InVivo Reoxidation Kinetics of Free Thiols in Multiple Domains of IgG1 Antibodies in Rats.

Abstract

While free thiols in monoclonal antibodies (mAbs) have been extensively characterized by invitro studies to probe its effect on antibody function and stability, their invivo biotransformation has not been comprehensively studied. In this study, a panel of five recombinant IgG1 mAbs with elevated free thiols in the VH, VL, and CH2 domains were intravenously administered into Wistar rats. Invivo biotransformation of thirty-five free thiol sites in total (7 disulfide pairs in VL, CL, VH, CH1, HH, CH2, CH3 domains across the 5 mAbs) were monitored using a denaturing differential isotopic tagging procedure on immunopurified timepoints followed by LC-MS of tryptic digests. The free thiol levels in two VH domain and one CH2 domain disulfide sites decreased invivo following first order kinetics. Free thiol levels of the remaining 32 sites were remarkably stable invivo. Further analytical characterization highlighted a positive association between a free thiol's solvent accessibility and a free thiol's reoxidation propensity. The data and discussion presented here shed valuable insights into the invivo fate of free thiols in several recombinant IgG1s and its implications for free thiols as a product quality attribute in therapeutic mAb products.