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

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...

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Bibliographic Details
Published in:Analytical chemistry (Washington) 2022-11, Vol.94 (43), p.15018-15026
Main Authors: Sarin, Deepika, Kumar, Sunil, Rathore, Anurag S.
Format: Article
Language:English
Subjects:
Analytical methods
Cation exchanging
Cation-exchange chromatography
Chemistry
Chromatography
Desalination
Fractionation
Heterogeneity
Hydrophobicity
Ions
Life cycle analysis
Liquid chromatography
Mass spectrometry
Mass spectroscopy
Monitoring
Monoclonal antibodies
Product life cycle
Quality management
Scientific imaging
Spectroscopy
Workflow
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Summary: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.
ISSN:0003-2700
1520-6882
DOI:10.1021/acs.analchem.2c02931