High-Throughput and Format-Agnostic Mispairing Assay for Multispecific Antibodies Using Intact Mass Spectrometry

Multispecific antibodies have gainedsignificant importance ina broad indication space due to their ability to engage multiple epitopessimultaneously and to thereby overcome therapeutic barriers. Withgrowing therapeutic potential, however, the molecular complexity increases,thus intensifying the demand for innovative protein engineering andanalytical strategies. A major challenge for multispecific antibodiesis the correct assembly of light and heavy chains. Engineering strategiesexist to stabilize the correct pairing, but typically individual engineeringcampaigns are required to arrive at the anticipated format. Mass spectrometryhas proven to be a versatile tool to identify mispaired species. However,due to manual data analysis procedures, mass spectrometry is limitedto lower throughputs. To keep pace with increasing sample numbers,we developed a high-throughput-capable mispairing workflow based onintact mass spectrometry with automated data analysis, peak detection,and relative quantification using Genedata Expressionist. This workflowis capable of detecting mispaired species of & SIM;1000 multispecificantibodies in three weeks and thus is applicable to complex screeningcampaigns. As a proof of concept, the assay was applied to engineeringa trispecific antibody. Strikingly, the new setup has not only provedsuccessful in mispairing analysis but has also revealed its potentialto automatically annotate other product-related impurities. Furthermore,we could confirm the assay to be format-agnostic, as shown by analyzingseveral different multispecific formats in one run. With these comprehensivecapabilities, the new automated intact mass workflow can be appliedas a universal tool to detect and annotate peaks in a format-agnosticapproach and in high-throughput, thus enabling complex discovery campaigns.