Chemical and Physical Instability of Monoclonal Antibodies Induced by Metal-catalyzed Carbonylation

Abstract

In the pharmaceutical industry, recombinant monoclonal antibodies (mAbs) have become a major focus of therapeutic drug development due to their broad applications in many disease areas. During manufacturing and storage of mAb drug products, metal-catalyzed carbonylation, a post-translational modification referring to the formation of carbonyls (aldehydes and ketones) on proteins after metal-catalyzed oxidation, is a relevant product variant of mAbs, which may impact mAb safety and/or efficacy. However, to date, very little is known about the occurrence of metal-catalyzed carbonylation during mAb manufacturing and storage or the effects of metal-catalyzed carbonylation on chemical and physical stability of mAbs. To answer those questions, this dissertation first focused on developing a new protein carbonylation assay with improved assay precision and robustness over the conventional assays. Using the new protein carbonylation assay, a study was performed to investigate various cell culture and formulation factors, which revealed that iron ion, hydrogen peroxide, and polysorbate 20 can critically impact mAb carbonylation during manufacturing or storage. This study also revealed that metal-catalyzed carbonylation positively correlated with increased acidic charge heterogeneity and aggregation propensity of mAbs. To elucidate the increased acidic charge heterogeneity, the acidic fraction of a model IgG1 mAb after metal-catalyzed oxidation was collected and characterized, which showed that metal-catalyzed carbonylation products (primarily threonine carbonylation products) directly contributed to the increased acidic charge heterogeneity. Several additional oxidation products, such as carboxylic acids (from further oxidation of aldehydes) and pyroglutamate (from oxidation of proline residues in the hinge region), were identified for the first time in mAbs, which provides further explanation to the increased acidic charge heterogeneity. To elucidate the increased aggregation propensity, this dissertation used a model stress system and anions from the Hofmeister series to investigate the effects of metal-catalyzed carbonylation on physical stability of the model IgG1 mAb. In particular, the investigation applied a site-specific carbonylation analysis, which revealed for the first time that mAb aggregation can be affected by carbonylation location, carbonylation type, and buffer type. These findings showed that effects of metal-catalyzed carbonylation on physical instability of mAbs can be much more complex than what have been previously proposed/suggested in the literature. Finally, this dissertation investigated formation of Schiff base crosslinks as a potential route of chemical instability of the metal-catalyzed carbonylation products. To facilitate the investigation, a new analytical methodology was developed to characterize crosslinked peptides. This new methodology addressed several key analytical challenges for crosslink analysis and successfully identified bissulfosuccinimidyl suberate (BS3)-crosslinked peptides in a BS3-treated mAb sample. Using the new analytical methodology, this study showed that Schiff base formation is a not major degradation pathway of mAbs even under a harsh condition by copper-catalyzed oxidation. In summary, this dissertation provided a comprehensive understanding of the chemical and physical instability of mAbs induced by metal-catalyzed carbonylation, which can help improve mAb stability against metal-catalyzed carbonylation in the future.