No abstract

Monitoring of critical quality attributes such as size and charge-related heterogeneities is essential for biopharmaceutical manufacturers. Size-exclusion chromatography (SEC) is the preferred analytical technique for the quantification of aggregates and fragments in the product, whereas weak-cation exchange chromatography (WCX) is widely used for the characterization of charge variants of biotherapeutic products, in particular monoclonal antibodies (mAbs). Multiattribute monitoring offers the ability to monitor these attributes in a single run flow using two-dimensional liquid chromatography (2D-LC). Typically, in this approach, only the second-dimension samples are directly analyzed through mass spectrometry, as the first dimension has limitations concerning direct coupling with mass spectrometry. In the present study, a novel 2D-SEC-MS/WCX-MS workflow has been proposed, in which chromatography of both dimensions (D 1 and D 2 ) was directly coupled with mass spectrometry, through which size-related and charge-related variants of monoclonal antibody mAb A were analyzed simultaneously in their native form. In comparison to stand-alone SEC and WCX methods, this method enables simultaneous analysis of size and charge variants in a single workflow without manual intervention, allowing analysis of low abundant variants. Further, this method has 75% less sample requirement and a shorter analysis time (25 min vs 90 min) when size and charge variants were analyzed individually. The proposed native 2D-LC-MS workflow was used to analyze a stressed sample of mAb A, in which D 1 analysis revealed the presence of aggregates (8−20%), which were primarily dimers, whereas D 2 analysis showed an increment in acidic variants (9−21%).

Monoclonal antibodies are tetrameric complex proteins, primarily produced using mammalian cell culture. Attributes such as titer, 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 titer estimation, whereas size exclusion chromatography is employed in the second dimension to characterize size variants using native mass spectrometry. The present workflow offers a 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 min 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 h 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.