Multiattribute Monitoring of Charge-Based Heterogeneity of Recombinant Monoclonal Antibodies Using 2D HIC-WCX-MS

Abstract

Charged heterogeneity of monoclonal antibody (mAb) products is regarded as a critical quality attribute (CQA) depending on its impact on the safety and efficacy profile of the product. Hence, manufacturers are expected to perform a comprehensive characterization of the charge heterogeneity to ensure that the manufactured product meets its specifications. Further, monitoring is also expected during the product lifecycle to demonstrate consistency in product quality. However, conventional analytical methods for characterization of hydrophobic and charge variants are nonvolatile salt-based and require manual fraction collection and desalting steps before analysis through mass spectrometry can be performed. In the present study, a workflow of a two-dimensional liquid chromatography method using mass spectrometry (MS)-compatible buffers coupled with native mass spectrometry was performed to characterize hydrophobic variants in the first dimension and charge variants in the second dimension without any need for manual fractionation. This novel two-dimensional (2D) hydrophobic interaction chromatography (HIC)-weak cation-exchange chromatography (WCX)-MS workflow identified 10 variants in mAb A, out of which 2 variants are exclusive to the 2D orthogonal method. Similarly, for mAb B, a total of 11 variants are identified, including 5 variants exclusive to the 2D orthogonal workflow. When compared to stand-alone, HIC resolved only 4 variants for both mAbs and WCX resolved 7 variants for mAb A and 6 variants for mAb B. In addition, the proposed method allows direct characterization of hydrophobic/charge variant peaks through native mass spectrometry in a single-run workflow.

HPLC, MS, and CDS: The New Products to Watch—from Pittcon and Beyond

“…However, this approach requires extensive sample preparation, which, despite major advancements in automation, continues to be prone to artifacts such as induced modifications and a lack of reproducibility (11). On the other hand, a subunit or intact-mass analysis requires minimum sample preparation and can monitor multiple attributes-such as purity, size variants, glycosylation and charge variants-in a single LC-MS method (12)(13)(14). This approach can provide an enhanced control strategy for reporting attributes, such as titer, presence of dimer or fragments, or antibody-drug conjugations, something that would not be possible to monitor through a standard MAM.…”

“…MAMs are useful when residue-specific monitoring is not required, and when rapid analysis is desired for monitoring attributes such as aggregates, dimers or fragments, or charge variants. A recently published article showed a MAM workflow to monitor charge heterogeneity of mAbs, described here as a case study (14).…”

“…Introduction of a volatile-salt-based mobile phase overcomes the limitation of direct coupling of analytical separation techniques with mass spectrometry. In a recent study, an iMAM method was presented in which an online workflow of a 2D-LC method using an MScompatible mobile phase coupled with native mass spectrometry was performed to characterize hydrophobic variants in the first dimension and charge variants in the second dimension without needing manual fractionation (Figure 1) (14). As a result of this novel workflow, characterization of 10 variants for one mAb can be achieved, out of which a pair of variants was exclusive to the orthogonal 2D method.…”

“…These reported methods are time consuming, expensive, slow, less environment friendly, and show poor extraction efficiency for few analytes such as daminozide and others (7-10). In the past, we published our novel LC-MS/MS to meet regulations for pesticides and mycotoxins regulated by different U.S. states and Canada in different cannabis matrices (11)(12)(13)(14)(15). The aim of this study was to validate a novel, cost-effective, and fast LC-MS/MS method to analyze all 102 pesticides and five mycotoxins regulated by Colorado state in hemp matrix.…”

Revealing charge heterogeneity of stressed trastuzumab at the subunit level

“…Ion-exchange chromatography and electrophoretic separation methods have been widely used to separate charge variants of mAbs [7][8][9][10]. Other chromatographic approaches may also be employed for this purpose, such as hydrophobic interaction chromatography [11][12][13][14] and displacement chromatography [15,16].…”