Biosimilars are biological generics drugs.They undergo a rigorous evaluation to get approved.How to prove biosimilariy from analytical comparability is explained using a recently approved US FDA bio-similar monoclonal antibody.

1. By
Sada siva , Scientist,
Analytical Development,
Biologics/Bio-pharma professional.
Sadasivaibt@gmail.com
Role of Analytics in Biosimilars Approval
(mAb Case sudy)

2. This Discussion covers….
 Quick introduction on Biosimilars and Regulatory approval
bodies in India , Europe and USA
 Regulatory guidelines,Analytical requirements and
approaches used to show Biosimilarity
 Biosimilar mAb case study: Approved in USA & Europe
-Analytical data generated to show Biosimilarity
- Physico chemical / structural analysis (not functional assay)

3. What are these Biosimilars…
 A 'biosimilar‘ is a biological medicine that is similar to existing
biological medicine authorised .
 Biosimilars/ Follow on biologics/similar biologics/ subsequent
entry biologics/Similar biotherupetic product
 FDA: Biosimilarity is defined in section 351(i) of the PHSAct to
mean that the biological product is highly similar to the
reference product notwithstanding minor differences in
clinically inactive components and that there are no
clinically meaningful differences between the biological
product and the reference product in terms of the safety, purity,
and potency of the product

4. Why Biosimilars needed..??
 Bringing a new drug s into market s a very complex process
which includes large no. of .non-clinical and clinical studies.
 It is associated with high developmental Cost and long time,
Low success rates.
 Making Drugs cost higher to and low access to large patients.
 Regulators allow this to promote innovation in unmet
medical needs.

5. Evolution of the Concept of Abbreviated
medicines / Generics/ Biosimilarity
 Hence to overcome those constrains, Regulatorys came up wih
Abbreviated pathway for copy /similar drugs with reduced
design.( once the patent expire for new drugs)
 Generic small molecule drugs introduced “sameness”
 Biosimilarity (EU 2004,WHO 2009, US 2010) based on “highly
similar” to the reference and no clinically meaningful differences

6. Differences in Data requirements between
New and biosimilar molecules

7. Advantages of Biosimilars
 Similar drugs having same efficacy and safety comes to
market .
 Development cost and time are relatively less for Biosimilars
 It Helps in Cost reduction of Drugs
 It also Increased access to Patients for costly treatments.

8. Biosimilars approval in India
 Biosimilars approval is regulated by Drug controller general
India (DCGI) , Central Drugs Standard Control Organization
(CDSCO) and the Department of Biotechnology (DBT)
 Guidelines on Similar Biologic: Regulatory Requirements for
MarketingAuthorization in India (2015)
 First approved Biosimilar:
 Biosimilars approved :
 GCSF, Rituximab, Interferons, Bevacizumab, erytrhopointion,
Darbipoitin,Adalimumab, Etarnacept

9. Biosimilars approval in Europe
 Biosimilars approval is regulated by european medicines
agency (ema)
 Legal Basis: Directive 2001/83/EC
 First approved Biosimilar:
 Omnitrope (somatropin) from Eli Lilly (in 2006 )
 Others Biosimilars:Around 22 versons
 Growth hormone, EPO, GCSF, Infliximab, Eternacept, Insulin,
Interferon Etc..

10. Biosimilars Data requirement in Europe

11. Biosimilars approval in Europe

12. Biosimilars approval in USA
 Biosimilars approval is regulated by US FDA under PHS act.
 First approved Biosimilar:
 rGCSF from Sandoz (in 2015 )
 Others Biosimilars:
 Infliximab from Cellitron-First Biosimilar mAb in 2016
 Etarncept From Sandoz in 2016

13. Biosimilars approval in USA

14. Advantage of 351(K) Biosimilar
Pathway
 Quality Considerations in Demonstrating Biosimilarity of a
Therapeutic Protein Product to a Reference Product
 Scientific Considerations in Demonstrating Biosimilarity to a
Reference Product

15. Infliximab (by cellitron)–Case study
 First Biosimilar mAb in US and Europe
 Reference product: Remicade (Janssen )
 One of theTop 10 revenue generating biologics.
 Indications: Rheumatoid,Arthritis (RA), Psoriatic
Arthritis (PsA), Crohn’s Disease (CD) etc.

