Online Coupling of Asymmetrical Flow Field-Flow Fractionation with Native Mass Spectrometry for Monoclonal Antibody Characterization.

Integration of Asymmetrical Flow Field-Flow Fractionation (AF4) with online native mass spectrometry (MS) is an attractive concept that offers significant value for the analytical characterization of protein therapeutics. Its execution, however, is challenged by the incompatibility of mobile phase components and the difficulties of achieving sensitive MS detection without compromising the separation efficiency. Herein, we describe in detail the successful coupling of a commercial AF4 instrument with online native MS detection. By optimizing the operational conditions of AF4 using ammonium acetate-based mobile phases and fine-tuning the interface configurations, the established AF4-MS platform achieved uncompromised separation performance while enabling sensitive online native MS measurements. The feasibility of the method for studying protein analytes was first demonstrated by analyzing a protein mixture sample (17 kDa to 660 kDa), followed by size variant characterization of the NISTmAb. The AF4-MS method was subsequently compared with native SEC-MS using both protein mixture and mAb samples, highlighting their complementarity as size-based separation techniques and showcasing the quantitative advantages of AF4-MS. Applications of AF4-MS in supporting therapeutic mAb development were demonstrated using two case studies, including heterodimer quantitation in a coformulated mAb cocktail sample and antibody-antigen binding stoichiometry characterization. This developed AF4-MS method is easily implementable and could be a valuable addition to our analytical toolbox to support therapeutic mAb development.