Breakthrough MDLC–MS Method Enhances Characterization of Therapeutic Antibody Charge Variants

In the field of therapeutic antibody development, gaining a comprehensive understanding of charge heterogeneity is vital for ensuring product safety and efficacy. Scientists from Henlius Biologics Co., Ltd. in Shanghai, China, have made a significant breakthrough with the development of an innovative online multidimensional liquid chromatography-mass spectrometry (MDLC–MS) method, enabling in-depth characterization of monoclonal antibody (mAb) charge variants.

Antibody binding to human cell receptors | Image Credit: © Tatiana Shepeleva - stock.adobe.com

MDLC-MS combines the capabilities of liquid chromatography (LC) and mass spectrometry (MS) to separate and identify individual components within a mixture. In MDLC-MS, the sample undergoes multiple stages of chromatographic separation using different columns with varying separation mechanisms. The eluted components are then introduced into the mass spectrometer for ionization and subsequent mass analysis, providing valuable information about their identity and structure. This integrated approach enables the precise characterization of complex mixtures. Its online nature ensures a streamlined and automated workflow, enhancing efficiency and accuracy in the analysis process.

The study, recently published in Analytical Chemistry, introduces a novel approach that combines MDLC and MS techniques to facilitate the detection and analysis of charged species present at low abundance (1). By implementing 96-well plate fractionation and on-column preconcentration through multi-injection, the researchers were able to overcome the challenges associated with identifying rare charge variants.

To demonstrate the efficacy of the method, the team focused on characterizing the charge variants of mAb-A. Initial analysis using 2D-LC (CEX × RP-C₄)–MS revealed the presence of TRVHS and RVHS signal peptide variants within the basic peaks of the cation exchange (CEX) profile. These signal peptide-containing variants, which could potentially trigger immunogenic responses, were successfully eliminated through an optimized purification process, enhancing the overall safety profile of the therapeutic antibody.

ND-LC (CEX × RP-C₄)–MS refers to an N-dimensional liquid chromatography-mass spectrometry method that combines the use of cation-exchange (CEX) and reversed-phase (RP) chromatographic separations followed by mass spectrometric analysis. In this technique, the sample is initially subjected to cation-exchange chromatography, where the components with different charge properties are separated based on their interactions with the stationary phase. The eluted fractions from the first dimension are then transferred to a second dimension, or higher N dimension, which involves reversed-phase chromatography using an RP-C₄ column. In this step, the analytes are further separated based on their hydrophobicity and molecular properties. Finally, the eluted fractions are directed to the mass spectrometer for ionization and detection, allowing for the identification and quantification of the separated components. This multidimensional approach provides enhanced separation power and resolution compared to traditional one-dimensional LC–MS methods, enabling a more comprehensive analysis of complex samples. It is particularly useful for the characterization of biomolecules, such as proteins and peptides, in fields like proteomics and biopharmaceutical analysis.

Further investigations using different N dimensions and columns, such as 4D-LC (CEX × RP-C₄ × Trypsin × RP-C₁₈)–MS unveiled additional post-translational modifications, including deamidation, cyclization of N-terminal glutamine, C-terminal lysine truncation, proline amidation, and methionine oxidation. Through this comprehensive characterization, the researchers were able to evaluate the potential risks associated with these modifications, providing valuable insights for optimizing the antibody development process.

To assess the biological activity of the identified charge variants, 2D-LC (CEX × FcγRIIIa) was employed, revealing increased binding affinity to the FcγRIIIa receptor in the acidic variants. Notably, the MDLC-MS method offers significant advantages in terms of efficiency and resource utilization. With a detection time of 72 hours and a sample requirement of only 1.25 mg of mAb, it proves to be sample-economic, time-effective, and labor-saving, meeting the aggressive timelines associated with antibody development. These findings highlight the importance of charge variant analysis in understanding the functional properties of therapeutic antibodies.

The groundbreaking MDLC-MS approach developed by the research team opens new possibilities for in-depth charge variant characterization in the field of therapeutic antibody development. By providing a powerful and timely tool, this innovative method enhances the quality control and optimization of therapeutic antibodies, ultimately improving patient outcomes in the field of biopharmaceuticals.

Reference

(1) Liu, Z.; Cao, Y.; Zhang, L.; Xu, Y.; Zhang, Z. In-Depth Characterization of mAb Charge Variants by On-Line Multidimensional Liquid Chromatography-Mass Spectrometry. Anal. Chem. 2023, 95 (20), 7977–7984. DOI: https://doi.org/10.1021/acs.analchem.3c00791