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Characterization of Antibody Drug Conjugates Prepared on Magnetic Protein A and G Particles by MS after IdeS Digestion

Part # PS216


Chris Hosfield1, Philip Compton2, Becky Godat1, Sergei Saveliev1, Archer Smith2, Nidhi Nath1, Paul Thomas2,Neil L. Kelleher2, Michael Rosenblatt1 and Marjeta Urh1
1Promega Corporation, Madison, WI; 2Proteomics Center of Excellence, Northwestern University, Evanston, IL

Rituximab was conjugated with a fluorescent dye on magnetic particles using an efficient and scalable protocol. Dye incorporation was monitored by MS using intact and middle-up approaches. IdeS & PNGase F are ideal for preparing antibodies for intact and middle-up analysis. On-bead conjugation facilitates labeling at all 8 interchain cysteine residues. Labeling is not impeded by immobilization. “Native” (non-denaturing) MS protocols are critical to obtain intact mass spectra of cysteine-conjugated antibodies. Trypsin/LysC improves reproducibility of digestion for bottom-up approaches.

Printed in USA.