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Day 4, June 25（Wed.）
Room P (Maesato East, Foyer, Ocean Wing)
- 4P-CP-01
COMPLETE CHARACTERIZATION OF KADCYLA USING A MODIFIED HYBRID QUADRUPOLE-ORBITRAP MASS SPECTROMETRY WITH ELECTRON-TRANSFER/HIGHER-ENERGY COLLISION DISSOCIATION (ETHCD) FRAGMENTATION
(1Thermo Fisher Scientific Shanghai, 2Thermo Fisher Scientific Singapore)
Xiaoxi Zhang1, oNicole Zhang2
In this study, comprehensive characterization of lysine-conjugated ADC Kadcyla at multiple levels were achieved using a modified hybrid Quadrupole-Orbitrap mass spectrometry. At native intact level, mAb bearing 0-8 conjugated drugs were detected. The software auto-calculated average DAR is 3.46, which is in agreement to previous publication. In reduced intact mass analysis, LC D0 and HC D1 were the main payloads, which agrees with native intact MS results. LC D1 and D2, HC D0 and D2 were identified with high quality spectra. Minor LC D3 and HC D3 were also detected. N-glycoforms on HC and other modifications, like oxidation and succinimidation were also observed.
In subunit analysis, in addition to N-glycoforms distribution in Fc region and payload distribution in light chain and Fab region, isomers carrying same payloads were chromatographic separated and detected. In peptide mapping analysis, HCD fragmentation achieved 100% sequence coverage. The EThcD fragmentation helped us to successfully identified 43 conjugation sites out of 46 potential conjugation sites at the sequence, including some close located lysines, such as K183/K188/K190 on the light chain. Other common modifications in biotherapeutic products, such as deamidation, succinimidation, glycation and oxidation were identified and quantified. The most abundant N-glycoform is A2G0F (54.59%).