.S.-licensed 
599  comparator product lots using the same reference standard for those methods that report the 
600  result relative to the reference standard.  Data supporting the stability of the reference product 
601  and non-U.S.-licensed comparator product beyond the expiration date under these conditions 
602  should be included in the submission. 
603   
604  In summary, analytical studies carried out to support the approval of a proposed product should 
605  not focus solely on the characterization of the proposed product in isolation.  Rather, these 
606  studies should be part of a broad comparison that includes, but is not limited to, the proposed 
607  product, the reference product, and, where applicable, a non-U.S.-licensed comparator, 
608  applicable reference standards, and consideration of relevant publicly available information. 
609    
610  H.  Finished Drug Product  
611   
612  Product characterization studies of a proposed product should be performed on the most 
613  downstream intermediate best suited for the analytical procedures used.  The attributes evaluated 
614  should be stable through any further processing steps.  For these reasons, characterization studies 
615  are often performed on the drug substance.  However, if a drug substance is reformulated and/or 
616  exposed to new materials in the finished dosage form, the impact of these changes should be 
617  considered.  Whenever possible, if the finished drug product is best suited for a particular 
618  analysis, the sponsors should analyze the finished drug product.  If an analytical method more 
619  sensitively detects specific attributes in the drug substance but the attributes it measures are 
620  critical and/or may change during manufacture of the finished drug product, comparative 
621  characterization may be called for on both the extracted protein and the finished drug product. 
622   
623  Proteins are very sensitive to their environment.  Therefore, differences in excipients or primary 
624  packaging may affect product stability and/or clinical performance.  Differences in formulation 
625  and primary packaging28 between the proposed product and the reference product are among the 
626  factors that may affect whether or how subsequent clinical studies may take a selective and 
                                                 
28 See the ICH guidance for industry Q8(R2) Pharmaceutical Development (November 2009). 
 
 
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627  targeted approach.29  Sponsors should clearly identify excipients used in the proposed product 
628  that differ from those in the reference product.  The acceptability of the type, nature, and extent 
629  of any differences between the finished proposed product and the finished reference product 
630  should be evaluated and supported by appropriate data and rationale.  Additionally, different 
631  excipients in the proposed product should be supported by existing toxicology data for the 
632  excipient or by additional toxicity studies with the formulation of the proposed product.  
633  Excipient interactions as well as direct toxicities should be considered.   
634   
635  I.   Stability  
636   
637  As part of an appropriate physicochemical and functional comparison of the stability profile of 
638  the proposed product with that of the reference product, accelerated and stress stability studies, 
639  as well as forced degradation studies, should be used to establish degradation profiles and to 
640  provide a direct stability comparison of the proposed product with the reference product.  These 
641  comparative studies should be conducted under multiple stress conditions (e.g., high 
642  temperature, freeze thaw, light exposure, and agitation) that can cause incremental product 
643  degradation over a defined time period.  Results of these studies may reveal product differences 
644  that warrant additional evaluations and also identify conditions under which additional controls 
645  should be employed in manufacturing and storage. 30  Sufficient real time, real-condition stability 
646  data from the proposed product should be provided to support the proposed shelf life.  
647   
648  VI.   COMPARATIVE ANALYTICAL ASSESSMENT 
649   
650  A thorough understanding of the reference product is critical for a successful biosimilar 
651  development program.  The Agency recommends that sponsors approach the comparative 
652  analytical assessment by first understanding the physicochemical and biological characteristics 
653  of the reference product.  A full characterization of the reference product, in addition to 
654  consideration of publicly available information, will form the basis of product understanding.  As 
655  described previously, protein products are complex molecules that generally are manufactured in 
656  living cells and purified using a variety of technologies; therefore, they have a certain degree of 
657  inherent lot-to-lot variability in terms of quality characteristics.  The observed lot-to-lot 
658  variability may derive from manufacturing conditions and from analytical assay variability.  
659  Factors that contribute to lot-to-lot variability in the manufacture of a protein product include the 
660  source of certain raw materials (e.g., growth medium, resins, or separation materials) and 
661  different manufacturing sites.  Therefore, the comparative analytical assessment, it is important 
662  to adequately characterize the lot-to-lot variability of the reference product and the proposed 
663  biosimilar product.  
                                                 
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 (April 2015). 
 
