Capillary Electrophoresis – Mass Spectrometry for Intact Mass Analysis of Antibodies and Antibody-Drug-Conjugates Aran Paulus, Chien-Hsun Chen, Andreas Krupke, Stephane Houel, Michael Wenz, and Daniel Lopez-Ferrer 45th International Symposium on High Performance Liquid Phase Separations and Related Techniques Prague, Czech Republic, June 19, 2017 The world leader in serving science Overview • Introduction/Motivation • Antibodies and Antibody-Drug-Conjugates and other “bodies” • Workflows and Instrumentation • CEMS of NIST mAb and NIST mAb derived ADC • Top-Down Proteomics with CEMS • Conclusions • Acknowledgements 2 Paradigm Shifts in Pharmaceutical Industry Small molecule drug Antibodies and ADC’s First introduction to market: 1899 1986 Aspirin MW 180D Transtuzumab MW 145’531D 3 Paradigm Shifts in Pharmaceutical Industry Acetyl Salicylic Acid ~1320 - 1540 amino acids 2 heavy and 2 light chains MW: ~150 000 D 4 Cys-Cys bonds Minimum of 2 glyco sites 4 Structures of mAb Derived Biopharmaceuticals 5 Intact mAb and mAb Fragments 6 NIST Monoclonal Antibody as Universally Available Standard with Charge Variants NISTmAb, recombinant humanized IgG1κ Number of amino acids: 1326 Disulfide bonds: 14(inter-chain), 2(intra-chain) High-abundance PTM: N-terminal pyroglutamination C-terminal lysine clipping glycosylation 7 Analytical Problems for mAb and ADC Comprehensive Characterization • Primary Structure Assessment • Charge Variants • Separation (HPLC/CE) with MS (peptide mapping, middle down and top down MS) • AA sequence and variants • Intact mass (ESI-MS) • Bottom-up peptide maps • Middle-down (reduction, heavy/light chain, IDeS) • Top down (ETD/ECD, UVPD, CID, etc) • Glyco variants • (released glycans, glycoprotein/peptides) • Cystein-linked variants • Higher Order Structures, Aggregates and mAb/Ag • PK/Quantification 8 Proteomics Workflows for mAb Characterization Bottom-Up, Middle-Down, Top-Down •Proteomics workflows depend on combination of • high resolving separation methods with • sensitive and high resolution MS •Bottom-up combines nanoLC with MS •Misses PTM information •Top down combines sample prep with MS •No good LC method for proteins 9 CEMS on Microfluidic Devices (ZipChip) 10