17. Infliximab biosimilar- Regulatory
History and FDA Interactions….
 Proposed Product development began in 2008 for Europe
market, was guided by EMA biosimilar guidelines and scientific
advice interactions.
 Based on these data, Proposed Product became the first
licensed biosimilar monoclonal antibody in the world.
Proposed Product is currently licensed for use in 67 countries.
 Following 2012 FDA draft biosimilar guidance, FDA provided
input on the development program for Proposed Product at
Biosimilar Biological Product Development meetings held on
2013 (BPDType 3) and 2014 (BPDType 4)

18. Infliximab biosimilar- Regulatory
History and FDA Interactions….
 FDA suggested that adequate analytical and PK
bridging data could be sufficient to demonstrate
Biosimilarity between Proposed Product ,US Remicade and
EU Remicade
 CELLTRION conducted analysis of the structural and
physicochemical attributes and testing of biological
activities using US Remicade, EU Remicade and
Proposed Product to demonstrate analytic biosimilarity
 In addition, CELLTRION conducted a 3-way PK Study to
bridge the EU Remicade PK data with that of US Remicade.

19. Biosimilarity – Approach….
 Biosimilarity is comparison exercise between a proposed and
reference product.
 The aim of the Biosimilar exercise is to demonstrate that the
Proposed product and the reference medicinal product are
similar at the level of the finished product.
 It is not expected that all quality attributes of the
biosimilar product will be identical to RMP.
 However, where qualitative and/or quantitative
differences are detected, such differences should be
justified and, where relevant, demonstrated to have no
impact on the clinical performance of the product.

20.  Analytical comparison plays main basis for Biosimilarity.
 Results of analytical comparison , helps to decide further
to the number of of Non clinical and clinical
comparability studies required to prove Biosimilarity.

21. Factors for consideration in assessing
Biosimilarity (FDA)….
 Reference Product and Reference Standards
 Expression system
 Manufacting process
 Assessment of Physicochemical Properties
 Functional Activities
 Receptor Binding and Immunochemical Properties
 Impurities
 Stability

22. Reference medicinal Product (RMP)….
 RMP used in Biosimilarity should be approved and licensed
by respective regulatory authority.
 Single sourced RMP should be used all the studies (Quality,
Non Clinical and Clinical studies )
 However, with the aim of facilitating the global development
of Biosimilars , it is allowed to compare the biosimilar with a
other regulatory authorised comparator.
 In this Case ,A bridging comparability study (Analytical and
PK and or PD ) has to be performed between the two RMP
products along with Proposed Biosimilar product.

23. Reference Medicinal Product….
 The reference medicinal product details should be captured
properly (Complete details like lot, source, presentation)
 Multiple different batches of the reference medicinal product
should be used and justified.
 The age of the different batches of reference medicinal
product (Wide range over expiry is recommended )
 Publicly available reference standards (e.g. Ph. Eur.) cannot
be used as the reference medicinal product for demonstration
of biosimilarity

26. Reference Medicinal product Details
 Side by side analysis performed between the 3 products in all
the tests
 Comparative stabilities performed.
Samples Number of
Batches
Age at the
Analysis
Proposed product 7-23 9 -21 M
US Remicade 7-32 2 – 29 M
EU Remicade 6-32 4-36 M

27. Expression system….
 Careful selection should be done to produce High similar
product .
 Expression system differences may results in undesired
consequences like atypical Glycosylation pattern, a
different impurity profile compared to RMP
 Better to choose the same expression system

28. Manufacturing process….
 Develop a process to produce a Product highly similar to
RMP
 QualityTarget for product profile (QTPP) should be
established at the early Development phase by RMP analysis
and publicly available in formation.
 Performance and consistency of the manufacturing process of
he biosimilar .
 Proper formulation should be selected .better to have the
same as RMP does not need to be identical.
 If a different formulation and/or container & closure system
, its impact on safety and efficacy should be justified

31. Assessment of Physicochemical
Properties….
 Extensive State of the art analytical tests should be used to
show Biosimilarity between the proposed product and the
reference product.
 Lots used for the analyses should support the Biosimilarity of
both the clinical material used in the clinical study(ies)
intended to support a demonstration of Biosimilarity
 Multiple representative lots should be used understand
the lot-to-lot variability of both products
 A risk-based assessment should be performed to
understand the impact and criticality of each
quality attribute on safety and efficacy.

