Characterizing and understanding the formation of cysteine conjugates and other by-products in a random, lysine-linked antibody drug conjugate

mAbs

Naresh Chennamsetty¹  Yande Huang²  Difei Qiu²  Michael Hay²  Scott A. Miller² 

[1] Biologics Development, Global Product Development and Supply, Bristol Myers Squibb Company, New Brunswick, NJ, USA;Chemical Process Development, Global Product Development and Supply, Bristol Myers Squibb Company, New Brunswick, NJ, USA;

关键词: Antibody drug conjugate;    lysine-linked conjugation;    cysteine-linked conjugation;    mass spectrometry;   

DOI  :  10.1080/19420862.2021.1974150

来源: DOAJ

【 摘 要 】

This study describes the characterization of conjugation sites for a random, lysine conjugated 2-iminothiolane (2-IT) based antibody-drug-conjugate synthesized from an IgG1 antibody and a duocarmycin analog-based payload-linker. Of the 80 putative lysine sites, 78 were found to be conjugated via tryptic peptide mapping and LC-HRMS. Surprisingly, seven cysteine-linked conjugated peptides were also detected resulting from the conjugation of cysteine residues derived from the four inter-chain disulfide bonds during the reaction. This unexpected finding could be attributed to the free thiols of the 2-IT thiolated antibody intermediates and/or the 4-mercaptobutanamide by-product resulting from the hydrolysis of 2-IT. These free thiols could cause the four inter-chain disulfide bonds of the antibody to scramble via intra- or inter-molecular attack. The presence of only pair of non-reactive (unconjugated) lysine residues, along with the four intact intra-chain disulfide bonds, is attributed to their poor accessibility, which is consistent with solvent accessibility modeling analysis. We also discovered a major by-product derived from the hydrolysis of the amidine moiety of the N-terminus conjugate. In contrast, the amidine moiety in lysine-linked conjugates appeared stable. Based on our results, we propose plausible formation mechanisms of cysteine-linked conjugates and the hydrolysis of the N-terminus conjugate, which provide scientific insights that are beneficial to process development and drug quality control.

【 授权许可】

Unknown