Optimize Non-Reduced CE-SDS Analysis with the SCIEX Low pH SDS Sample Buffer

Importance of the Topic

Capillary electrophoresis sodium dodecyl sulfate (CE-SDS) is widely used in biopharmaceutical R&D and QC to assess protein size heterogeneity, detect fragments, and support process development, ensuring safety and efficacy of therapeutic proteins.

Objectives and Study Overview

This study evaluated the impact of sample buffer pH on fragmentation artifacts in non-reduced CE-SDS analysis of monoclonal antibody biosimilars. It compared a standard pH 9.0 buffer with a low pH 6.8 buffer to optimize robustness and improve correlation with orthogonal methods.

Methodology

Monoclonal antibodies (adalimumab, infliximab, drozitumab) were prepared at 0.5 mg/mL by combining 50 μL protein solution with 5 μL 500 mM iodoacetamide and 45 μL SDS sample buffer (pH 9.0 or 6.8), followed by heating at 70°C for 5 minutes. Samples were centrifuged and injected using pressure (5 PSI, 65 seconds). Separations were carried out with 15 kV at 25°C, including capillary preconditioning and automated reagent replenishment.

Used Instrumentation

• PA 800 Plus Pharmaceutical Analysis System (SCIEX)
• 50 μm ID × 10 cm bare fused-silica capillaries

Main Results and Discussion

Lowering the sample buffer pH from 9.0 to 6.8 increased main peak purity by 1.3–2.1% across all antibody biosimilars. Heavy-heavy-light chain fragment peaks and total fragment percentages were significantly reduced, while high molecular weight species showed slight decreases. Electropherograms at low pH exhibited sharper main peaks and fewer artifacts, indicating reduced disulfide scrambling.

Benefits and Practical Applications

• Enhanced method robustness with reduced artificial fragmentation
• Improved agreement with orthogonal assays such as SEC
• Reliable quantitation of protein purity and stability for batch release

Future Trends and Potential Applications

• Application of low pH buffer approaches to diverse biotherapeutic formats
• Integration into automated, high-throughput QC workflows
• Development of novel buffer chemistries to further suppress disulfide exchange

Conclusion

Optimizing sample buffer pH is essential in non-reduced CE-SDS workflows to minimize disulfide bond scrambling and analytical artifacts. The low pH (6.8) SDS sample buffer significantly improves purity measurements and aligns results more closely with orthogonal methods, supporting robust biopharmaceutical characterization.

References

1. Good D. et al., Methods in Molecular Biology, vol.276: Capillary Electrophoresis of Proteins and Peptides, Humana Press, 2019.
2. Salas-Solano O. et al., Anal. Chem. 2006, 78, 6583–6594.
3. Rustandi R. et al., Electrophoresis 2008, 29, 3612–3620.
4. Dada O. et al., J. Pharm. Biomed. Anal. 2017, 145, 91–97.
5. Zhang J. et al., J. Pharm. Biomed. Anal. 2010, 53, 1236–1243.
6. Leer H., J. Immunol. Methods 2000, 234, 71–81.
7. Wang S. et al., J. Pharm. Biomed. Anal. 2018, 154, 468–475.