Evaluation and application of salt- and pH-based ion-exchange chromatography gradients for analysis of therapeutic monoclonal antibodies

Importance of the Topic

Monoclonal antibodies are complex therapeutics that frequently exhibit charge microheterogeneity due to enzymatic and chemical modifications. Accurate charge variant profiling is critical for ensuring safety, efficacy, and regulatory compliance during development and manufacturing of both innovator and biosimilar mAbs.

Goals and Study Overview

The study evaluated a ready-to-use pH gradient buffer system (Thermo Scientific CX-1) combined with MAbPac SCX-10 columns on a Vanquish Flex UHPLC to simplify IEX analysis of charge variants. Objectives included demonstrating improved reproducibility, faster method optimization, and broad applicability across multiple therapeutic mAbs compared to conventional salt-based gradients.

Methodology and Instrumentation

Ion-exchange chromatography was conducted with three buffer systems: phosphate-based salt gradients, MES-based salt gradients, and pH gradients generated either by four-solution quaternary mixing or CX-1 pH buffer kits. A Thermo Scientific Vanquish Flex UHPLC equipped with a quaternary pump, diode array detector, and a MAbPac SCX-10 column (4×250 mm, 10 µm) was used. Carboxypeptidase B digestion assigned peaks corresponding to C-terminal lysine variants.

Main Results and Discussion

Reproducibility tests revealed significant retention time shifts for salt buffers prepared on different days, whereas MES salts showed minor variation. CX-1 pH gradient buffers delivered the highest reproducibility and resolved up to 12 charge variants versus 10 with salt gradients. Comparison with quaternary mixing showed similar separation but required complex pump tuning and longer optimization. Application to bevacizumab, cetuximab, infliximab, and trastuzumab using a full pH 5.6–10.2 gradient followed by shallow pH range adjustments (<1.5 h optimization) enhanced variant resolution. Enzymatic digestion confirmed assignment of basic peaks to C-terminal lysine residues in three mAbs and absence of such variants in trastuzumab.

Benefits and Practical Application

• Rapid, generic method development for most therapeutic mAbs.
• Superior reproducibility and reduced buffer preparation variability.
• Enhanced resolving power and peak capacity for detailed charge profiling.

Future Trends and Opportunities

Integration with automated method development tools and coupling with mass spectrometry are expected to further expedite and deepen charge variant characterization. Extension of the platform to bispecific antibodies, antibody-drug conjugates, and other complex biotherapeutics will broaden its utility in both biopharma development and QC laboratories.

Conclusion

The CX-1 pH gradient buffer system paired with MAbPac SCX-10 columns on a Vanquish Flex UHPLC offers a robust, easily optimized, and widely applicable approach for mAb charge variant analysis, outperforming traditional salt gradients in reproducibility and resolution.

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