1. Comparability Study (for Biosimilar)
Wisit Tangkeangsirisin, PhD
Faculty of Pharmacy, Silpakorn University

2. Content: Biosimilar Definition and Concept Regulations
Quality Assessment of Biosimilar
Example and Cases
Assignment

3. Monoclonal Antibodies
Biologics
Biopharmaceuticals
Recombinant Proteins
Monoclonal Antibodies
Etc.
Others
Gene therapy
Cell therapies
Tissue therapy
Allergens
Blood
&
Blood Products
Vaccines
&
Toxoids

4. Monoclonal Antibodies
Therapeutic Proteins
Biopharmaceuticals

5. Required only for Biosimilarity

6. Major Biopharmaceuticals
Recombinant Proteins
Insulin, Epoetins, Interferons, Somatropin , Filgrastim
Monoclonal Antibodies
Trastuzumab, Rituximab, Infliximab
Immunoglobulin Fusion Proteins
Etanercept

7. Biopharmaceuticals First generation Native Proteins, Unengineered
murin antibodies, simple replacement proteins
Frequent Injection
Second generation
(biobetter)
Engineered, modified, alteration of amino acid sequence, alteration of glycosylation, PEGylation
Suitable PK

8. The complexity of biologics relates to structure and function
Monoclonal antibodies belong to the most complex classes of biologics
CDRs (complementarity determining regions)
Selected for optimum target mediated effects (light chain and heavy chain variable binding domains)
Polypeptide subunits
Carbohydrate
For comparison – the size and complexity of aspirin
Fc region:
Different isotypes with different effector functions
Contains 2 glycosylation sites (1 in each of the 2 polypeptide chains)
In total more than atoms
All parts contribute to the efficacy and safety in a cooperative way
Slide notes taken from original slides
Monoclonal antibodies belong to the most complex classes of biotherapeutics
– atoms, MW ca (IgG)
4 subunits, 2 light and 2 heavy chains
Glycosylated: 2 Fc glycosylation sites plus optional Fab glycosylation
Multifunctionality
Antigen (target) binding through unique CDRs in Fab part
multiple interactions of Fc part influencing effector functions and pharmacokinetics
Immunoglobulin G1 (IgG1) molecule: Padlan EA. Mol Immunol 1994;31(3):

9. Monoclonal Antibodies
Structurally much more complex than other proteins
Complexity
More complex than currently developed biosimilars
Biological Activity
glycosylation patterns are critical-smalll differences correlate to changes in biological activity
Predictability
multi-functionlity (binding and immuneffector functions) coupled with an often limited understanding of structure-function relationship will limit predictability of in vitro data
Extrapolation
complexity and diversity of the mechanisms of action will be of particular challenge for indications and line extensions

10. Quality Attributes to Consider - Mab
Functional characteristics
Physico-chemical characteristics
Fab Fc
Antigen binding
Effector functions
complement interaction
Fc recepter interaction
N-terminal heterogeneity
pyroglutamate formation
Other modifications
AA modifications
deamidation, oxidation, glycation, isomerization
Fragmentation
Cleavage in hinge region, Asp-Pro
Oligosaccharides
Fucosylation, sialyation, galactosylation…
Disulfide bonds
Free thiols, disulfide shuffling, thioether
C-terminal heterogeneity
Lysine processing, proline amidation

12. Biologics are hard to copy

13. Significance and Needs
Why Biosimilar??

14. Why Biosimilar? Ensuring safety and effectiveness
Accessibility of high cost medicine
Safety
Effectiveness
Cost of Medicine
(Biologics)

15. Definition
Global Approval Pathways
Key Issues
biosimilars

16. NDA vs. ANDA Review Process
New Drug
NDA Requirements
Generic Drug
ANDA Requirements
Chemistry
Manufacturing
Controls
Labeling
Testing
Animal Studies
Clinical Studies
Bioavailability
Chemistry
Manufacturing
Controls
Labeling
Testing
Bioequivalence

17. Generics vs. Biosimilars
Patent
Expired
Active Pharmaceutical Ingredients
Identical
Never identical
(Highly Similar)
Proof Needed
Bioequivalence
Comparability

18. What Biosimilar are…. And are not?
They are similar …… They are not the same
They are different from innovator’s product.
They are not generic biologics

19. Which biologics are in the scope of Biosimilar?
Vaccines
Blood Products
Recombinant Proteins
Monoclonal Antibodies
Transgenic cells/ Cell therapy
EMA guidelines – can be adapted to all biologics including blood products, vaccines
WHO guidelines and others – adapted only well established and well characterized biotherapeutic products

20. BioMarket Trends Jun 15, 2012 (Vol. 32, No
BioMarket Trends Jun 15, 2012 (Vol. 32, No. 12) Biosimilar Market Posts Steady Gains
Actual Performance Varies between Regulated and Semi- and Unregulated Environments.

