Convenient Customization of Your Cation Exchange Analysis

Importance of the Topic

Cation exchange chromatography is the gold standard for analyzing charge variants of monoclonal antibodies, crucial for ensuring safety and efficacy of biopharmaceuticals due to microheterogeneity arising from post-translational modifications.
Use of pH gradients streamlines method development and enhances resolution compared to conventional salt gradients.

Objectives and Study Overview

This application note evaluates high-resolution charge variant analysis of two mAbs, trastuzumab and the NIST mAb reference standard, using different pH gradients on an Agilent 1260 Infinity II Prime Bio LC System combined with Agilent Buffer Advisor Software.

Methodology and Instrumentation

• Instrumentation:
  • Agilent 1260 Infinity II Prime Bio LC System with iron-free MP35N flow path
  • Agilent 1260 Infinity II Bio Flexible Pump
  • Agilent 1290 Infinity II Bio Multisampler with thermostat
  • Agilent 1290 Infinity II Multicolumn Thermostat
  • Agilent 1290 Infinity II Variable Wavelength Detector at 280 nm
• Software:
  • Agilent OpenLab CDS v2.5+
  • Agilent Buffer Advisor
• Columns and reagents:
  • Bio MAb NP5, 2.1×250 mm, PEEK
  • LC-grade water, phosphate and salt buffers prepared via Buffer Advisor recipes
• Samples:
  • Trastuzumab (Herceptin) in 30 mM phosphate buffer, pH 6.8
  • Agilent NISTmAb reference standard
• Methods:
  • Quaternary phosphate-based salt and pH gradients
  • Wide-range pH gradients (pH 7–8.4 and pH 8.3–10) with optimized linearity

Results and Discussion

• Comparison of flat salt gradient versus phosphate-buffered pH gradient (pH 7–8.4) for trastuzumab showed slight resolution improvement with pH gradient.
• High reproducibility over seven runs: retention time RSD <0.085%, area RSD <1% for major peaks.
• Wide-range pH gradient (pH 8.3–10) resolved six acidic and two basic variants of trastuzumab with RT RSD ≤0.135% and area RSD ≤5%.
• Modified wide-range pH gradient (pH 8.9–10.5) successfully separated three acidic and two basic variants of NISTmAb.
• Buffer Advisor’s Optimize Gradient function ensures linear pH profiles by inserting additional steps.

Benefits and Practical Applications

• pH gradients reduce buffer preparation time and simplify method development.
• Enhanced resolution allows detection of minor charge variants relevant for QC and biopharmaceutical characterization.
• Reproducible results support robust quality control workflows.

Future Trends and Applications

The integration of dynamic buffer calculators with high-performance LC systems will enable more automated and adaptive chromatographic methods. Further expansion into multi-dimensional separations and real-time pH profiling will accelerate biopharmaceutical analytics and personalized medicine applications.

Conclusion

pH gradient ion-exchange chromatography on the Agilent 1260 Infinity II Prime Bio LC System combined with Buffer Advisor simplifies high-resolution charge variant analysis of monoclonal antibodies, offering superior resolution, reproducibility, and streamlined method development compared to traditional salt gradients.

References

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4. Rea J, et al. J Pharm Biomed Anal. 2011;54:317–323.
5. Lingg N, et al. J Chromatogr A. 2014;1373:124–130.
6. Turner A, Schiel JE. Anal Bioanal Chem. 2018;410(8):2079–2093.
7. Schneider S. Agilent application note 5994-2692EN. 2020.