Effectively utilizing Post Translational Modification analysis to fast track process development for Biosimilars

Transcription

1 Effectively utilizing Post Translational Modification analysis to fast track process development for Biosimilars Sanjeev Gupta DGM-Advanced Biotech (R&D) Ipca Laboratories Ltd., Mumbai, India March 04, 2015 BioSimilars World India-2015, 4-5 March,Pune, India

2 AGENDA Introduction Product development approaches-best practices Accelerating process development & Quality assessment Factors affecting Glycosylation Case Study-Effect of Upstream process parameters

3 Ipca at a glance.

4 INDIA MUMBAI

5 Company Overview Incorporation : 1949 Present Management : Since 1975 Total Sales (F.Y ) : US$ 538 Mn. Number of Employees : ~12700 Business Model : Fully integrated pharma company producing branded generic formulations, APIs and intermediates New Business Model : Ventured Into Bio-pharma development ( )

6 Inception of Biosimilar R&D Established state of the art R&D in Nov-14 Lashed with modern equipments & HTP devices Capable of In-house development from clone to clinic Product Type-Cell culture based mabs Our Collaborator: USA based Biosimilar company- Oncobiologics Inc., NJ

7 Introduction

8 Product Modification :mab Post-translational Modification Chemical Modification Glycosylation Oxidation Lysine Variants Deamidation Disulphide Isoforms Cyclization Signal processing Isomerization Glycation

9 Glycosylation The key therapeutic properties of Mabs are strongly related to the posttranslational Process of glycosylation. Glycan confirmation can influence: Therapeutic properties Protein Secretion Solubility Antigenicity Receptor recognition Stability and Bioactivity

10 N-Glycosylation:IgG Antibody FC Glycosylation: 2-3% of the IgG Mass Highly dependent on clones & production processes. Impact on immunogenicity & effecter functions..

11 Productivity :Current Trends How about Quality? Glycosylation Charge variants & Aggregates Distribution of current titers for commercial biologics (g/l) Ref: by Ronald A. Rader Eric S. Langer Friday, February 6, 2015

12 A Desirable Production Process Simple process developed in a short time High & Uniform productivity Good Product Quality Regulatory compliance Commercially Viable Can be achieved by developing a robust process.

13 A Robust Process depends on Suitable host & clone selection USP & DSP processes Bio-analytical capability Consistency Commercial scale Clone Clone USP USP & &D D Fate of Product Analytical Analytical Potency

14 Product Development & Quality Attributes Maximum Product Variability at Project Start

15 Quality Assessment from Clone to Clinic Pool Generation Clone Screening Shake Flask/AMBR studies Transfection, selection & amplification Screen as many clones as possible Media and Feed Optimization Bioreactor Studies Large Scale Bioreactor Studies Process Optimization CQA Based Clone & Process CQA based Clone & Process Development Development Replicating the behavior of cells from small scale

16 First Check Point Host selection

17 Protein Production Host & Glycosylation Approx. 250 genes are involved in Glycosylation modification The abundance of glycosylation enzymes differs among the production strains Maximum heterogeneity occurs due to glycosylation pattern

18 Glycan composition: CHO Vs Sp2/0,NS0 S N Glycosylation CHO cells Very Less Sp2/0 or NS0 1. Terminal NANA 2. Third GlcNac bisecting arm Present(~10%) 3. Additional Gal α-1, 3gal Absent (~ 2-4%) Present 4. Terminal NGNA Traces Present (~ 1-2%) 5. Oligo Mannose Present Present Implication Absent Decreased ADCC Absent Increases ADCC Immunogenic and Low half life Glycoforms

19 IgG1 Glycan Profile: CHO Vs Murine IgG1 profile Murine cell Product quality vary strain to strain (CHO-S, NS0 etc.) Always recommended to use the same host as Innovator s Product quality to be assessed CAREFULLY if changing the host IgG1 profile CHO Cells