21. Biosimilarity requirements
Can we use
the same approval pathway
of generic
for biologic?

22. Same Approval Pathway as Generics for Biologics?
For Blood Products and Vaccines
No, the approval pathway treats them as New biologics.
For Recombinant Proteins
Yes and No
Yes  Generic Approval
No  New biologics Approval
Somewhat between  Biosimilar

23. Biosimilarity is built on five indispensible pillars.
Modified from lecture of Dr. Thomas Schreitmueller

24. Approval of Biosimilar vs. Originator Biologics
Pharmacovigilance
Clinical
(PI,II,III for each indication)
Clinical
(with extrapolation)
Non-clinical
Non-Clinical
Comparability exercise in Quality
Chemistry, Manufacturing and Control
Chemistry, Manufacturing and Control
Originator Biologics
Biosimilars

25. Establishment of biosimilar guidelines increased, driven by WHO
But: Variability in adoption by National Regulatory Authorities
2010
2015
Biosimilar pathways in place
Biosimilar pathways under development

26. Global regulatory environment
In general, regulators require:
Comparative analytical program
Clinical program
Post-approval commitment to Pharmacovigilance
High degree of analytical similarity
High degree of pre-clinical and clinical similarity
Regulators may permit extrapolation when scientifically justified.

27. Key Issues in Biosimilar Dossier
Quality Comparisons
The most critical and extensive step
Cell culture, impurities, glycosylations ……..
Non-clinical Comparisons
In vitro receptor binding & cell based assay are fundamental
Clinical Comparative Studies
Most sensitive population and endpoints
6-12 months safety data
Extrapolation of indications

29. Current Thailand Biosimilar Guidelines
คู่มือ และหลักเกณฑ์การขึ้นทะเบียนตำรับยาชีววัตถุคล้ายคลึง 2556
Defines philosophy and principles
Biotechnology-derived proteins
General guidelines
Quality
Non-
Clinical
EPO Review
Unorthodox
Pathway ????
2557
2558
2559
Epoetin
IFN-α
Somatropin
GCSF
mAbs
IFN-β
FSH
Annex guidelines- specific data requirements
Non-
Clinical
Non-
Clinical
Non-
Clinical
Non-
Clinical
Non-
Clinical
Non-
Clinical
Non-
Clinical
Immunogenicity
Guideline for biosimilars

30. ประเภทของยาชีววัตถุในประเทศไทย
Previous
New Regulations
ยาชีววัตถุใหม่
ยาชีววัตถุที่ไม่ใช่ยาใหม่
ยาชีววัตถุใหม่
ยาชีววัตถุคล้ายคลึง

31. Extrapolation of indications
key to the biosimilar concept
needs to be justified in all cases

32. Extrapolation of Indications
The Key Consequence of Biosimilar is the EXTRAPOLATION of INDICATIONS
Justification of the extrapolated indication is on a case-by-case basis.
Decision based upon mechanism of action, type of receptors, Affinity etc.

33. Interchangeability
In EU, the biosimilar approval do not automatically allow interchangeability
Interchangeability/switching remains a national decision.
Later, after 10 yrs experience of biosimilar in the market, several countries change regulations.

35. Production of Biosimilar Limitations
Limit access in manufacturing process information
Need to Proof Similarity in Certain Items
Product Monograph
Specific Guidelines
Scientific Evidences

36. Dossier requirements for a biosimilar vs. an originator
Dossier requirements for a biosimilar compared with an originator (stand-alone) dossier. For quality, a more extensive dossier is required, which includes the comparability exercise to the originator, whereas the nonclinical and clinical parts of the dossier cross-reference to parts of the reference mAb dossier whenever possible.
FromSetting the stage for biosimilar monoclonal antibodies
Christian K Schneider,
Camille Vleminckx,
Iordanis Gravanis,
Falk Ehmann,
Jean-Hugues Trouvin,
Martina Weise
& Steffen Thirstrup
Nature Biotechnology 30, 1179–1185 (2012) doi: /nbt.2447
The State of the Art in the Development of Biosimilars
Mark McCamish and Gillian Woollett Nature Biotechnology 30, 1179–1185 (2012)

37. Manufacturing Process (1)
Demonstrate the Consistency and Robustness of manufacturing process
cGMP implementation
Modern QA/QC procedures
In-process controls
Process validation
Minimize Differences b/w SBP vs. RBP
Reduction clinical testing requirements of SBP
Minimize any predictable impact on clinical safety and efficacy of SBP
Differences are expected and may acceptable
Appropriate justification  lack of impact on clinical performance