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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664   
 
665  A.  Considerations for Reference and Biosimilar Products 
666   
667  1.  Reference Product  
668   
669  To ensure that the full range of product variability is accurately captured, sponsors should 
670  acquire multiple reference product lots throughout the development program of a proposed 
671  biosimilar in sufficient quantity to conduct multiple physiochemical and functional assays.  
672  Considering the inherent heterogeneity present in protein products and the expected lot-to-lot 
673  variability stemming from manufacturing processes, the Agency recommends that a sponsor 
674  include at least 10 reference product lots (acquired over a time frame that spans expiration dates 
675  of several years), in the analytical assessment to ensure that the variability of the reference 
676  product is captured adequately.  The final number of lots should be sufficient to provide adequate 
677  information regarding the variability of the reference product.  In cases where limited numbers of 
678  reference product lots are available (e.g., for certain orphan drugs), alternate flexible comparative 
679  analytical assessments plans should be proposed and discussed with the Agency.  
680   
681  2.  Proposed Product  
682   
683  The Agency recommends that a sponsor include at least 6 to 10 lots of the proposed product in 
684  the comparative analytical assessment, to ensure 1) adequate characterization of the proposed 
685  product and understanding of manufacturing variability, and 2) adequate comparison to the 
686  reference product.  These should include lots manufactured with the investigational- and 
687  commercial-scale processes, and may include validation lots, as well as product lots 
688  manufactured at different scales, including engineering lots.  These lots should be representative 
689  of the intended commercial manufacturing process.  If there is a manufacturing process change 
690  during development, it may be possible, with adequate scientific justification, to use data 
691  generated from lots manufactured with a different process.  However, data should be provided in 
692  the 351(k) BLA to support comparability of drug substance and drug product manufactured with 
693  the different processes and/or scales.  The extent of process development design (as described in 
694  guidelines ICH Q8 (R2) Pharmaceutical Development and ICH Q11 Development and 
695  Manufacture of Drug Substances) and process understanding should be used in support of the 
696  number of proposed biosimilar product lots proposed for inclusion in the comparative analytical 
697  assessment in the 351(k) application.  
698   
699  To the extent possible, proposed biosimilar lots included in the comparative analytical 
700  assessment described in section VI.B, Considerations for Data Analysis, should be derived from 
701  different drug substance batches to adequately represent the variability of attributes inherent to 
702  the drug substance manufacturing process.  Drug product lots derived from the same drug 
703  substance batch(es) are not considered sufficiently representative of such variability, except for 
704  use in testing certain drug product attributes for which variability is mostly dependent on the 
705  drug product manufacturing process (e.g., protein concentration).  Although it may be preferable 
706  to compare the proposed product lots to the reference product lots, it may be acceptable to also 
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707  include independent drug substance batches (if the drug substance was not used to make drug 
708  product), if needed, to attain a sufficient number of lots for the comparative analytical 
709  assessment. 
710   
711  3.  Accounting for Reference Product and Proposed Product Lots 
712   
713  Sponsors should account for all the reference product lots acquired and characterized.  The 
714  351(k) BLA should include data and information from all reference product and proposed 
715  product lots that were evaluated in any manner, including the specific physicochemical, 
716  functional, animal, and clinical studies for which a lot was used.  When a lot is specifically 
717  selected to be included in or excluded from certain analytical studies, a justification should be 
718  provided.  The date of the analytical testing as well as the product expiration date should be 
719  provided in the application.  In general, expired reference product lots should not be included in 
720  the comparative analytical assessment because lots analyzed beyond their expiration date could 
721  lead to results outside the range that would normally be observed in unexpired lots, which may 
722  result in overestimated reference product variability.  Testing of lots past expiry may be 
723  acceptable if samples are stored under long term conditions (e.g., frozen at -80C) provided that 