32. Extensive analytical testing should
cover….
 Primary structures, such as amino acid sequence
 Higher order structures, including secondary, tertiary, and
quaternary structure (including aggregation)
 Enzymatic posttranslational modifications, such as
glycosylation and phosphorylation
 Other potential variations, such as protein deamidation and
oxidation
 Intentional chemical modifications, such as PEGylation sites
and characteristics

33. Analytical methods selection…
 Selection of tests chosen for comparability is mainly based on
characteristics of the protein product and its
potential impurities.
 Method to be used should have appropriate accuray,
sensitivity and specificity to provide meaningful
information as to whether the proposed product and the
reference product are highly similar.
 Unlike routine quality control assays, tests used to
characterize the product do not necessarily need to be
validated. Qualification is needed for characterization tests.

34. Analytical methods selection…
 It is often necessary to apply more than one analytical
procedure to evaluate the same quality attribute.
 Because they provide independent data to support the
quality of that attribute (e.g., orthogonal methods to assess
aggregation).
 In addition, the use of complementary analytical techniques
in series is encouraged , should provide a meaningful and
sensitive informaion for comparing products. (such as
peptide mapping combined with mass spectrometry of the
separated molecules)

35. Product specific tests…

37. Risk and criticality analysis….

39. Primary Structure analysis….
 Peptide mapping by HPLC showedt hat Proposed Product
has a highly similar chromatographic peak profile to US and
EU Remicade . No missing or additional significant peptides
were detected and peaks had comparative retention times.

40. Structure analysis-Data….

41. Charge analysis….
 The same charge variant peaks were detected in Proposed
Product, US Remicade, and EU Remicade. Proposed Product
contains higher levels of Peak 1 and Peak 4 than US or EU
Remicade.The difference is attributable to C-terminal lysine
variability.
 Further analysis of the impact of C-terminal lysine variability was
obtained from in vitro studies of biological activities, which showed
that C-terminal lysine variability had no bearing on
biologicalactivities in vitro.
 Additionally,incubation with IgG-free human serum resulted in rapid
clipping of C-terminal lysine residues shown as reductions in Peaks
4, 5, and 6 which contain forms with one or two C-terminal lysine
residues

42. IEX profile….

43. Glycan analysis….
 Glycan site analysis by LC-MS
 Glycan analysis by NP-HPLC
 Sialic acids (Charged glycans) analysis by HPAEC-PAD
 Monosaccharide analysis
 lower amounts of afucosylated glycans (G0 and Man5) in
Proposed Product had previously been observed
 As there is no association of G0 with immunogenicity and G0
is present on endogenous antibodies, there appears to be no
safety impact of the lower level.

44. Glycation….
 Although high similarity in percent glycation of Proposed Product
and Remicade was notobserved as Proposed Product contained
higher levels of glycated forms
 No differences in biological activities between Proposed Product
and Remicade are expected due to glycation, as was supported by
data from similarity studies of biological assays includingTNFα
binding and neutralization assays.
 Data from samples of Proposed Product and US Remicade with
artificially created levels of glycation showed that glycation has no
impact on FcγRIIIa binding affinity of Proposed Product or US
Remicade.
 No impact on immunogenicity is expected as antibodies are
glycated on incubation with serum and in vivo

45. Impurities…..
 The sponsor should characterize, identify, and quantify
impurities in the proposed product and the
reference product, to the extent feasible
 If a comparative physicochemical analysis reveals comparable
product-related impurities at similar levels between
the two products, no additional studies required.
 If proposed product different or higher levels of impurities
than in the RMP, then additional pharmacological
/toxicological or other studies may be necessary to
understand their impact.

