Sasidhar N. Nirudodhi, Lin Tzihsuan, Justin Sperry, Jason C. Rouse, James A. Carroll

Transcription

1 Impact of Sequence Variants on the Higher-Order Structure (HOS) of Monoclonal Antibodies Using an Optimized HDX- MS Method with a Dual-Protease Co-Immobilized Column Format Sasidhar N. Nirudodhi, Lin Tzihsuan, Justin Sperry, Jason C. Rouse, James A. Carroll BTX PHARMACEUTICAL SCIENCES, ANALYTICAL RESEARCH AND DEVELOPMENT, PFIZER CASSS MASS SPEC 2015, BROOKLYN, NY

2 Overview HDX-MS Introduction HDX-MS Method Optimization Introduction to Sequence Variants (SVs) Application of Optimized HDX-MS method for SV analysis Summary 2

3 Higher Order Structure: H/D Exchange for Conformation Reference Experiment D 2 O H/D exchange at amides Deuterated Reference Deuterated Experiment Key conditions ph and temp Quench, digest, LC/MS Compare D uptake Peptide Number Comparison Plot of Samples Houde, D., et al., J. Pharm. Sci. 2011, on-line. 3

4 Need for Optimizing HDX-MS Predominantly used for protein-ligand binding studies in a academic setup Recent interest by bio-pharma to address the heightened HOS characterization of bio-therapeutics Several publications on IgG1 mabs There were some challenges Large peptides Incomplete sequence coverage Lack of redundancy Interest in IgG2 mabs more difficult to proteolyze 4

5 Method Optimization Peptide level HDX-MS workflow highlighting the regions for optimization: 1. Quench buffer conditions (8M Urea, 1M TCEP-HCl, 100 mm ph 2.5 PBS ) 2. Proteolytic digestion for better digestion efficiency 3. UPLC gradient for better peptide separation and elution (5 min trapping, 15 min gradient) 5

6 Digestion Optimization for IgG2 Subclass mab Type of Protease Column Sequence Coverage/Redundancy HC LC Schematic of the Digestion Column Optimization Pepsin 86.9% / % / 7.8 Pepsin Column Type % / % / 9.3 Type XIII column Pepsin, Type XIII (Split Flow) 88.2% / % / 8.0 Splitter Type XIII column Pepsin Column Splitter Pepsin, Type XIII (Tandem Flow) 87.3% / % / 8.1 Pepsin Column Type XIII column [Pepsin, Type XIII] in 1 Column 94.9% / % / 11.0 Pepsin Column + Type XIII Immobilized Column 6

7 Cleavage Specificity of Dual Protease Column Versus Pepsin and Type XIII protease Enzymatic cleavage specificity of pepsin, type XIII and dual protease column is presented. The dual protease column has enhanced cleavage specificity compared to the individual protease columns. The table presents the enzymes cleavage sites after each amino acid residue. IgG Subclass Sequence Coverage/Redundancy HC LC IgG2 94.9% / % / IgG1 95.5% / % / 7.19 IgG4 97.6% / % / 8.16 Optimized HDX-MS method with dual protease column approach resulted in enhanced sequence coverage and redundancy for IgG1, IgG2 and IgG4 mabs. Amino acid Residues Pepsin Type XIII Dual protease column A R N D C Q E G H I L K M F P S T W Y V

8 Sequence Variants in Biotherapeutics Sequence variants may occur during production DNA replication: 10-8 / base DNA-RNA transcription: 10-5 / base RNA-protein translation: / codon Sensitivity improvements in MS technology have led to the detection and quantitation of variants Need SVA methods to monitor 10-5 to 10-3 level SVs may impact the HOS, efficacy and immunogenicity MS based method to characterize HOS 8

9 SV and Product Quality Analysis of mabs for Clone Selection 9

10 Antibody Subunit Domain Mapping: Clone Selection Subunit/domain mapping with Waters UPLC-MS was implemented for clone selection to enable rapid and definitive characterization of possible point mutations in the L chain and H chain scfc and Fd domains. scfc L Chain Fd Subunit/Domain Mapping UPLC- MS Clone A Clone B scfc T304P ~31% scfc L Chain Fd F151Y ~38% Fd F151Y: Da T304P: Da Point mutated residue (T304P) N-linked glycosylation site scfc T304P Amino Acid Sequence GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVL PVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 10

11 Example 1: Differential Deuterium Uptake Plots of an IgG2 mab for RM Vs SV sample Above the axis: High D uptake in RM F151Y RM T304P SV Below the axis: High D uptake in SV Differential deuterium uptake (%) of RM-SV for each peptide is plotted according to the sequence HC : LVKDYFPEPVT HC : TVVHQDWL 11 Deuterium uptake profiles of peptides over labeling time points

12 Example 2: Differential Deuterium Uptake Plots of an IgG1 mab for RM Vs T363N SV (~50%) (CH3 domain) Above the axis: High D uptake in RM RM T363N Below the axis: High D uptake in SV SV No significant changes were observed in the peptides encompassing SV region 353_ _ Deuterium Uptake (Da) T363_SV T363_RM T363N Deuterium Uptake (Da) T363_SV T363_RM T363N Time Points (min) Time Points (min) 12

13 Example 3: Differential Deuterium Uptake Plot of an IgG1 mab for RM Vs A332G SV (~45%) (CH2 Domain) Above the axis: High D uptake in RM Below the axis: High D uptake in SV RM A332G SV Significant changes were observed in the peptides encompassing SV region 13

14 Summary and Conclusion Amino Acid Change SV Level Impact on HOS Model based calculations mab1 IgG2 H Chain: F151Y (C H 1) H Chain: T304P (C H 2) ~31% ~38% No Change Significant change No change in molecular conformations mab2 IgG1 H Chain: T363N (C H 3) ~30-50% No change No change in molecular conformations mab3 IgG1 H Chain: A332G (C H 2) 35-45% Significant change Significant change in molecular conformations Optimized HDX-MS method was used to evaluate different sub-class of mabs Impact of SVs on HOS was evaluated by HDX-MS Impact of SV on HOS depends on the type of amino acid substitution and its location Each of the SVs have to be independently evaluated to understand their impact on HOS 14

15 Acknowledgements Mass Spectrometry and Biophysical Characterization Group (MSBC) Justin Sperry Jason C. Rouse James A. Carroll Paul W. Brown Olga V. Friese Jacquelynn Smith Kathleen Cornelius Ying Zhang Andrew Dawdy Thomas Wesley Powers Lisa Marzilli All MSBC members Mass Spectrometry and Cell Line Development Jennifer Lin 15