724  sponsors submit data and information demonstrating that storage does not impact the quality of 
725  the product (see section V.G). 
726   
727  The same type of information and data described above to be collected for reference product lots 
728  should also be provided on every manufactured drug substance and drug product lot of the 
729  proposed product.  
730   
731  Reference product and proposed product lots used in the clinical studies (e.g., PK and PD, if 
732  applicable, similarity, and comparative clinical study) should be included in the comparative 
733  analytical assessment. 
734   
735  4.  Reference Product and Non-U.S.-Licensed Comparator Products 
736   
737  As described in other guidances, a sponsor that intends to use a non-U.S.-licensed comparator in 
738  certain studies should provide comparative analytical data and analysis for all pairwise 
739  comparisons (i.e., U.S.-licensed product versus proposed biosimilar product, non-U.S.-licensed 
740  comparator product versus proposed biosimilar product, and U.S.-licensed product versus non-
741  U.S.-licensed comparator product). 
742   
743  The acceptance criteria used to support a demonstration that a proposed biosimilar product is 
744  highly similar to the reference product should be derived from data generated from a sponsors 
745  analysis of the reference product.  The comparative analytical assessment should be based on a 
746  direct comparison of the proposed product to the reference product.  As a scientific matter, 
747  combining data from the reference product and non-U.S.-licensed comparator product to 
748  determine the acceptance criteria or to perform the comparative analytical assessment to the 
749  proposed product would not be acceptable to support a demonstration that the proposed product 
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750  is biosimilar to the reference product.  For example, combining data from the reference product 
751  and non-U.S.-licensed products may result in a larger range and broader similarity acceptance 
752  criteria than would be obtained by relying solely on data from reference product lots.  Sponsors 
753  are encouraged to discuss with FDA, during product development, any plans to submit data 
754  derived from products approved outside of the U.S. in support of a 351(k) application. 
755   
756  B.  Considerations for Data Analysis 
757   
758  Sponsors should develop a comparative analytical assessment plan and discuss the approach with 
759  the Agency as early as practicable.  A final comparative analytical assessment report should be 
760  available at the time a 351(k) BLA is submitted.   
761   
762  The Agency recommends development of a comparative analytical assessment plan using a 
763  stepwise approach.  The first step is a determination of the quality attributes that characterize the 
764  reference product in terms of its structural/physicochemical and functional properties.  These 
765  quality attributes are then ranked according to their risk to potentially impact activity, PK/PD, 
766  safety, efficacy, and immunogenicity.  Finally, the attributes are evaluated using quantitative 
767  analysis, considering the risk ranking of the quality attributes, as well as other factors.  It should 
768  be noted, however, that some attributes may be highly critical (e.g., primary sequence) but not 
769  amenable to quantitative analysis. 
770   
771  1.  Risk Assessment 
772   
773  FDA recommends that sponsors develop a risk assessment tool to evaluate and rank the reference 
774  product quality attributes in terms of potential impact on the mechanism(s) of action and function 
775  of the product.  Certain quality evaluations of the reference product (e.g., its degradation rates, 
776  which are determined from stability or forced degradation studies) generally should not be 
777  included in the risk ranking.  However, these evaluations should still factor into the comparative 
778  analytical assessment of the proposed biosimilar and reference product. 
779  Development of the risk assessment tool should be informed by relevant factors, including: 
780   
781    Potential impact of an attribute on clinical performance:  Specifically, FDA recommends 
782  that sponsors consider the potential impact of an attribute on activity, PK/PD, safety, 
783  efficacy, and immunogenicity.  Sponsors should consider publicly available information, 
784  as well as the sponsors own characterization of the reference product, in determining the 
785  potential impact of an attribute on clinical performance.  
786   
787    The degree of uncertainty surrounding a certain quality attribute:  For example, when 
788  there is limited understanding of the relationship between the degree of change in an 
789  attribute and the resulting clinical impact, FDA recommends that that attribute be ranked 
790  as having higher risk because of the uncertainty raised.  
791   
  21 Contains Nonbinding Recommendations 
Draft  Not for Implementation 
 
