This document discusses regulatory analysis and approval of biosimilars. It begins by noting key differences between biosimilars and generics, including the greater size and complexity of biologics. It then outlines guiding principles for biosimilar development and approval, including analytical studies demonstrating biosimilarity, animal studies assessing toxicity, and clinical studies of immunogenicity, pharmacokinetics, and efficacy or safety. The document discusses specific assay requirements and considerations for pharmacokinetic, structural, protein characterization, immunogenicity, and neutralizing antibody testing. It emphasizes the need to demonstrate biosimilarity through a totality of evidence from characterization, animal, and clinical studies.

1. Regulatory Analysis &
Approval of Biosimilars
Plenary Lecture at Ganpat University
Mehsana, Gujarat, July 20, 2012
Dr. Bhaswat S. Chakraborty
20.07.2012

2. Contents
 Differences of Biosimilars from Generics of small mol
drugs
 Guiding Principles for Overall Biosimilars
 Brief Description of Biosimilar Mfg.
 PK/TK Assays
 Examples
 Immunogenicity Assays
 Antidrug Antibody Assays (ADA)
 Neutralizing Antidrug Antibody Assays (NAbA)
 Examples
 Risk Management
 Conclusions

3. What are Biosimilars?
 Biosimilars are often called follow-on biologics,
generic biologics or follow-on proteins
 Biosimilars are new versions of existing trade-name
biological products whose patents have expired
 Highly similar biosimilars are not “identical” to the
reference product
 They do not utilize the same living cell line,
production process, or raw material as the innovator
drug

4. Size & Complexity
Big Guys

5. How are Biopharmaceuticals Made?
 Upstream & Downstream

7. Overview of EMEA Guidelines for Biosimilars
Mellstedt H et al. Ann Oncol 2007;19:411-419
© The Author 2007. Published by Oxford University Press on behalf of the European Society for
Medical Oncology. All rights reserved. For permissions, please e-mail:
journals.permissions@oxfordjournals.org

8. Overview of USFDA Guidelines for Biosimilars
Integration of Information to Biosimilarity

9. General Regulatory Approach for
Assessment
 A risk-based, totality-of-the-evidence approach to
evaluate all data and information provided by a sponsor to
support a demonstration of biosimilarity
 Sponsors must use a stepwise approach in their
development of biosimilar products
 The type and amount of analyses and testing required to
demonstrate biosimilarity will be on a product-specific
basis
 General scientific principles in conducting comparative
analyses will be followed

US FDA

10. Recombinant protein production: sources of variation between manufacturers.
Mellstedt H et al. Ann Oncol 2007;19:411-419
© The Author 2007. Published by Oxford University Press on behalf of the European Society for
Medical Oncology. All rights reserved. For permissions, please e-mail:
journals.permissions@oxfordjournals.org

11. Reasons of Biosimilars’
Heterogeneity
 Reasons of Biosimilars’ heterogeneity (~ potential
differences between the biosimilar and the innovator
drug):
 Biological therapeutics are a complex mixture consisting of the
parent drug, multimers, truncated fragments
 The components may or may not exhibit biological activity,
post-translational modifications of the parent and/or truncated
fragments, host cell proteins as well as process related
impurities
 Any one of these can cause differences in the way these
drugs behave in the immunoassay, bioassay and
electrophoresis

12. The General Requirements are:
 Analytical studies demonstrating that the biological
product is “highly similar” to the reference product
 Animal studies (including the assessment of toxicity); and
 Clinical studies
 assessment of immunogenicity and pharmacokinetics (PK)
 PD studies or RCTs to demonstrate
 efficacy & safety
 purity, and potency
 in 1 or more appropriate conditions of use for which the
reference product is licensed.
 Overall Guiding Principles

13. Requirements for Approval

14. PK/TK: Same Platform Technology,
if possible
 Since the assay will quantitate both biosimilar (B) and
innovator (R) compounds
 Preferable to develop an assay using the same platform
technology (RIA, ELISA, TOF)
 However, it is not necessary to utilize the same assay
platform
 Use a comparability test for quantitation of both B & R
 To demonstrate comparability, at a minimum, accuracy
and precision tests should be conducted using B as CC
 When comparable, use one assay for both B & R
 Assays can be developed and validated using either B or R
 Often B is used for CC

15. PK/TK contd.
 Use both B and R QCs throughout the entire assay range (from
ULOQ to LLOQ)
 The same assay acceptance criteria should apply for both
 Meeting the accuracy and precision acceptance criteria will
demonstrate that both compounds are comparable, since one standard
curve is used to quantify both.
 Make Calibration (CC) samples with R [or B]
 Analyze QCs at least of 3 levels of both B & R
 Acceptance criteria: Intra- and inter-batch imprecision (%CV) and
inaccuracy (%RE) ≤20% except at LLOQ where up to 25% can be
allowed
 Method total error (sum the % of the CV and absolute %RE) < 30%
 Demonstrate absence of matrix effect

16. Dilutional Linearity
 Dilutional linearity must be tested
 For single dilutions, back-calculated concentration for each
diluted sample be <20% of the nominal within the linear range
(< 25% at ULOQ and LLOQ).
 For multiple dilutions, the back-calculated conc. for cumulative
diluted samples should be within < 20% of the nominal
original value.
 The precision of the cumulative back calculated concentration
should be < 20% (< 25% at ULOQ and LLOQ).
 The presence or absence of hook (or prozone) effect should
also be evaluated at the higher QC conc. (>1000×).

