Post translational Modifications of Biologics: Impact on clinical safety and efficacy

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1 Post translational Modifications of Biologics: Impact on clinical safety and efficacy Narendra Chirmule, PhD Senior Vice President Head of R&D Biocon, Bangalore, India 1

2 Extensive post translational modifications in mabs Product Attribute Profile Hot Spots: Cyclizing of N terminal E Truncation des ES of LC Deamidation (NG) Deamidation (NS) Oxidation (Met, Trp) Glycosylation of Fc Cleavage (Asp Pro) C terminal Lys of HC 2

3 Product Characterization Intens. x10 7 Peptide Mass fingerprinting m /z, a m u Intact molecule by Mass spectrometry Time [min] NP-HPLC of Glycan Size by SEC Heterogeneity due to charge, size Glycosylation analysis by MALDI, NPHPL MALDI-MS of Glycan IEX-HPLC for monitoring charge variant Lys-0 Lys-1 Lys-2 Circular Dichroism CD [mdeg] Functional and higher order structure List of Bioassays in vitro Potency assay in vitro Binding assay Fc potency assay Binding to FcRn Binding to FcgR1 Binding to FcgR2a Binding to FcgR2b Binding to FcgR3a Binding to FcgR3b Binding to C1q 3

4 Example of Risk Assessment and Mitigation Plan (QbD perspective) Aggregate (HM W) Iso-Asp (LC92) gal-a-gal NG-HC Oxidized Species Dimer Solubility/Precipitation Non-main Peaks/ Fragments (per CE-SDS) Other IsoAsp potential sites Glycation Sub-visible Particles Cystine Adduct Pre- mo no mer Free cysteine Extent of High Sialylation Ext ent o f High M anno se Extent of Fucosylation Deamidated Species Extent of gallactose Free light chain Primary Sequence 3 D Structure Unfolded/ Perturbed (conformation) Disulfide M odification/ Rearrangement N-terminal M odification (Glutamine to pe) C-terminal Lysine Severity x Likelihood

5 Aggregates Glycosylation 5

6 Questions Do aggregates of protein therapeutic (mabs) induce immunogenicity in humans? Do all aggregated mabs induce a reactive immune response irrespective of sequence? Does size of particles matter? 6

7 The Morphology of Aggregates from Distinct IgG Molecules mab1 IgG 2 mab2 IgG 2 mab3 IgG 1 IVIG intravenous IgG mab1 untreated 10 µm mab2 untreated 10 µm mab3 untreated 10 µm IVIG untreated 10 µm mab1 stir 3d 80 µm mab2 stir 3d 80 µm mab3 stir 3d 80 µm IVIG stir 3d 80 µm mab1 stir 20h 25 µm mab2 stir 20h 25 µm mab3 stir 20h 25 µm IVIG stir 20h 25 µm mab1 65C/pH µm mab2 65C/pH µm mab3 65C/pH µm IVIG 65C/pH µm mab1 syringe so + 40 µm mab2 syringe so + 40 µm mab3 syringe so + 40 µm IVIG syringe so + 40 µm Particle images of were captured on a liquid borne particle Micro Flow Imaging (MFI) System DPA4100. Representative images of the largest particles detected are shown. The size threshold indicates the lower size limit of the particles that were used for comparison. MK Joubert, Q Luo, Y Nashed Samuel, J Wypych, and LO Narhi Classification 7 and characterization of therapeutic antibody aggregates J Biol Chem 2011; advanced online publication (doi: /jbc.m ).

8 Aggregated mab2 Aggregated mab1 mab WITH anon tolerant Epitope (n=10) mab WITHOUT anon tolerant Epitope (n=6) MHC II MHC II MHC II Joubert et al, J Biol Chem. 2012; 287: IL 1β, IL 6, IL 10, MCP 1, MIP 1α, MIP 1β, MMP 2 and TNF α IL 1β, IL 6, IL 10, MCP 1, MIP 1α, MIP 1β, MMP 2 and TNF α 8

9 mab2 (non tolerant epitope) CD4+ T cell TLR Costimulatory Molecules, e.g. CD86 src/shc MyD88 PLC 1 PI IP3 Ca ++ DAG CN PKC NFATc/n NF B TRAF6 TAB TAK IRAK ECSIT/MEK MEKK ERK/JNK/p38 IL 1.TNF NF B IRF3,7 Fos Jun Gene Transcription 9

10 in vivo induction of B cell responses the aggregated therapeutic protein in the Xeno x WT mouse model XenoHet mice: 95% mouse BCR+; 2.5% human BCR+ Secrete human IgM and IgG (10 fold less than WT Xenomice) 2 weeks 8 weeks 16 weeks ADA T ADA T ADA T ASC Monomer mab TCE KLH mab Stir 20h, Stir 3 day mab 20 um (microspheres) mab 5 um (nanospheres) Bi et al, J Pharm Sci. 2013; 102:

11 Summary of Results Aggregated mabs, with non tolerant epitopes, induce a quantifiable response: in vitro cytokine signature (from PBMC [~30% of the donors] and THP 1 cell line) in vitro T cell cytokine (IL2/IFN ) and proliferative response [30% of the donors] in vivo induction of transient B cell responses the aggregated therapeutic protein in the Xeno x WT mouse model 11

12 THE IMMUNE SYSTEM MAKES DIFFERENT TYPES OF RESPONSES TO DIFFERENT TYPES OF AGGREGATES Innate response Cytokine signature Adaptive response Polyclonal CD4+ T cell Oligoclonal CD4+ T cell No T cell epitope Primary B cell Mature B cell Aggregated No T cell epitope T cell epitope Aggregated T cell epitope Inflammation at site of injection Infusion reaction Delayed type HSR Immune complex disease Immune complex disease Allergic response (IgE) 12

13 Next steps Determine potential role of TLR and aggregates interactions Understand mechanism of aggregate induced immunogenicity Transient B cell response Potential polyclonal T cell responses Correlation of clinical immunogenicity and adverse events with lot# (2 10 um) 13

14 Glycosylation Glycosylation is an intrinsic PTM that is required for the function of proteins The mechanism of regulation of immune responses to glysocyl residues have not been extensively studies The role of non classical antigen processing and presentation (CD1, Qa) Induction of tolerance and immune responses 14

15 Glycosylation NP-HPLC of Glycan Glycosylation analysis by MALDI, NPHPL MALDI-MS of Glycan Functional and higher order structure List of Bioassays in vitro Potency assay in vitro Binding assay Fc potency assay Binding to FcRn Binding to FcgR1 Binding to FcgR2a Binding to FcgR2b Binding to FcgR3a Binding to FcgR3b Binding to C1q 15

16 CD1 MHC class I m 2 -m

17 Examples of immune responses to glycosyl residues in biologics Immune Responses Different glycosylation's on EPO biosimilars Immune Tolerance Lack of observed immune responses to glycosyl residues on EPO (AraNESP) Anti IgE responses to (nonhuman) GalC residues in cetuzimab Mannosylated mab results in increased clearance through mannose receptors Observation that increased mannosylated mabs does not induce increased immunogenicity (despite inducing increased clearance) Glycosyl residues may either induce an adaptive immune response or immune tolerance 17

18 Summary All proteins undergo extensive PTM Not all PTM induce immune responses Mechanisms of PTM mediated immune response to proteins can include: i. induction T and/or B cell activation, OR ii. immune tolerance Future studies development of sensitive diagnostics that can predict immunogenicity mediated adverse events in subjects that develop clinically relevant anti drug antibodies 18