20 Second Check Point Cell Pool & Clone Stage

21 Product Quality Assessment During Clone Selection Reference: Feng Li et. al, 2010

22 Quality Check at Pool Stage Transfection 6-8 pools Shake flask Studies Stable Pools Selected Cells Material For Titer & Quality Dilution Cloning & Screening Non Producer Low Producer Medium Producer High Producer Charge Variants Glycosylation N-terminal seq for Signal peptide

23 HTP devices for efficient cloning & Selection Manual Cloning in 96 well plate Verified Stable Pools HTP Screening by ClonePix or FACS Cell Imagine-96wp Labchip for quality96wp Octet for titer-96wp AMBR for clone selection Screening of clones/pool Selection of Top Clones Titer & Quality Assessment

24 Clone Selection Criteria Cells growth behavior qp and yield Genetic Stability Process Feasibility Clonality and Purity Product quality Bio-activity/Potency Final clone

25 Charge Variants-CEX analysis Charge heterogeneity of Top 4 selected Pools Acidic Variants K 0 Basic Variants K 1 K 2

26 Sequence Variant Identification at Clone stage Sequence variants can be generated by DNA/RNA mutation or amino acid mis-incorporation Single base-pair mutation of TAA (Stop codon) to GAA (Glu) in LC Figure : Overlay of tryptic peptides maps of IgG1 derived from four clones. The new peak at 83 min in clone B is absent from other clones. Ref: Taylor Zhang et. al, mabs 4:6, ; 2012

27 CZE of Market Samples-Charge Variants Basic Variants Acidic Variants Ref. Standard Biosimilar-1 Ref. standard Biosimilar-2 Biosimilar-3 Same Host & Sequences but different clones and processes

28 Glycan Analysis of Market samples:hplc Ref Stand Biosimilar-1 Biosimilar-3 Ref. stand Biosimilar-2 Waters Glycan std Same Host & Sequences but different clones and processes

29 Third Check Point Upstream Process Cell Culture Process & Product Quality

30 Cell Culture Parameters & Product Quality Causes glycosylation changes

31 Use of HTP devices to speed up the product development Selected clones AMBR for Process Opt-DOE Octet for titer & quality Analytical tools for quality assessment Process Scale up Akta Avant for Purification & DOE HPLC for Charge & Glycan MALS for aggregates CE -charge & Glycan Labchip for Charge & Glycan Mass -Glycan, peptide

32 Effect of upstream process on Glycosylation

33 Effect of cell culture Nutrients on Glycosylation Product : Monoclonal Antibody Host : CHO cells Mode : Batch mode in Shake flask Study : Effect of Glutamine, Lactate, Glucose & Ammonia on mab glycosylation

34 Cell Culture Profile at various conc. of Glutamine Four different conc. of Glutamine is used to study the effect of Bi-product ammonia On mab Glycosylation Maximum: Cell 4mM Glu Glu Glut Ammonium, Lactate & mab titer@8mm Glu

35 Glycan Profile at different conc. of Glutamine GI: Galactosylation Index SI: Sialylation Index

36 Effect of ph on Glycosylation of IgG1 expressed in murine cells

37 Growth & Expression Profile at different ph

38 Glycan Profile at different condition ph shifted culture to h 54h 80h Reduced G1F & G2F at ph 7.8 (shifted ) & Increased G0F compared to standard cond. Maximum GI (0.43) & SI (0.15) were achieved at low ph 6.8 Reduced GI & SI with increased ph, while FI remains almost same. 26h 54h 80h

39 Future Perspective Glyco-engineering to improve the effecter function CHO FUT8 gene knock-out Yeast engineering Afucosylated mab Human like /Afucosylated mab Enhanced ADCC Enhanced ADCC Ex. Obintuzumab-Gazyva (Anti-CD20) launched by Roche in 2013 Rituximab with more Efficacy than CHO derived mab

40 SUMMARY Glycosylation is the key therapeutic properties of the mab which can affect the Potency and Immunogenicity. The product quality assessment shall be initiated at the project start (Host selection, Clone & Process Development). The clone and upstream process affects the product quality most. Implementation of HTP devices with high end analytical tools allows speedy process and product development.

41 Thank You! Visit us at - Write to us at-sanjeev.gupta@ipca.com Questions?