17. Selectivity (Non-interference from Matrix)
 Matrix interference should be performed using B QC
spiked samples
 spiked at high and low concentrations into at least 10
individual matrix samples
 It should also include the blank individual controls that
will be tested at the minimum required dilution (MRD).
 Acceptable non-interference should be seen in >80%
matrices tested.

18. Sample Stability
 Stability experiments should mimic, as best as
possible
 the conditions under which study samples will be
collected, stored and processed
 The duration during which….
 The effect of freeze-and-thaw cycles should also be
assessed.

19. Structural Analysis
 Sponsors should use an appropriate analytical methodology
with adequate sensitivity and specificity for structural
characterization of the proteins. Generally, such tests include
the following comparisons of the drug substances of the
proposed product and reference product:
 Primary structures, such as amino acid sequence
 Higher order structures, including secondary, tertiary, and quaternary
structure (including aggregation)
 Enzymatic post-translational modifications, such as glycosylation and
phosphorylation
 Other potential variants, such as protein deamidation and oxidation
 Intentional chemical modifications, such as PEGylation sites and
characteristics

20. Protein Characterization Assays
 Use validated bioassays or receptor-binding assays;
quantitative PCR would be excellent
 Show equivalency of potency and batch consistency
 Usual acceptance criteria: 80-125% but could be wider for
bioassays
 When wider, this assay may not be used for PK/TK comparability
 Isotyping – significant issue in characterizing assays
 It is important to evaluate if assay is indeed due to
immunoglobulin and, if so, what type of antibody
 If not IgG but IgE class, it could have potentially serious
safety outcomes.

21. Biosimilar EPO

22. Human PK

23. Immunogenicity Assays
 The immunogenicity of therapeutic proteins must be assessed
for safety and efficacy concerns
 small process changes during the production can change immunogenicity
rate & extent
 Immunogenicity rate is difficult to measure, particularly at low
incidence
 e.g., from autoimmune reactions to self proteins
 Large sample size would be required if the rate of immunogenicity
incidence is low
 It is critical to assess the immunogenicity of the B relative to R
 An assay using the same platform technology, the same
reagents under the same assay conditions to evaluate antidrug
antibodies (ADAs) would be desirable to assess reactogenicity

25. Immunogenicity Assays..
 Initiate very early during development of B, immunization of
animals to develop a positive control (against both B & R)
 Evaluate the two ADA positive controls (ADA B & R)
 Differences in the starting titers of the positive control antisera
against either the B or are possible due to the individual immune
response of each animal
 Assay platform could be ELISA, bridging assays, electrochemi-
luminescence (ECL) or RIA addressing:
 Can the assay reagents detect both B & R comparably?
 Can the assay tolerate both biosimilar and B & R conc.
comparably?
B = Biosimilar; R = Reference Innovator

27. Bioassay practices
Assessing “linearity” and similarity
Significance testing versus equivalence testing Laboratory B
0.8
p = 0.08 (p > 0.05, i.e., not Standard Data
Test Data
significantly different) 0.4 Standard Line
Test Line
Log10 Response
Conclude parallel! 0
0.5 1 1.5 2 2.5
Rewarded for poor assay -0.4
performance
-0.8
-1.2
Log10 Concentration
Laboratory A
0.8
Standard Data
Test Data
p = 0.02 (p < 0.05, i.e., 0.4
Standard Line
Test Line
significantly different)
Log10 Response
0
0.5 1 1.5 2 2.5
Conclude nonparallel!
-0.4
Penalized for good assay
performance -0.8
-1.2
Log10 Concentration

28. Non-comparable (Non-similar) Assays
 If comparability is not demonstrated, separate assays
should be validated for B & R Immunogenicity Assays
 If separate assays are to be used for future preclinical or
clinical comparability studies, interpretation is difficult
 samples from different arms of the study will be tested using
different assays
B = Biosimilar; R = Reference Innovator

29. Neutralizing-antibody (NAb) Assays
 For clinical studies, once a test sample is confirmed to be ADA
positive, evaluate it for Nab assay
 to see if it is neutralizing the biologic activity of the drug (B or R)
 Regulatory agencies usually prefer to have a cell-based NAb
assay
 but other assay formats (e.g., immuno-based assays) are OK when
appropriate cell-lines are not available during development
 If a cell-based assay exists for R, use the same platform for
NAb of B
 Validating cell-based NAb assays is technically difficult
 due to higher variability and a longer turnaround time for these
assays
B = Biosimilar; R = Reference Innovator

31. Patients with NAb can Develop PRCA
PRCA = Pure Red Cell Aplasia or Aplastic Anemia

32. Post-approval Commitment [example]

33. Thus
 Biosimilars are not like small molecule generics
 Differences between B & R would affect the B’s
potency, Clinical & PK characteristics and safety
profile
 A particular B might never be interchangeable with R
 Assays are complex, challenging but doable
 Validations are not only based on drug conc. alone but also on
biologic activity especially immunogenicity
 Demonstrate highly similar first in characterization and
animal studies (including the assessment of toxicity); then
clinical biosimilarity through immunogenicity, PK & PD
and clinical outcomes

34. Acknowledgments:
Dr. Nirav Desai
Mr. Chintan Patel
Thank you Very Much