38. Manufacturing Process (2)
Current manufacturing process following current guidelines (ICHs) and extensive quality control is encourage
Potential impact of Changing of on Quality Safety and Efficacy should be identified.
Some Differences are expected and may acceptable
Appropriate justification (based on scientific and clinical experience  lack of impact on clinical performance

39. Comparability Exercises in Quality of Drug substance
General Principles
Manufacturing Processes
To ensure the similarity of drug substance in SBP vs. RBP (vs. Reference standard)
Products should be expressed and produced in the same host cell type
Expression vectors
Cell banks
Cell culture/ Fermentation
Harvest
Purification
Modification reaction
Filling / Storage
Dosage form
Container closure system

40. Analytical Techniques
Using appropriate, state-of-the-art in Biochemical, Biophysical, Biological
Provided on
Primary and higher-order structures
Post translational modifications
Biological activity
Purity
Impurities
Product-related substance
Immunochemical properties

41. Justification of Analytical Methods
Suitability
State-of-the-art (Current and up-to-date)
Able to detect any difference between SBP vs. RBP
Method Validations

42. Physicochemical Assessment
A comprehensive understanding and description of the RBP vs. SBP to support development throughout the manufacturing process to
Understand impact of manufacturing changes
Ensure continued and consistent biosimilar properties to the reference product
Build a database to support the Quality by Design (QbD) program
Orthogonal methods/multiple techniques employed to fully elucidate structure (as far as possible) including microheterogeneities

43. Physicochemical Assessment
Extensive enough to fully characterize primary and higher order structure including post translational modifications
Primary structure, sequence and bridging (disulphide, thioether)
Higher (secondary, tertiary and quaternary) order structure
C and N terminal variants
Post translational modifications
Protein concentration
Surface charge
Protein size
Aggregation
Bioassay, potency
Purity and impurities

44. Wisit Tangkeangsirisin, Biosimilars Guideline Public Hearing
Biosimilar filgrastim compared to reference product (Neupogen) using three state-of-the-art analytical techniques
Biosimilar filgrastim compared to reference product (Neupogen) using three state-of-the-art analytical techniques.
The State of the Art in the Development of Biosimilars
Mark McCamish and Gillian Woollett
The State of the Art in the Development of Biosimilars
Mark McCamish and Gillian Woollett
2/22/2013
Wisit Tangkeangsirisin, Biosimilars Guideline Public Hearing

45. Biological Activity Assessment
Biological activity based on clinically relevant assays are required
The extent is, to some degree, predicated by the physicochemical data
All clinically relevant functional attributes to be assessed where possible
E.g., for a mAb it may be necessary to test for both antigen binding and effector binding in appropriate in-vitro bioassays
Relevance may determine extent of further nonclinical (and clinical studies)
Animal toxicity studies not useful if no suitable model
Studies should be comparative and provide a meaningful toxicological comparison
Stand-alone toxicology study can be justified
Safety pharm, repro toxicity, and carc studies not generally warranted
Single-dose PK and PD study in animals can be useful

46. Specifications
ICH Q6B : Specifications: Test Procedures and Acceptance Criteria for Biotechnological/Biological Products
Data from batch-to-batch consistency, pharmaceutical development, comparability exercise in quality, safety and efficacy profile
Range of acceptance should not wider than RBP

47. Possible biophysical and clinical outcomes for biosimilars
Full Comparability Studies for Safety and Efficacy
Biophysical Comparison
No significant differences
Significant differences
Clinical Results
No detectable difference
Bioequivalence Based Approach
Possible biophysical and clinical outcomes for biosimilars

48. Biosimilar Development
Understand a Reference Product
Characterization
Identify Critical Quality Attributes (CQA)
Creating and Producing a Biosimilar
Identify the suitable clones
Production process development
Throughout Comparison of the Reference Product vs Biosimilar
Analytical testing and Clinical study
Totality of Evidence for similarity

49. Biosimilar Development

50. Data requirements for comparability exercises for a moderate risk and a high risk manufacturing change by the same manufacturer compared with a biosimilar manufacturing process.

51. http://www. amgenbiosimilars

52. Therapeutic Mab Critical Quality Attributes
Charge Variants
Glycolysation
Immunogenicity
Aggregation & Degradation
Primary & High Order Structures

53. RED = always very high criticality
BLUE= at least high criticality
BLACK = variable criticality

55. Why do we need state of the art analytics?

56. Quality: Analytical
Analytical comparison is more than “Just Meets Specs”
Possible Analytical Comparison Outcomes
Analytical Methods Alone Confirm High Similarity
Analytical Methods Not Sufficient or Adequately Discerning
Analytical Methods Confirm Differences, but Differences Are Shown to Be Not Practically Significant
Eg. HX575-higher phosphorylated mannose type glycan
Analytical Methods Confirm Differences, but Significance of Differences on Safety and Efficacy Cannot Be Ruled out
Analytical Methods Confirm Not Comparable
Analytical Methods is better

