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 (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%).

An Online Two-Dimensional Approach to Characterizing the Charge-Based Heterogeneity of Recombinant Monoclonal Antibodies Using a 2D-CEX–AEX–MS Workflow

“…Hence, identification of charge variants was proposed based on observed mass differences with respect to the main peak. Earlier, researchers have extensively characterized mAb A by the CEX–MS method and identified variants that were mainly results of the deamidation of Asn or isomerization of Asp. , In line with the published literature, MS analysis of mAb A revealed acidic variants such as the deamidation and isomerization of Asp, and basic variants were detected such as isomerization of Asp and pyroglutamate (pyroGlu) modifications.…”

“…These modifications affect protein characteristics by altering the molecular surface charge distribution of the protein, which results in the formation of acidic or basic variants of the protein. Modifications such as deamidation, glycation, cysteinylation, reduced disulfide bonds, and sialic acid contribute to the formation of acid variants. − On the other hand, methionine oxidation, C-terminal lysine, succinimide, or glycine amidation and incomplete disulfide bonds contribute to the creation of basic variants of mAb. − Ion-exchange chromatography (IEX) and capillary electrophoresis (CE) are two standard methods for monitoring charge heterogeneity of mAbs . Typically, these methods are performed under nonvolatile buffer conditions considered to be incompatible with mass spectrometry.…”