Tina S. Morris, Ph.D., Vice President Biologics & Biotechnology USP-NF

1. 6th Science and Standards SymposiumJanuary 16th, 2013Istanbul, TurkeyQuality Attributes of Monoclonal Antibodies Tina S. Morris, Ph.D., Vice President Biologics & Biotechnology USP-NF

2. Quality Control for Biotechnology Products - ICH • ICH Guideline Q6B - Test Procedure and Acceptance Criteria for Biotechnology/ Biologic Products • Quality Attributes • Identity • Purity • Impurity profile • Potency • Strength • Safety

3. Q6B on Analytical Characterization

4. Product Type versus Molecule Class – Platform Accessibility

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

6. Monoclonal Antibodies and Platform Assays • Quality Control Assays for Monoclonal Antibodies • Product Class Analytical Methods - platform assays • Examples • Size exclusion chromatography • Isoelectric Focusing • Oligosaccharide assay • Peptide Mapping • Process Related Impurity assays • Host cell protein • DNA • Protein A • Compendial Assays; Endotoxin, pH, Conductivity, Sterility

7. Capturing Platform Assays in a Compendial Chapter • <129> Analytical Procedures for Recombinant Therapeutic Monoclonal Antibodies • Will contain a collection of validated compendial procedures with established system suitability criteria for therapeutic MAbs • Will be accompanied by USP MAb System Suitability RS • Will not contain product or class specific acceptance criteria • Will be supported by multiple >1000 Information Chapters that discuss quality attributes, manufacturing and quality control aspects for MAbs

8. <129> Current Chapter Scope • Product Scope: • Murine, chimeric, and humanized IgGisotypeMabs and subtypes (e.g. IgG1 and IgG2) • MAbs for therapeutic, prophylactic and in vitro diagnostic use • EXCLUDES: MAbs used as manufacturing reagents or process materials • Included Procedures: • Size Exclusion Chromatography (SEC) • Capillary SDS Electrophoresis (reduced and non-reduced) • Oligosacchride Analysis • Sialic Acid Analysis

9. Round Robin Study in Support of <129> • Purpose: • Evaluate the proposed Size Variant and CE procedures • Test the proposed USP Monoclonal IgG system suitability standard • Gather batch data on MAbs currently made in commercial manufacturing as well as in development (clinical and non-clinical) Study Logistics and Time Line: • Study materials were dispatched in at the end of November, 2012, deadline for data submission is March 30th, 2013 • Large international study participation of 30 laboratories Chapter Time Line: • Will appear in PF39(3) with a comment deadline of July 31st, 2013

10. USP Monoclonal IgG System Suitability Standard • Compendial use • Needed for system suitability for proposed SEC and CE-SDS USP procedures • Material Description and Source • IgG1 MAb • Lyophilization protocol available • Will be developed and distributed as a USP reference standard in lyophilized presentation

11. SEC Profile of IgG System Suitability Standard

12. Antibody Glycosylation Analysis – fit for Common Assay(s)? CE Analysis of Released Oligosaccharides Mab Glycan standard G2F+1 NeuAc Glycans from polyclonal human IgG Data courtesy of <129>/<1260> expert panel

13. 0 0 0.5 0.5 1 1 1.5 1.5 2 2 Moles Galactose Mole Heavy Chain Glycosylation and Bioactivity Correlation Bioactivity Correlates with Galactose Content 200 150 Bioactivity Bioactivity 100  galactosidase treated 50

14. Galactosylation Profile of hIgG Glycans F-CE MS methods Fluorophore - HPLC HPAEC

15. Product-Specific Quality Attributes of MAbs Several Quality Attributes of MAbs can be highly product specific and should be addressed at the monograph level. Examples • Charge heterogeneity, analyzed by IEX chromatography or cIEF • Hydrophobic Interaction Chromatography • Ligand binding, e.g. by ELISA • Cell-based potency assay

16. 0.07 0.06 0.05 0.04 0.03 0.02 0.01 0 10 20 30 40 50 60 Quality Control for Biotechnology Products Ion-Exchange Profile of an Intact Antibody Q pE pE pE pE pE Q pE pE pE pE pE pE Q pE Peak 3 Peak 2 Q Peak1 pE Q pE Q mAu 1 Peak 4 Peak 5 1a 3 1b 2a 2 3a 2b 4 5 Time (min)

17. Quality Control for Biotechnology Products Ion-Exchange Profile of a Papain Digested Antibody 100 80 + + Papain H 60 F(ab) Fc mAu B 40 20 J K C M A L D N O P E I G F 0 0 12 24 36 48 60 Time (min)

18. Information Chapter(s) in Development • <1260> Therapeutic Monoclonal Antibodies – General Considerations • Product Class Overview • General Manufacturing and Quality Considerations <1260.1> Analytical Control Strategies for Recombinant Monoclonal Antibodies • Quality Attributes, their determination, control and measurement • General considerations and analytical considerations for quality attributes that are product specific • Comparability and post-approval quality issues

19. Acknowledgements • USP Monoclonal Antibody Expert Panel • Chair: Dr. Anthony Mire-Sluis • USP Biologics & Biotechnology Monographs 1 Expert Committee • Chair: Dr. Michael Mulkerrin • USP Staff • EC and EP Liaison: Dr. Anita Szajek • Reference Standard Scientist: Dale Schmidt

20. USP Headquarters, Rockville, Maryland