Develop A Highly Similar" Biosimilar Compound: Lessons Learnt

Transcription

1 Develop A Highly Similar" Biosimilar Compound: Lessons Learnt Hui-Chun Li, Ph.D. Sr. Manager of Process Science 2015 Feb 05 3rd Biologics World Taiwan 2015

2 SPIN OFF ACQUISITION Development Center for Biotechnology cgmp BioPharmaceutical Pilot Facility Formosa Laboratories A strategic Investor Investment Bank VC and NDF

3 20,060 3, 4, 6F 30,780 10,260 6F 5F 4F 3F 2F 1F B1 Staff Office Microbial R/D Laboratory Microbial CGMP Production Area HVAC units Staff Office Cell Line Development & Analytical Lab. Process Development & QC Lab. Central Control Systems HVAC Air Handling Units Cell Banking Area (I) Cell Banking Area (2) Cell Culture CGMP Production Area CGMP Warehouse Cleaning and Support Utilities Bio-waste Inactivation 11,000 sq. ft.

4 One-Stop-Solution from DNA to NDA Pre-Clinical Research IND Clinical Development NDA Customized Roadmap & Proposal Design Cell Line Development Process Development Analytical Development Protein characterization Biosafety Test & Toxicology cgmp Manufacturing (Mammalian cell & Microbial) Powered by EirGenix Project Management, Documentation and Regulatory Support, and Quality Management To provide the high quality and cost effective Contract Development & Manufacturing services for our clients, and to develop high quality and cost effective biologic products together with partner(s) to the benefit of our communities and society as a whole

5 Biosimilar Business Global biosimilars market expected to show dramatic growth rates Source: IMS Health

6 EirGenix Business Strategy CDMO BIOSIMILARS ME TOO / ME BETTER

7 Biosimilarity The Essentials Additional clinical studies Additional clinical studies Clinical Pharm Originator Clinical Pharm Nonclinical Biosimilar Nonclinical Analytical Analytical A stepwise approach to demonstrate biosimilarity between biosimilar and reference medicinal product with respect to structure, function, animal toxicity, human pharmacokinetics (PK) and pharmacodynamics (PD), clinical immunogenicity and clinical safety and effectiveness.

8 Establishing Biosimilarity Quality Non-clinical PK/PD studies VS Demonstrate similarity of biosimilar to reference product Pre-clinical toxicology Confirm dosing regimen by PK profiles Confirm the mechanism of actions by biomarkers (PD) Clinical safety and efficacy Confirm therapeutic index and safety profiles Qualify impurities by short-term animal studies Full animal toxicity studies are Confidential not necessary Immunogenicity and/or Hypersensitivity Comparison Confirmatory trials or other clinical trials for interchangeability Use of complementary biomarkers, or surrogate 8 endpoints in some cases

9 Knowing the Structure: Analytical Characterization + - Primary Structure Analysis HPLC, LC/MS/MS Peptide map; total sequence analysis N-/C-terminal variants Disulfide linkages Oxidation, deamidation, and other post-modifications Secondary and Higher Order Structures CD (Circular Dichroism) DSC (Differential Scanning Calorimetry) Fluorescence Spectrometer Heterogeneity Analysis IEF/cIEF CIX-HPLC Glycan Analysis N-, O-linked carbohydrates site and structure by LC/MS/MS N-glycan ratio by CE Monosaccharide analysis CIX-HPLC

10 Heavy Chain Knowing the Function: Biological and Preclinical Characterization Antigen Binding Cellular Binding Assay Surface Plasmon Resonance Fab Programmed Cell Death Cell Based Apoptosis Bioassay Fc Heavy Chain Effector Functions ADCC CDC ADCC Cell Based ADCC Bioassay Surface Plasmon Resonance CDC Cell Based CDC Bioassay Surface Plasmon Resonance, ELISA

11 Stepwise testing Similarity & Comparability Structure and Function tests provide full characterization based on: Molecular complexity Manufacturing process Degree of characterization Clinical indications Production history Availability of safety and clinical data Quality Studies Physicochemical tests Bioactivity/Potency assays Stability Non-clinical Studies PK/PD studies Toxicology Clinical Studies Efficacy Immunogenicity Biosimilar Products Comprehensive studies due to: Different cell line and production process Different characterization and release tests Short production history Extensive safety and clinical data not available

12 Confirm highest degree of similarity Matching the reference product Similarity/Equivalence Validation with Uncertainty management Clinical Trial PK/PD Design specification for product & process Pre-clinical Biological Characterization Analytics Physicochemical Characterization Process Development

13 Biosimilar Case Study

14 Biosimilar Development Clinical & Bioanalysis Human PK Study PK & Pivotal Trial Side-by-Side (comparability exercise) Physiochemical Biological Pre-Clinical Clinical Process development Cell culture in bioreactor Purification Cell line development Cell line Stability Titer improvement Protein Sequence Lys-C mapping by MS Gluc-C mapping and MS

15 Case 1 _ Amino acid sequence variant

16 Case 1 _ Amino acid sequence variant Extra peak Biosimilar Reference -- Extra peak is observed while analyzing biosimilar charge variants profile with CIX-HPLC.

17 Case 1 _ Amino acid sequence variant (L)K1-K2 +4 (L)K1-K2 +3 Biosimilar 1-XXAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAA-44 Reference -- Light chain analysis showed additional peaks (L)K1-K2 in biosimilar only, not in the reference via RP-HPLC/MS/MS analysis. Extra two amino acids were present in this peak.