 
792  FDA recommends that an attribute that is a high risk for any one of the performance categories 
793  (i.e., activity, PK/PD, safety, efficacy, and immunogenicity) be classified as high risk.  Ideally, 
794  the risk assessment tool should result in a list of attributes ordered by the risk to the patient.  The 
795  risk scores for attributes should, therefore, be proportional to patient risk.  The scoring criteria 
796  used in the risk assessment should be clearly defined and justified, and the risk ranking for each 
797  attribute should be justified with appropriate citations to the literature and data provided. 
798   
799  2.  Quantitative and Qualitative Data Analysis 
800     
801  Appropriate analyses of the comparative analytical data are necessary to support a demonstration 
802  that the proposed product is highly similar to the reference product notwithstanding minor 
803  differences in clinically inactive components.  One approach to data analysis would be the use of 
804  descriptive quality ranges for assessing quantitative quality attributes of high and moderate risk, 
805  and the use of raw data/graphical comparisons for quality attributes with the lowest risk ranking 
806  or for those quality attributes that cannot be quantitatively measured (e.g., primary sequence).  
807  The acceptance criteria for the quality ranges (QR) method in the comparative analytical 
808  assessment should be based on the results of the sponsors own analysis of the reference product 
809  for a specific quality attribute.  The QR should be defined as (  X ,  X ), where  is 
R R R R R
810  the sample mean, and  is the sample standard deviation based on the reference product lots.  

R
811  The multiplier (X) should be scientifically justified for that attribute and discussed with the 
812  Agency.  Based on our experience to date, methods such as tolerance intervals are not 
813  recommended for establishing the similarity acceptance criteria because a very large number of 
814  lots would be required to establish meaningful intervals.  The sponsor can propose other methods 
815  of data analysis, including equivalence testing.  
816   
817  The objective of the comparative analytical assessment is to verify that each attribute, as 
818  observed in the proposed biosimilar and the reference product, has a similar population mean and 
819  similar population standard deviation.  Comparative analysis of a quality attribute would 
820  generally support a finding that the proposed product is highly similar to the reference product 
821  when a sufficient percentage of biosimilar lot values (e.g., 90%) fall within the QR defined for 
822  that attribute.  The Agency recommends that narrower acceptance criteria of the QR method in 
823  the comparative analytical assessment (e.g., a lower X value) be applied to higher risk quality 
824  attributes.  
825   
826  In addition to risk ranking, other factors should be considered in determining which type of 
827  quantitative data analysis should be applied to a particular attribute or assay.  Some additional 
828  factors that should be considered when determining the appropriate type of data evaluation and 
829  analysis of results include:  
830   
831    Nature of the attribute:  Attributes that are known to be of high risk should be prioritized 
832  over attributes with unknown but potentially high risk (i.e., attributes with a high-risk 
833  ranking due to uncertainty).  
834   
  22 Contains Nonbinding Recommendations 
Draft  Not for Implementation 
 
