Simple charge variant profile comparison of an innovator monoclonal antibody and a biosimilar candidate

Significance of the Topic

Charge heterogeneity analysis of monoclonal antibodies is essential for ensuring product quality, safety and efficacy. Regulatory approval of biosimilars requires robust comparability studies to demonstrate similarity in charge variant profiles between innovator and biosimilar candidates. A reliable, reproducible separation method simplifies quality control and reduces method development time.

Objectives and Overview of the Article

The primary objective is to showcase a straightforward cation-exchange chromatography (CEX) workflow using a linear pH gradient to profile charge variants of an innovator cetuximab mAb and a biosimilar candidate. The study evaluates method reproducibility, resolution, ease of optimization and suitability for comparability assessment.

Methodology and Instrumentation

A pH gradient from 5.6 to 10.2 was generated using Thermo Scientific CX-1 buffer system and applied on a MAbPac SCX-10 (4 × 250 mm, 10 µm) column. Key components:

• Vanquish Flex Quaternary UHPLC system with Quaternary Pump F, Column Compartment H, Split Sampler FT and Diode Array Detector HT (280 nm)
• Thermo Scientific CX-1 pH Gradient Buffers A (pH 5.6) and B (pH 10.2), diluted 1:10
• Mobile phase flow rate of 1.0 mL/min at 30 °C column temperature
• Sample preparation at 5 mg/mL, with optional carboxypeptidase B digestion for C-terminal lysine variant confirmation

Main Results and Discussion

The optimized shallow pH gradient (Gradient B) resolved ten distinct charge variant peaks for both innovator and biosimilar samples. Compared to the full pH range gradient, the shallow gradient increased resolution of acidic and basic species without extending run time. Retention time precision across six injections showed an average RSD of 0.14 %, ensuring reliable peak identification. Carboxypeptidase B treatment selectively removed peaks corresponding to one or two C-terminal lysine residues, confirming their assignment. Comparison of the two mAbs revealed minor differences in variant distribution, notably in early acidic and basic peaks, illustrating the method’s utility for comparability studies.

Benefits and Practical Applications

This pH-based CEX method offers:

• Rapid and robust charge variant profiling in a single run
• Elimination of complex mobile phase formulation and system corrosion risk
• High retention time precision and reproducible gradient preparation
• Ease of method optimization and broad applicability across diverse mAbs

Future Trends and Applications

Advancements may include coupling with mass spectrometry for direct variant identification, automation of gradient optimization, and integration with data analytics platforms for real-time comparability assessment. Expanding this platform to other protein therapeutics can streamline quality control workflows.

Conclusion

The combination of Thermo Scientific CX-1 pH gradient buffers, MAbPac SCX-10 column and Vanquish Flex UHPLC provides a simple, reproducible and high-resolution approach for charge variant analysis. This workflow supports efficient comparability studies between innovator mAbs and biosimilar candidates, meeting regulatory and quality control requirements.

References

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