Rapid analysis of titer, aggregate and intact mass of antibody therapeutics using automated multi-dimensional liquid chromatography coupled with native mass spectroscopy.

Monoclonal antibodies are tetrameric complex proteins, primarily produced using mammalian cell culture. Attributes such as titre, aggregates, and intact mass analysis are monitored during process development/ optimization. In the present study, a novel workflow such that the Protein-A affinity chromatography is performed in the first dimension for purification and titre estimation, whereas size exclusion chromatography is employed in the second dimension to characterize size variants using native mass spectrometry. The present workflow offers significant advantage over the traditionally used standalone Protein-A affinity chromatography followed by size exclusion chromatography analysis in that it can monitor these four attributes in 8 minutes while requiring a minimal sample size (10-15 μg) and not requiring any manual peak collection. In contrast, the traditional standalone approach requires manual collection of eluted peaks in Protein-A affinity chromatography followed by buffer exchange to a mass compatible buffer, which can take up to 2-3 hours with considerable risk of sample loss, degradation, and induced modifications. As the biopharma industry moves to make analytical testing efficient, we believe that the approach proposed here would be of significant interest due to its ability to monitor multiple process and product quality attributes in a single workflow and via rapid analysis. This article is protected by copyright. All rights reserved.