18 Case 1 _ Amino acid sequence variant O Pre-protein N-terminal XXXXXXXXX AAAAAA Protein N-terminal XXXXXXXXXAAAAAA 100% 100% C-terminal XXXXXXXXXAAAAAA N-terminal C-terminal C-terminal

19 Case 1 _ Amino acid sequence variant O Pre-protein N-terminal XXXXXXXXX AAAAAA Protein N-terminal XXXXXXXXXAAAAAA 100% 100% 80% C-terminal XXXXXXXXXAAAAAA N-terminal C-terminal X C-terminal 0% ~20% N-terminal XXXXXXX XXAAAAAA C-terminal -- Cryptic signal peptide cutting site on light chain is the possible cause for amino acid sequence variant.

20 Case 1 _ Amino acid sequence variant Steps P1 P2 P3 P4 P5 P6 P7 P8 Step Recovery Total Recovery Protein A % 84% CIX % 48% Mix-mode % 40% AIX % 39% -- Purification efforts were made to reduce the level of extra peak from 27% to ~0%. The total recovery rate is less than 50%.

21 Case 1 _ Amino acid sequence variant Observation: - Extra peak presence on CIX-HPLC chromatogram - Extra amino acids presence on light chain by LC/MS/MS Impact: - Sequence fidelity - Product impurity - It can be removed by purification process modification at the expense of recovery rate Solution: - Start over with a different signal peptide

22 Case 2 _ Glycan profile variation The impact of afucosylation Sialic acid Galactose GlcNAc Mannose Fucose Asn297

23 afucosylated N-glycan ratio (%) Case 2 _ Glycan profile variation Afucosylation ratio _ Clone and Medium 20 clone A clone B clone C clone D Reference Culture media -- In order to find the cell line with similar afucosylation profile, N-glycan analysis was applied during clone selection. Different afucosylation level was observed in different clones.

24 afucosylated N-glycan ratio (%) Case 2 _ Glycan profile variation Afucosylation ratio _ Clone and Medium 20 clone A clone B clone C clone D Reference Culture media -- Different afucosylation level was observed as a combinational effect of clones and media. -- Clone B was selected for further fed-batch process optimization.

25 afucosylated N-glycan ratio (%) Case 2 _ Glycan profile variation Afucosylation ratio _ Supplement Reference Supplement concentration -- The base line level of afucosylation of clone B in fed-batch condition is still lower than reference.

26 afucosylated N-glycan ratio (%) Case 2 _ Glycan profile variation Afucosylation ratio _ Supplement Reference Supplement concentration -- Supplement showed positive effect on clone B s afucosylation profile.

27 Case 2 _ Glycan profile variation Typical process Productivity Productivity Productivity clones clones 1-5 clones 1 clones Process Development - Productivity - Quality

28 Case 2 _ Glycan profile variation Typical process Productivity Productivity Productivity clones clones 1-5 clones Modified process for biosimilar 1 clones Process Development - Productivity - Quality Productivity Quality clones clones 1-5 clones Productivity Quality

29 Case 2 _ Glycan profile variation Observation: - Variation of afucosylation level derived from clones - Variation of afucosylation level derived from culture media Impact: - Physicochemical similarity of biosimilar - Biological activity similarity of biosimilar Solution: - Cautiously select clones and media from early process development

30 Case 3 _ Charge variants variation

31 acidic variant ratio (%) Case 3 _ Charge variants variation 60 original modified 40 Reference 20 0 a b c Culture time (days) -- Culture time showed impacts on product acidic variants ratio of biosimilar.

32 acidic variant ratio (%) Case 3 _ Charge variants variation 60 original modified 40 Reference 20 0 a b c Culture time (days) -- Modified biosimilar cell culture process under certain culture time can mimic the level of acidic variant level of reference.

33 Case 3 _ Charge variants variation Typical process Productivity Productivity Productivity clones clones 1-5 clones Modified process for biosimilar 1 clones Process Development - Productivity - Quality Productivity Quality clones clones 1-5 clones Productivity Quality

34 Case 3 _ Charge variants variation Observation: - Higher acidic variants level observed derived from cell culture process Impact: - Physicochemical similarity of biosimilar - Biological activity and other quality attributes similarity of biosimilar Solution: - Modification of cell culture process to modulate the acidic variants level of biosimilar

35 Summary Comparability and Similarity: - The "comparability" is to compare physicochemical, biological and in-vivo properties of a product before and after the major changes of manufacturing process or manufacturing site change. - The "similarity" is to compare physicochemical, biological and in-vivo properties of a biosimilar to the innovator's product. Comparability exercise for biosimilars: - The biosimilar approach is to perform a stepwise process from physicochemical and biological characterization to non-clinical in vitro/ in vivo studies and clinical studies. - To demonstrate quality characteristics, biological activity, safety and efficacy based on a comprehensive comparability exercise.

36 Summary Eliminating product related impurity as amino acid sequence variants derived from cryptic cleavage of the signal peptide is critical to maintain product homogeneity. Fine-tune product physicochemical properties such as glycan and charge variant level starting from clone selection stage has higher probability to minimize similarity issue between biosimilar and reference medicinal product.

37 Acknowledgement EirGenix Process Science Group Quality Control Group

38 Client s Success is Our Priority