46. Impurities…..
 It is preferable to rely on purification processes to remove
impurities . . . rather than to doing additional tesing.
 Differences that may have a safety advantage (e.g. lower
levels of impurities) should be explained.
 The process-related impurities in the proposed product are
not expected to match those observed in the reference
product. But they should be minimised by process.
 The potential impact of the differences in the impurity
profile upon safety should be addressed and supported by
appropriate data.

47. Aggregate analysis
 SEC-HPLC detects monomer, molecular weight forms(HMW).
Proposed Product lots had a higher HMW content (mean value
0.8%) than US &EU Remicade lots (0.2%)
 The clinical relevance of the higher HMW and lower monomer
content in relation to immunogenicity was assessed through the
repeat-dose studies in RA andAS and was shown not to be
clinically meaningful.
 SEC-MALS also detects monomer, HMW, and their sizes.
 Analytical Ultracentrifugation (AUC) is an alternative method to
measure monomer and HMW forms.All 3 products contained
monomer as the predominant sedimenting species.

51. Stability Data….
 Proposed Biosimilar & RMP degradation profiles should be
established .
 Accelerated and stress stability studies, as well as forced
degradation studies should be performed side by side.
 These comparative studies should be conducted under multiple
stress conditions (e.g., high temperature, freeze thaw, light
exposure, and agitation)
 Results should be presented and if these studies may reveal
product differences that warrant additional studies .
 Shelflife should be determined from the Realtime stability data of
the Proposed Biosimilar and extrapolation is no recommended.
(RMP in real time stability is not required for Biosimilarity)

52. Stability studies performed

53. Functional Activities
 Functional assays are imp part of Biosimilarity as an essence
step establishing complete characterization profile.
 These tests act to complement physicochemical analyses and
are a qualitative measure of the function of the protein
product.
 For complex proteins, the available analytical technology, the
physicochemical analysis may be unable to confirm the
integrity of the higher order structures. In his case
functional assays are very helpful.

54. Receptor Binding Properties
 Receptor Binding and Immunochemical Properties
 When binding or immunochemical properties are part of the
activity attributed to the protein product, analytical tests
should be performed to characterize the proposed product in
terms of these specific properties, (e.g., if binding to a
receptor is inherent to protein function, this property should
be measured and used in comparative studies)

58. Statistics recommended…

61. `

66. Some important points to consider
about Biosimilars are….
 Biosimilars don’t have to be proved for every indication.
 Other than looking at the guidelines there are no
requirements for development / trials of biosimilars.
 Biosimilars must have comparable quality, safety and efficacy.
 Each Biosimilar will have gone through the appropriate
regulatory pathway.
 Every Biosimilar product is different and each should be
considered an individual brand in their own right.
 Because every biosimilar is different – no one set of
regulatory rules will work for all developing the need for
each to be considered on a case by case basis.

67. Other related topics for discussion….
 Functional studies comparison of this case study
 Nonclinical and Clinical studies of this case study
 Other Biosimilar Case studies (GCSF and Etarnacept)

68. Learning starts from questioning
??????????

69. For giving this opportunity

70. References….
 EMEA:Guideline on similar biological medicinal products
 EMEA:Guideline on similar biological medicinal products containing biotechnology-
derived proteins as active substance: non-clinical and clinical issues
 EMEA:Guideline on similar biological medicinal products containing monoclonal
antibodies – non-clinical and clinical issues)
 FDA:Quality Considerations in Demonstrating Biosimilarity of aTherapeutic Protein
Product to a Reference Product
 FDA:Scientific Considerations in Demonstrating Biosimilarity to a Reference Product
 FDA:Clinical Pharmacology Data to Support a Demonstration of Biosimilarity to a
Reference Product
 ICH guideline Q5E: Note for guidance on biotechnological/biological products
subjected to changes in their manufacturing process
 ICH guideline Q5C: Note for guidance on quality of biotechnological products: Stability
testing of biotechnological/biological products
 ICH guideline Q6B: Note For Guidance on Specifications:Test Procedures and
Acceptance Criteria for Biotechnological/Biological Products
 India DCGI: Guidelines on Similar biologics by CDSCO and DBT