Capillary electrophoresis (CE) has been demonstrated to be effective for analyzing complex biological samples, such as monoclonal antibodies (mAbs) and other protein based therapies. When interfaced with mass spectrometry (MS) analysis the integrated techniques present a powerful platform for characterizing biomolecules. Utilizing microfluidic technology to perform CE-MS analysis enables faster and more efficient separations as compared to what is achieved using capillary tubes. The work described here utilizes the ZipChip microfluidic CE-MS interface coupled to an orbitrap MS for high resolution accurate mass (HRAM) analysis of mAbs and antibody drug conjugates (ADCs).<br/>Results will be presented analyzing the mAb products at multiple levels all with on-line MS analysis. Using near native conditions with the microfluidic CE-MS interface, near baseline separation of intact charge variants differing in net charge by as little as +1 can be achieved, with total run times under 3 minutes. For mAb conjugates the average DAR can be calculated by utilizing both the separation data and MS information generated during the analysis. Further characterization of the molecules was performed by analyzing antibody and ADC fragments under denaturing conditions after reduction and limited proteolysis. This provided general conjugation localization on the mAb structure and additional DAR information. Finally, rapid peptide mapping was performed to generate site specific information about modifications to the mAb structure. Overall, the microfluidic CE-MS interface is a simple and rapid way to assess mAb based therapeutics.<br/>

Characterization of monoclonal antibodies and antibody drug conjugates using microfluidic CE-MS technology

256th National Meeting (2018)

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