Characterization of therapeutic monoclonal antibodies reveals differences between in vitro and in vivo time-course studies

Abstract

Purpose: To examine and determine the sites and the kinetics of IgG1 mAb modifications from both in vitro (rat plasma and PBS) and in vivo (rat model) time-course studies.

Methods: A comprehensive set of protein characterization methods, including RPLC/MS, LC-MS/MS, iCIEF, capSEC, and CE-SDS were performed in this report.

Results: We demonstrate that plasma incubation and in vivo circulation increase the rate of C-terminal lysine removal, and the levels of deamidation, pyroglutamic acid (pyroE), and thioether-linked (lanthionine) heavy chain and light chain (HC-S-LC). In contrast, incubation in PBS shows no C-terminal lysine removal, and slower rates of deamidation, pyroE, and HC-S-LC formation. Other potential modifications such as oxidation, aggregation, and peptide bonds hydrolysis are not enhanced.

Conclusion: This study demonstrates that in vivo mAb modifications are not fully represented by in vitro PBS or plasma incubation. The differences in modifications and their rates reflect the dissimilarities of matrices and the impact of enzymes. These observations provide valuable evidence and knowledge in evaluating the criticality of modifications that occur naturally in vivo that might impact formulation design, therapeutic outcome, and critical quality attribute assessments for therapeutic mAb manufacturing and quality control.