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

Monitoring of critical quality attributes such as sizeand charge-relatedheterogeneities is essential for biopharmaceutical manufacturers.Size-exclusion chromatography (SEC) is the preferred analytical techniquefor the quantification of aggregates and fragments in the product,whereas weak-cation exchange chromatography (WCX) is widely used forthe characterization of charge variants of biotherapeutic products,in particular monoclonal antibodies (mAbs). Multiattribute monitoringoffers the ability to monitor these attributes in a single run flowusing two-dimensional liquid chromatography (2D-LC). Typically, inthis approach, only the second-dimension samples are directly analyzedthrough mass spectrometry, as the first dimension has limitationsconcerning direct coupling with mass spectrometry. In the presentstudy, a novel 2D-SEC-MS/WCX-MS workflow has been proposed, in whichchromatography of both dimensions (D-1 and D-2) was directly coupled with mass spectrometry, through which size-relatedand charge-related variants of monoclonal antibody mAb A were analyzedsimultaneously in their native form. In comparison to stand-aloneSEC and WCX methods, this method enables simultaneous analysis ofsize and charge variants in a single workflow without manual intervention,allowing analysis of low abundant variants. Further, this method has75% less sample requirement and a shorter analysis time (25 min vs90 min) when size and charge variants were analyzed individually.The proposed native 2D-LC-MS workflow was used to analyze a stressedsample of mAb A, in which D-1 analysis revealed the presenceof aggregates (8-20%), which were primarily dimers, whereasD(2) analysis showed an increment in acidic variants (9-21%).