 
835    Distribution of the attribute:  In general, the Agency recommends that sponsors develop 
836  the manufacturing process to target the centers of distribution of the quality attributes of 
837  the reference product as closely as possible.  Therefore, the QR, which assumes that the 
838  population mean and standard deviation are similar, is an appropriate approach to 
839  demonstrate that the proposed product is highly similar to the reference product.  If there 
840  are concerns with the distribution, additional information or analyses may be needed to 
841  support the QR method or to support a different analysis approach.  For example, the 
842  distribution of an attribute in the proposed biosimilar product that is biased towards one 
843  side of the reference product distribution may raise concerns depending on the nature of 
844  the attribute and the role the attribute plays in, for example, the mechanism of action of 
845  the product.  If such a distribution is observed, appropriate justification may be needed, 
846  as a scientific matter, to support the comparative analytical assessment of the products.  
847  In cases where an attribute in the reference product is not normally distributed, sponsors 
848  should consult with the Agency.   
849   
850    Abundance of the attribute:  Because of the inherent heterogeneity present in protein 
851  products, an attribute of the reference product that may pose a high risk when the 
852  attribute is present in high abundance (e.g., percent aggregation or percent oxidation) 
853  may pose a significantly lower risk (or negligible risk) if the attribute is low-abundance.  
854  The abundance of the attribute should be confirmed in both the reference product (as 
855  determined by the proposed product sponsors analysis of the reference product) and the 
856  proposed product.  Limit assays do not necessarily need to be evaluated using QR; 
857  however, the selected limits regarding the amount of an attribute should be defined and 
858  justified.  The justification should also include consideration of how the amount of the 
859  attribute changes over time.   
860   
861    Sensitivity of assay used for assessing an attribute:  Although multiple, orthogonal assays 
862  are encouraged for assessing an attribute, not all assays assessing the attribute need to be 
863  evaluated in the same manner.  While the most sensitive assay for detecting product 
864  differences should be evaluated using QR, it may be appropriate to evaluate the results of 
865  other assays for the same attribute using a graphical comparison.  A justification should 
866  be provided for the method of evaluation used for each type of assay.   
867   
868    Types of attributes/assays:  Quantitative analyses may not be applicable to some 
869  attributes, (e.g., protein sequence or certain assays used for higher order structure 
870  evaluation, or to assays that are only qualitative).  The comparative analytical assessment 
871  plan should clearly define specific assays where quantitative data analyses would not be 
872  applied, and the rationale for that decision. 
873   
874    Publicly available information:  Publicly available information may be relevant to the 
875  appropriate type of data analysis and acceptance criteria in the comparative analytical 
876  assessment.  A sponsor should seek additional advice from the Agency on the inclusion 
877  of any publicly available information in the comparative analytical assessment.   
  23 Contains Nonbinding Recommendations 
Draft  Not for Implementation 
 
 
878   
879  For qualitative analyses of lower risk attributes, FDA recommends side-by-side data presentation 
880  (e.g., spectra, thermograms, graphical representation of data), to allow for a visual comparison of 
881  the proposed product to the reference product.   
882   
883  The final comparative analytical assessment plan should include the risk ranking of attributes, 
884  the type of data evaluation to be used for each attribute/assay, and the final data analysis plan.  
885  The plan should specify the anticipated availability of both proposed biosimilar and reference 
886  product lots for evaluation of each attribute/assay and should include a rationale for why the 
887  proposed number of lots should be considered sufficient for the evaluation.  The comparative 
888  analytical assessment plan should be discussed with the Agency as early in the biosimilar 
889  development program as possible so that agreement can be reached on which attributes/assays 
890  should be evaluated.  The final comparative analytical assessment plan should be submitted to 
891  the Agency prior to initiating the final analytical assessments; typically, this occurs in a meeting 
892  with the Agency. 
893   
894  C.  Comparative Analytical Assessment Conclusions 
895   
896  In the comparative analytical assessment, risk ranking and data analysis are used to evaluate a 
897  large number of attributes, often using multiple orthogonal assays.  FDA evaluates the totality of 
898  the analytical data; if the results of a particular assay do not meet pre-specified criteria, this alone 
899  does not preclude a demonstration of high similarity.  For example, if differences between 
900  products are observed as part of the comparative analytical assessment (including the 
901  components of the assessment that were not included in the risk ranking), the sponsor may 
902  provide additional scientific information (risk assessment and additional data) and a justification 
903  for why these differences do not preclude a demonstration that the products are highly similar.  
904   
905  In certain situations, changes to the manufacturing process of the biosimilar product may be 
906  needed to resolve differences observed in the comparative analytical assessment.  Data should be 
907  provided demonstrating that the observed differences were resolved by any manufacturing 
908  changes, and that other quality attributes were not substantially affected.  If other attributes were 
909  affected by the manufacturing change, data should be provided to demonstrate that the impact of 
910  the change has been evaluated and addressed. 
911   
912  VII.  CONCLUSION 
913   
914  The foundation for an assessment and a demonstration of biosimilarity between a proposed 
915  product and its reference product includes analytical studies that demonstrate that the proposed 
916  product is highly similar to the reference product notwithstanding minor differences in clinically 
917  inactive components.  The demonstration that the proposed product is biosimilar to the reference 
918  product thus involves robust characterization of the proposed product, including comparative 
919  physicochemical and functional studies with the reference product.  The information gained from 
920  these studies is necessary for the development of a proposed product as a biosimilar.  In addition, 
  24 Contains Nonbinding Recommendations 
Draft  Not for Implementation 
 
