Charge Heterogeneity Analysis of Intact Infliximab Using CESI-MS and Neutral OptiMS Cartridge

Significance of the Topic

Monoclonal antibody charge heterogeneity influences therapeutic safety and efficacy by altering isoelectric point, affecting product stability, potency and immunogenicity. Detailed profiling of charge variants is essential for quality control across biopharmaceutical development and manufacturing.

Objectives and Study Overview

This study aims to demonstrate a unified CESI-MS method using a neutral OptiMS cartridge on the SCIEX CESI 8000 system to achieve high-resolution separation and concurrent mass spectrometric identification of intact infliximab charge variants. The performance of this approach is compared to established UV-based CZE and cIEF platforms, and applied to both unstressed and thermally stressed samples to assess analytical robustness.

Methodology

Infliximab was prepared in 5 mM ammonium acetate pH 6.0 with 20 % methanol at 0.3–0.5 mg/mL. Stressed samples were generated by incubation at 35–40 °C for three days. Separation employed a CESI 8000 High Performance Separation and ESI Module with a neutral OptiMS cartridge at 20 °C, 30 kV separation voltage, using 0.3 % acetic acid as background electrolyte. MS detection was performed on a SCIEX TripleTOF 6600 with NanoSpray III source, scanning 2000–6000 m/z, collision energy 70 eV, declustering potential 190 V. Data analysis involved deconvolution via SCIEX PeakView 2.2 and BioTool Kit within the 140–160 kDa mass window.

Used Instrumentation

• SCIEX CESI 8000 High Performance Separation and ESI Module with Neutral OptiMS cartridge
• SCIEX TripleTOF 6600 System with NanoSpray III Source and CESI Adapter
• Amicon 10K centrifugal filters for buffer exchange

Key Results and Discussion

CESI-MS separation resolved six distinct peaks corresponding to basic and acidic variants of intact infliximab, matching profiles from CZE-UV and cIEF methods. Deconvolution identified Peak 1 and Peak 2 as variants retaining two and one C-terminal lysine residues (+258 and +129 Da), respectively. Peak 3 was assigned as a deamidated single-lysine variant (+131 Da shift). Peaks 5 and 6 displayed additional mass increases consistent with further deamidation. Glycan profiling revealed G0F/G1F as the predominant glycoforms across variants. Analysis of stressed samples showed elevated deamidation levels and low-level heavy chain fragmentation near 47 kDa, demonstrating the method’s sensitivity for stability studies.

Benefits and Practical Applications

Combining ultralow-flow CE with MS offers simultaneous high-resolution charge variant separation and molecular identification in a single assay, reducing analysis time and method complexity. This approach enables sensitive detection of low-abundance variants and detailed glycoform characterization, facilitating rapid quality assessment during mAb development, comparability studies and stability testing.

Future Trends and Opportunities

Advances in CE-MS integration and cartridge design will further enhance throughput and robustness, supporting automation in biopharmaceutical analytics. Coupling with high-resolution MS and advanced data processing will deepen PTM characterization, expand applicability to other antibody formats and ADCs, and improve real-time monitoring in manufacturing environments.

Conclusion

The CESI-MS method using a neutral OptiMS cartridge provides comparable charge variant resolution to established UV-based platforms while delivering direct mass confirmation and glycoform profiling. Its high sensitivity and streamlined workflow make it a valuable tool for comprehensive mAb charge heterogeneity analysis in research and QC settings.

References

1. SCIEX. Capillary Isoelectric Focusing (cIEF) Analysis for the PA 800 Plus Pharmaceutical Analysis System Application Guide.
2. SCIEX. Analysis of Monoclonal Antibody Charge Variants by Capillary Zone Electrophoresis.
3. He Y, et al. Rapid analysis of charge variants of monoclonal antibodies with capillary zone electrophoresis in dynamically coated fused-silica capillary. Journal of Separation Science. 2011;34:548–555.
4. SCIEX. Charge Heterogeneity Analysis of Intact NIST mAb Using CESI-MS and Neutral OptiMS Cartridge. RUO-MKT-02-9295-A.
5. Liu H, et al. Characterization of the stability of a fully human monoclonal IgG after prolonged incubation at elevated temperature. Journal of Chromatography B. 2010;837(1–2):35–45.