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
AGENDA Introduction Product development approaches-best practices Accelerating process development & Quality assessment Factors affecting Glycosylation Case Study-Effect of Upstream process parameters
Ipca at a glance.
INDIA MUMBAI
Company Overview Incorporation : 1949 Present Management : Since 1975 Total Sales (F.Y.2013-14) : 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 (2013-14)
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
Introduction
Product Modification :mab Post-translational Modification Chemical Modification Glycosylation Oxidation Lysine Variants Deamidation Disulphide Isoforms Cyclization Signal processing Isomerization Glycation
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
N-Glycosylation:IgG Antibody FC Glycosylation: 2-3% of the IgG Mass Highly dependent on clones & production processes. Impact on immunogenicity & effecter functions..
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
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.
A Robust Process depends on Suitable host & clone selection USP & DSP processes Bio-analytical capability Consistency Scalability @ Commercial scale Clone Clone USP USP & &D D Fate of Product Analytical Analytical Potency
Product Development & Quality Attributes Maximum Product Variability at Project Start
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
First Check Point Host selection
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
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
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
Second Check Point Cell Pool & Clone Stage
Product Quality Assessment During Clone Selection Reference: Feng Li et. al, 2010
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
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 1000-2000 clones/pool Selection of Top 10-20 Clones Titer & Quality Assessment
Clone Selection Criteria Cells growth behavior qp and yield Genetic Stability Process Feasibility Clonality and Purity Product quality Bio-activity/Potency Final clone
Charge Variants-CEX analysis Charge heterogeneity of Top 4 selected Pools Acidic Variants K 0 Basic Variants K 1 K 2
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, 694 700; 2012
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
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
Third Check Point Upstream Process Cell Culture Process & Product Quality
Cell Culture Parameters & Product Quality Causes glycosylation changes
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
Effect of upstream process on Glycosylation
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
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 count @ 4mM Glu Glu cons @4mM Glut Ammonium, Lactate & mab titer@8mm Glu
Glycan Profile at different conc. of Glutamine GI: Galactosylation Index SI: Sialylation Index
Effect of ph on Glycosylation of IgG1 expressed in murine cells
Growth & Expression Profile at different ph
Glycan Profile at different ph @Standard condition ph shifted culture to 7.8 26h 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
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
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.
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