Development of a platform method for rapid detection and characterization of domain-specific post-translational...

Charge heterogeneity is inherent to all therapeutic antibodies and arises from post-translational modifications (PTMs) and/or protein degradation events that may occur during manufacturing. Among therapeutic antibodies, the bispecific antibody (bsAb) containing two unique Fab arms directed against two different targets presents an additional layer of complexity to the charge profile. In the context of a bsAb, a single domain-specific PTM within one of the Fab domains may be sufficient to compromise target binding and could potentially impact the stability, safety, potency, and efficacy of the drug product. Therefore, characterization and routine monitoring of domain-specific modifications is critical to ensure the quality of therapeutic bispecific antibody products. We developed a Digestion-assisted imaged Capillary isoElectric focusing (DiCE) method to detect and quantitate domain-specific charge variants of therapeutic bispecific antibodies (bsAbs). The method involves enzymatic digestion using immunoglobulin G (IgG)-degrading enzyme of S. pyogenes (IdeS) to generate F(ab)₂ and Fc fragments, followed by imaged capillary isoelectric focusing (icIEF) under reduced, denaturing conditions to separate the light chains (LCs) from the Fd domains. Our results suggest that DiCE is a highly sensitive method that is capable of quantitating domain-specific PTMs of a bsAb. In one case study, DiCE was used to quantitate unprocessed C-terminal lysine and site-specific glycation of Lys⁹⁸ in the complementarity-determining region (CDR) of a bsAb that could not be accurately quantitated using conventional, platform-based charge variant analysis, such as intact icIEF. Quantitation of these PTMs by DiCE was comparable to results from peptide mapping, demonstrating that DiCE is a valuable orthogonal method for ensuring product quality. This method may also have potential applications for characterizing fusion proteins, antibody-drug conjugates, and co-formulated antibody cocktails.