57. Changes in Process: Changes in Products Quality Attributes
ARANESP

58. State of the Art Analytics

59. RITUXAN

60. ENBREL

61. Case studies on biosimilar approval in EU

62. Omnitrope (somatropin)
RBP = Genotropin
During development, changes by increase complexity of comparability demonstration
Esp. Additional steps to reduce Host Cell Protein
V. high level of non-neutralizing antibodies (about 60%, during clinical trials (old process))

63. Binocrit (Epoetin alpha)
RBP = Eprex
Extensive characterization and comparability exercise in quality
Structure comparisons
Qualitative similar
Quantitative differences found (increase in high mannose-6-phosphate, Decrease in N-glycolyl-neuraminic acid (NGNA)
Differences was justified
PRCA concerns
SC route contraindication in CKD

64. Silapo (epoetin zeta) RBP = Eprex
Extensive characterization and comparability exercise in quality
Structure comparisons
Qualitative similar
Quantitative differences found (increase in des O-glycan forms, Decrease in NGNA
Differences was justified
SC route initially contraindicated
SC indications added post-authorization upon further clinical data

65. Zarzio (filgrastim) RBP = Neupogen
Extensive characterization and comparability exercise in quality
Clinical data
Phase I: Comparative PK/PD studies in healthy volunteers. Efficacy endpoints: neutrophil and CD34 cell counts
Phase III: Non-comparative (single arm) clinical trial in cancer patients undergoing chemotherapy. Safety focus
G-CSF Guideline states:
“The recommended clinical model for the demonstration of comparability of the test and the reference medicinal product is the prophylaxis of severe neutropenia after cytotoxic chemotherapy in a homogenous patient group (…). Alternative models, including pharmacodynamic studies in healthy volunteers, may be pursued for the demonstration of comparability if justified”

66. Valtropin (somatropin)
RBP = Humatrope
Different expression system compared to reference medicinal product (S. cerevisiae vs. E.coli). Process specific HCP (yeast) assay required
Changes during development subject to additional comparability
Clinical trial:
Initially calculated to demonstrate non-inferiority
US sourced RBP used (supportive data)
Indication differ from Omnitrope
Pediatric indication for Omnitrope only: Small gestational Age, Prader-Willi Syndrome
May 2012 Voluntarily withdrawn for commercial reasons

67. Insulin Marvel (Withdrawal)
RBP = Humulin S
Three forms (Short, Intermediate (30/70), Long acting)
Quality Concerns
Incomplete comparability exercise, esp Drug Product
Inadequate process validation
Missing batch traceability
More data required for extended release forms
Clinical issues
Comparative PK/PD: euglycaemic clamp – most sensitive model
Similar PK Parameters, but not similar PD profiles (faster glucose absorption
Applicant resorted to efficacy trial with HbA1C end-point, not sufficiently sensitive
Limit Immunogenicity data

68. Alpheon (rhIFNalpha-2a; Negative)
RBP = Roferon-A
Quality concerns
Stability and impurities for drug substance and drug product profile also not matching RBP
Inadequate process validation in DP
No comparability studies between clinical trial batches and commercial batches
Non-clinical studies were inadequate and indicated differences
Clinical issues
Difference in virological relapse rates
Inconclusive data in response rate for Genotype 1 patients
Different rate of adverse events
Inadequate immunogenicity documentation

69. Some Concerns About Biosimilars
Route of Administration for approved indications of biosimilar may not be the same as Reference Biological Product
Eg. Binocrit initially contraindicated in CRF by SC route

70. Different glycan pattern--Not biosimilar REALLY????????
HX575 has different glycan profiles from RBP.
But the differences are acceptable and justified.

71. EPO Isoforms among Biosimilar vs. RBP are similar.
Not Identical

72. “ More recently, two cases of neutralizing antibodies were reported during a pre-marketing clinical trial of a biosimilar epoetin (HX575) in subcutaneous administration in chronic renal failure subjects (9). In this paper, we present data showing that contamination by tungsten during the manufacture of the syringes used for primary packaging is the most likely cause of protein denaturation and aggregation, which may have led to the occurrence of two cases of neutralizing antibodies in the investigational clinical trial.

73. Conclusion
Quality assessment is the primary and major part of biosimilar registrations.
Comparability studies with RBP determine the degree of intensity in non-clinic and clinical assessment.
State-of-the-Art technology should be used to detect any potential differences.
How to demonstrate product comparability is challenging
Case studies about EU biosimilar approval confirmed case-by-case varies

74. Any questions, comments and suggestions are welcomes
Thank you
Any questions, comments and suggestions are welcomes