In-capillary (electrophoretic) digestion-reduction-separation: A smart tool for middle-up analysis of mAb

Comprehensive characterization of physicochemical properties monoclonal antibodies (mAbs) is a critical process to ensure their quality, efficacy, and safety. For this purpose, mAb analysis at different levels (bottom-up, middle-up) common approach that includes rather complex multistep sample preparation (reduction, digestion). To high performance, the development fully integrated methodologies highly valuable. Capillary zone electrophoresis particularly well-adapted technique for implementation analytical strategies from detection. This feature was employed develop novel middle-up level. Multiple in-line reactions (simultaneous reduction digestion) were performed first time in separation capillary. Tris (2-carboxyethyl) phosphine hydrochloride (TCEP) used as an effective reducing agent under broad pH range IdeS (Immunoglobulin degrading enzyme Streptococcus) specific digestion. Transverse diffusion laminar flow profile (TDLFP) applied reactants mixing. Both parameters optimized non-denaturing denaturing conditions. The developed provided good reproducibility higher peak efficiencies comparing with off-line assays. They successfully mAbs.