 
921  a 351(k) application for a proposed product must contain, among other things, information 
922  demonstrating biosimilarity based on data derived from animal studies (including the assessment 
923  of toxicity) and a clinical study or studies (including the assessment of immunogenicity and PK 
924  or PD), unless the Agency determines that an element is unnecessary in a particular 351(k) 
925  application.31  A sponsors ability to discern and understand the impact of relevant analytical 
926  differences between the proposed product and its reference product is critical to determine 
927  whether the statutory standard for biosimilarity can be met.   
928   
929   
930  VIII.  RELEVANT GUIDANCES  
931   
932  The following draft and final guidance documents may be relevant to sponsors developing or 
933  considering development of a proposed biosimilar product.  All Agency guidance documents are 
934  available on FDAs web page  
935  (https://www.fda.gov/regulatory-information/search-fda-guidance-documents).     
936   
937  1.  Guidance for industry Scientific Considerations in Demonstrating Biosimilarity to a 
938  Reference Product (April 2015) 
939   
940  2.  Guidance for industry Questions and Answers on Biosimilar Development and the BPCI 
941  Act (December 2018)  
942   
943  3.  Draft guidance for industry New and Revised Draft Q&As on Biosimilar Development 
944  and the BPCI Act (Revision 2) (December 2018) 
945   
946  4.  Draft guidance for industry Formal Meetings Between the FDA and Sponsors or 
947  Applicants of BsUFA Products (June 2018) 
948   
949  5.  Guidance for industry Clinical Pharmacology Data to Support a Demonstration of 
950  Biosimilarity to a Reference Product (December 2016) 
951   
952  6.  Demonstration of Comparability of Human Biological Products, Including Therapeutic 
953  Biotechnology-derived Products (April 1996) 
954   
955  7.  Points to Consider in the Manufacture and Testing of Monoclonal Antibody Products for 
956  Human Use (February 1997) 
957   
958  8.  Guidance for industry for the Submission of Chemistry, Manufacturing, and Controls 
959  Information for a Therapeutic Recombinant DNA-Derived Product or a Monoclonal 
960  Antibody Product for In Vivo Use (August 1996) 
961   
                                                 
