1. 6 th Science and Standards Symposium January 16 th, 2013 Istanbul, Turkey Quality 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 Product TypeMolecule ClassSourceLegacy Products in this Class? Accessible to analytical platform approach? Cell TherapyWhole CellPatient-autologousNo Clotting FactorProtein/EnzymePlasma and recombinantYesto some extent Cytokine or growth factor Peptide or small protein Mostly recombinantYes - hormones Yes EnzymeProteinBoth recombinant and naturally derived Yes Monoclonal antibody Protein, IgG1RecombinantNoYes Polyclonal antibody Protein, Ig mixturePlasma-derivedYesto some extent ToxinProteinBoth recombinant and naturally derived Yes VaccineVariousBoth recombinant and naturally derived Yesto some extent Other – what about heparin? Polydisperse glycosaminoglycan Naturally derivedYes

5. Which Quality Attributes to Consider? Biological characteristicsPhysico-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

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 o Host cell protein o DNA o Protein A Compendial Assays; Endotoxin, pH, Conductivity, Sterility

7.  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 7 Capturing Platform Assays in a Compendial Chapter

8.  Product Scope:  Murine, chimeric, and humanized IgG isotype Mabs 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 8 Current Chapter Scope

9.  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 30 th, 2013  Large international study participation of 30 laboratories Chapter Time Line:  Will appear in PF39(3) with a comment deadline of July 31 st, 2013 9 Round Robin Study in Support of

10.  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 USP Monoclonal IgG System Suitability Standard

11. SEC Profile of IgG System Suitability Standard

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

13. Glycosylation and Bioactivity Correlation 00.511.52 Bioactivity Moles Galactose Mole Heavy Chain  galactosidase treated 50 100 150 200 Bioactivity 00.511.52 Bioactivity Correlates with Galactose Content

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

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. Quality Control for Biotechnology Products 0 0.01 0.02 0.03 0.04 0.05 0.06 0.07 102030405060 mAu Time (min) 2 3 4 1 5 3a 2b 2a 1b 1a pE Peak 1 Q pE Peak 2 QpE Q Peak 4 Q pE Q Peak 5 Q pE Peak 3 pE Ion-Exchange Profile of an Intact Antibody

17. Quality Control for Biotechnology Products 0 20 40 60 80 100 01224364860 mAu Time (min) A B C D F G H I J K L M NO P E Ion-Exchange Profile of a Papain Digested Antibody + + Fc F(ab) Papain

18.  Therapeutic Monoclonal Antibodies – General Considerations  Product Class Overview  General Manufacturing and Quality Considerations 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 18 Information Chapter(s) in Development

19.  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 19 Acknowledgements

20. USP Headquarters, Rockville, Maryland