Balancing speed and separation: A high-throughput native mass spectrometry platform for screening complex bispecific antibody variants.

High-throughput liquid chromatography coupled to mass spectrometry (LC-MS) workflows are increasingly required to support the structural characterization of complex antibody formats such as multispecific (e.g. 2 + 1 and 1 + 1+1) and bispecific antibodies (bsAb) where misassembled product related variants pose significant challenges to analytical process development and patient safety. Conventional size-exclusion chromatography coupled to native mass spectrometry (SEC-MS) remains limited by long run times, manual sample preparation, and susceptibility to operator-dependent variability. Here, we report a semi-automated, 96 well plate-based SEC-MS platform that integrates liquid-handler assisted sample preparation, with two rapid chromatography strategies coupled to native mass spectrometry to enable high-throughput assessment of light-chain mispairing and related product variants. Automated liquid-handler sample preparation demonstrated comparable analytical reproducibility to manual workflows, minimal intraplate positional and concentration effects, and consistent performance across independent automated runs. Rapid chromatography approaches showed distinct performance characteristics, with high-throughput size exclusion chromatography coupled to native mass spectrometry (HT-SEC-MS) closely matching benchmark SEC-MS for quantitative agreement, while online buffer-exchange coupled to native mass spectrometry (OBE-MS) enabled rapid elimination of low-quality clones with high levels of LC-mispairs. Integration of an automated data-processing pipeline further standardized peak detection, quantitation, and reporting. Collectively, this native-state LC-MS platform reduces end-to-end processing timelines four fold - from multiple days to just 24 h per 96-sample batch - and decreases analyst hands-on time by more than 50%, providing a flexible and analytically rigorous framework for the high-throughput screening of complex bispecific antibody formats.