Native State Charge Variant Analysis of Commercialized Monoclonal Antibodies in Minutes

Importance of the Topic

Monoclonal antibodies are critical biotherapeutics whose performance and stability depend on post-translational modifications. Charge variants arising from these modifications can impact efficacy and safety, making rapid and reliable charge variant analysis essential in drug development and QA/QC workflows.

Objectives and Study Overview

This study evaluates a native‐state capillary zone electrophoresis (CZE) method using the SCIEX CZE Rapid Charge Variant Analysis Kit on the PA 800 Plus Pharmaceutical Analysis System. The goal is to achieve high‐resolution separation of charge variants in commercially available monoclonal antibodies with minimal method development and in under ten minutes per sample.

Methodology

Sample Preparation:

• Therapeutic antibodies (Herceptin®, Rituxan®, Remicade®) diluted to 1 mg/mL in CE‐grade water.

Separation Conditions:

• PA 800 Plus system equipped with a pre‐assembled fused silica capillary cartridge.
• CZE Rapid Charge Variant Analysis Kit buffer.
• Separation voltage: 1000 V/cm; pressure injection: 0.5 PSI for 10 s.
• UV detection at 214 nm.
• Data acquired and analyzed with 32 Karat software V10.

Used Instrumentation

• PA 800 Plus Pharmaceutical Analysis System (SCIEX).
• Pre‐assembled fused silica capillary cartridge (SCIEX P/N A55625).
• 214 nm bandpass UV detector (SCIEX P/N 144437).

Main Results and Discussion

The assay demonstrated high reproducibility, with percent relative standard deviations below 2% for acidic variants, main peak, and basic variants across ten injections of each antibody. Electropherograms showed clear separation of charge variants within 5–8 minutes. Average variant distributions included approximately 28.7% acidic, 66.1% main, and 5.2% basic for trastuzumab; 11.1%, 38.4%, and 50.5% for infliximab; and 24.2%, 72.1%, and 3.7% for rituximab.

Benefits and Practical Applications

• Native‐state analysis provides stability‐relevant data.
• Minimal method development accelerates assay deployment.
• High throughput: total cycle time under 10 minutes.
• Platform versatility across a broad pI range above 7.0.

Future Trends and Possibilities

Ongoing developments include fine‐tuning buffer pH and additives to enhance resolution, extending capillary length to improve separation, and integrating automated workflows for even higher throughput. Adaptation to diverse therapeutic formats, such as antibody‐drug conjugates, presents further opportunities for rapid charge profiling.

Conclusion

The CZE Rapid Charge Variant Analysis Kit on the PA 800 Plus system delivers fast, reproducible native‐state charge variant profiling of monoclonal antibodies, reducing development timelines and supporting robust quality assessment in biopharmaceutical labs.

References

1. He Y., Isele C., Hou W., Ruesch M. J. Sep. Sci. 2011, 34, 548–555.
2. Moritz B. et al. J. Chromatogr. B 2015, 983–984, 101–110.
3. Moritz B. et al. Electrophoresis 2017, 38, 3136–3146.