31 Section 351(k)(2)(A)(i)(I) of the PHS Act. 
  25 Contains Nonbinding Recommendations 
Draft  Not for Implementation 
 
 
962  9.  Guidance for industry Cooperative Manufacturing Arrangements for Licensed Biologics 
963  (November 2008) 
964   
965  10. ICH guidance for industry M4:  The CTD Quality (ICH M4Q) (August 2001) 
966   
967  11. ICH guidance for industry Q1A(R2) Stability Testing of New Drug Substances and 
968  Products (ICH Q1A(R2)) (November 2003) 
969   
970  12. ICH guidance for industry Q2(R1) Validation of Analytical Procedures:  Text and 
971  Methodology (ICH Q2(R1) (November 2005) 
972   
973  13. ICH guidance for industry Q2B Validation of Analytical Procedures:  Methodology (ICH 
974  Q2B) (May 1997) 
975   
976  14. ICH guidance for industry Q3A(R) Impurities in New Drug Substances (ICH Q3A(R)) 
977  (June 2008) 
978   
979  15. ICH guidance for industry Q5A Viral Safety Evaluation of Biotechnology Products 
980  Derived from Cell Lines of Human or Animal Origin (ICH Q5A) (September 1998) 
981   
982  16. ICH guidance for industry Q5B Quality of Biotechnological Products:  Analysis of the 
983  Expression Construct in Cells Used for Production of r-DNA Derived Protein Products 
984  (ICH Q5B) (February 1996) 
985   
986  17. ICH guidance for industry Q5C Quality of Biotechnological Products:  Stability Testing 
987  of Biotechnological/Biological Products (ICH Q5C) (July 1996) 
988   
989  18. ICH guidance for industry Q5D Quality of Biotechnological/Biological Products:  
990  Derivation and Characterization of Cell Substrates Used for Production of 
991  Biotechnological/Biological Products (ICH Q5D) (September 1998) 
992   
993  19. ICH guidance for industry Q5E Comparability of Biotechnological/Biological Products 
994  Subject to Changes in Their Manufacturing Process (ICH Q5E) (June 2005) 
995   
996  20. ICH guidance for industry Q6B Specifications:  Test Procedures and Acceptance Criteria 
997  for Biotechnological/Biological Products (ICH Q6B) (August 1999) 
998   
999  21. ICH guidance for industry Q7 Good Manufacturing Practice Guidance for Active 
1000  Pharmaceutical Ingredients (ICH Q7) (September 2016) 
1001   
1002  22. ICH guidance for industry Q8(R2) Pharmaceutical Development (ICH Q8(R2)) 
1003  (November 2009) 
1004   
  26 Contains Nonbinding Recommendations 
Draft  Not for Implementation 
 
 
1005  23. ICH guidance for industry Q9 Quality Risk Management (ICH Q9) (June 2006) 
1006   
1007  24. ICH guidance for industry Q10 Pharmaceutical Quality System (ICH Q10) (April 2009) 
1008   
1009  25. ICH guidance for industry Q11 Development and Manufacture of Drug Substances (ICH 
1010  Q11) (November 2012) 
1011   
1012  26. ICH guidance for industry S6(R1) Preclinical Safety Evaluation of Biotechnology-
1013  Derived Pharmaceuticals (ICH S6(R1)) (May 2012) 
1014     
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Draft  Not for Implementation 
 
 
1015  GLOSSARY32 
1016   
1017  For the purpose of this document, the following definitions apply: 
1018   
1019  Biosimilar or biosimilarity means the biological product is highly similar to the 
1020  reference product notwithstanding minor differences in clinically inactive components, 
1021  and there are no clinically meaningful differences between the biological product and 
1022  the reference product in terms of the safety, purity, and potency of the product.33 
1023   
1024  Chemically synthesized polypeptide means any alpha amino acid polymer that (a) is made 
1025  entirely by chemical synthesis and (b) is less than 100 amino acids in size.  
1026   
1027  Product, when used without modifiers, is intended to refer to the intermediates, drug 
1028  substance, and/or drug product, as appropriate.  The use of the term product is consistent 
1029  with the use of the term in ICH Q5E. 
1030   
1031  Protein means any alpha amino acid polymer with a specific defined sequence that is 
1032  greater than 40 amino acids in size. 
1033   
1034  Reference product means the single biological product licensed under section 351(a) of 
1035  the PHS Act against which a biological product is evaluated in a 351(k) application.34 
1036   
1037   
                                                 
32 For additional information on the Agencys interpretation of certain terms relevant to implementation of the BPCI 
Act, see the draft guidance for industry New and Revised Draft Q&As on Biosimilar Development and the BPCI Act 
(Revision 2) (December 2018).  When final, this guidance will represent FDAs current thinking on this topic. 
 
33 Section 351(i)(2) of the PHS Act. 
 
34 Section 351(i)(4) of the PHS Act. 
 
  28