Multiattribute Monitoring of Aggregates and Charge Variants of Monoclonal Antibody through Native 2D-SEC-MS-WCX-MS.

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 (D1 and D2) 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 D1 analysis revealed the presence of aggregates (8-20%), which were primarily dimers, whereas D2 analysis showed an increment in acidic variants (9-21%).