Middle-up Analysis of Monoclonal Antibodies using FabRICATOR®
With their molecular specificity for biological targets, monoclonal antibodies (mAbs) have revolutionized the field of research and medicine. Due to numerous post-translational modifications causing heterogeneity in the structure of mAbs, a comprehensive characterization of the physiochemical properties is necessary to ensure the quality, efficacy and safety of mAb-based therapeutic drugs.
The characterization of mAbs usually involves separation techniques that provide complementary information about oxidation, glycosylation, deamidation and other critical quality attributes. Efforts has been invested in the development of chromatographic separation methods, and alternative separation techniques such as capillary zone electrophoresis (CZE) have recently shown improved/complementary separation abilities. In fact, recent developments have highlighted the potential of automation of CZE for bottom and middle-up analyses, since enzymatic reactions can be performed inside the capillary with the possibility of temperature control.
In this work, the development of new automated CZE methodologies for the quality control of mAbs at the middle-up level is described, where the FabRICATOR (IdeS) enzyme plays an important part. FabRICATOR is a cysteine protease that digests antibodies at a specific site below the hinge, generating a homogenous pool of F(ab’)2 and Fc/2 fragments within 30 minutes. Multiple in-line reactions (simultaneous reduction and FabRICATOR digestion) were performed in the separation capillary with high performance and good reproducibility, and the method has been successfully applied to several mAbs.
To conclude, the study demonstrates the utility of CZE to integrate multiple reactions of sample preparation and separation steps together with its high flexibility that allows for analyses under diverse conditions.
Dadouch et al., 2021. In-capillary (electrophoretic) digestion-reduction-separation: A smart tool for middle-up analysis of mAb. Journal of Chromatography. doi.org/10.1016/j.chroma.2021.462213
FabRICATOR – Below hinge digestion of IgG.