High-Throughput Native HRMS ProA-MS Analysis of mAb Variants in Cell-Culture Samples

Importance of the Topic

The rapid and accurate characterization of monoclonal antibodies (mAbs) during biopharmaceutical development is critical for ensuring product quality and safety. Native Protein A–mass spectrometry (ProA-MS) analysis directly from cell culture filtrates streamlines workflows by combining affinity capture, intact mass measurement, and titer determination in a single high-throughput method.

Objectives and Overview of the Study

This application note evaluates a one-minute Protein A elution gradient coupled to high-resolution mass spectrometry (HRMS) for the direct analysis of mAb variants in clarified cell culture fluid (CCCF). Key aims include assessing method speed, sensitivity, variant identification capability, and simultaneous titer determination via UV absorbance at 280 nm.

Methodology and Instrumentation

Sample preparation involved NISTmAb reference material and trastuzumab-spiked CHO CCCF. Chromatography was performed on a BioResolve™ Protein A Affinity Column (3.5 µm, 2.1 × 20 mm) at ambient temperature with 100 mM ammonium acetate for loading and 200 mM formic acid for elution at 0.20 mL/min. UV detection at 280 nm provided titer information. Mass analysis used a Xevo™ G3™ QTof with a mass range of 400–8000 m/z, 1 s scan time, source at 120 °C, desolvation at 500 °C, and cone voltage of 150 V.

Main Results and Discussion

• The 4-minute ProA-MS workflow delivered clear elution peaks for neat and CCCF-diluted mAb samples.
• Native HRMS spectra resolved low-abundance singly glycosylated variants with mass accuracy within 20 ppm.
• Signal-to-noise was slightly reduced in CCCF samples, likely due to residual matrix components.
• Titer measurements showed linear UV responses (–0.39 % to 0.39 % residual) and 105 % average recovery when comparing CCCF to neat samples.

Benefits and Practical Applications

The method offers ultra-fast affinity capture, intact mass identification of mAb variants, and concurrent titer quantitation without extensive sample cleanup. It is suitable for high-throughput screening in process development, QC laboratories, and comparability studies.

Future Trends and Potential Applications

• Optimization of mobile phases and wash protocols to enhance MS response for complex matrices.
• Extension to other Fc-fusion proteins and biotherapeutic classes.
• Integration into two-dimensional LC workflows for multi-attribute analysis.
• Automation of data processing within informatics platforms for real-time monitoring.

Conclusion

This study demonstrates that a high-efficiency Protein A column combined with native HRMS enables 4-minute analysis of mAb variants directly from cell culture, while delivering accurate titer data. The approach enhances throughput and insight into product heterogeneity for biopharmaceutical development.

References

1. Dunham W.H., Mullin M., Gingras A.-C. Proteomics 12:1576–1590 (2012).
2. Jakes C. et al. Anal. Chem. 93:13505–13512 (2021).
3. Cotham V.C. et al. J. Pharm. Biomed. Anal. 228:115337 (2023).
4. Koza S.M., Shiner S., Lauber M.A. Waters Application Note (2025).
5. Koza S.M. et al. Waters Application Note (2025).
6. Koza S.M. et al. Waters Application Note (2025).
7. Formolo T. et al. ACS Symp. Ser. 2:1–62 (2015).
8. Koza S.M., Shiner S., Lauber M.A. Waters Application Note (2025).