Salt gradient analysis of Protein G using a 3 μm monodisperse SAX chromatography column

Importance of the Topic

Protein therapeutics such as Protein G play a crucial role in biopharmaceutical research and quality control due to their therapeutic and diagnostic applications

Objectives and Study Overview

This application note demonstrates the development of high-resolution analytical and rapid QC salt gradient methods for charge variant analysis of Protein G using a 4 × 100 mm, 3 µm monodisperse ProPac 3R SAX column

Methodology and Instrumentation

The study used strong anion exchange chromatography under controlled salt gradients and buffer pH to resolve acidic and basic charge variants of Protein G

• UHPLC system: Thermo Scientific Vanquish Flex Quaternary UHPLC (System Base, Quaternary Pump, Column Compartment H, Split Sampler FT)
• Detector: Vanquish Variable Wavelength Detector (UV 280 nm)
• Stationary phase: ProPac 3R SAX 3 µm monodisperse divinylbenzene resin, PEEK hardware
• Software: Chromeleon 7.2.10 CDS
• Reagents: Protein G (5 mg/mL), 20 mM Tris buffer pH 8.0, NaCl for gradient, deionized water

Main Results and Discussion

• Buffer pH effect: Optimal separation at pH 8.0, with increased retention and variant resolution compared to pH 7.5 or 8.5
• Starting salt concentration: 60 mM NaCl loading prevents isocratic elution and maximizes reproducibility
• Gradient slope: Longer gradients (up to 30 min) improve variant separation, while shorter (6–10 min) support rapid QC
• Flow rate and temperature: Lower flow rates (0.3 mL/min) enhance signal, higher temperatures increase retention but may affect sample stability
• Sample loading and carryover: Dynamic capacity up to ~10 µg for narrow peaks; carryover below 0.1% at 50 µg injection
• Lot-to-lot reproducibility: Three media batches delivered consistent chromatographic profiles
• Comparison studies: Monodisperse ProPac 3R SAX outperformed traditional 10 µm SAX columns in resolution and peak capacity
• Stress testing: Heat-stressed Protein G showed emerging acidic and basic variants clearly resolved by the optimized method

Benefits and Practical Applications

• High resolving power for complex protein variant profiling in R&D and QC
• Straightforward method development guided by systematic optimization steps
• Robust column performance with minimal variability across lots and low carryover
• Flexibility to adapt gradient slope, flow rate, and temperature to diverse analytical goals

Future Trends and Potential Applications

• Integration of monodisperse SAX phases with mass spectrometry for deeper proteoform analysis
• Automated method scouting and AI-driven optimization for accelerated analytical workflows
• Expansion of monodisperse media to multi-dimensional separations and high-throughput platforms
• Regulatory adoption for characterization of next-generation biopharmaceuticals with complex post-translational modifications

Conclusion

The ProPac 3R SAX 3 µm monodisperse column enables both rapid QC and high-resolution charge variant analysis of Protein G with excellent reproducibility, low carryover, and method flexibility, meeting the demands of modern biopharmaceutical characterization

References

1. Rivera B, Anspach JA, Rao SL. LCGC Supplements. 2018;36(6):24–29.
2. Taha M, da Silva F, Quental M, Ventura SM, Freire M, Coutinho JP. Green Chem. 2014;16(6):3149–3159.