METHOD DEVELOPMENT OF IONIC-STRENGTH GRADIENT CATION EXCHANGE CHROMATOGRAPHY FOR MONOCLONAL ANTIBODY CHARGE VARIANT ANALYSIS

Significance of the topic

Charge heterogeneity in therapeutic monoclonal antibodies can affect their efficacy and safety. Ionic-strength gradient cation exchange chromatography (CEX) is widely applied to resolve and quantify charge variants, ensuring reliable quality control and supporting biopharmaceutical development.

Objectives and overview of the study

This work aimed to develop and optimize an ionic-strength gradient CEX method for monoclonal antibody charge variant analysis. The study evaluated the effects of key parameters—mobile phase pH, salt concentration range, gradient time, flow rate, temperature, organic additives, column length and internal diameter—using trastuzumab, adalimumab, bevacizumab and cetuximab as model compounds.

Methodology and instrumentation

• Sample preparation: mAbs diluted in water to 1–5 mg/mL and analyzed post expiry.
• Chromatography system: Waters ACQUITY UPLC H-Class Bio.
• Columns: BioResolve SCX mAb (4.6×50 mm, 4.6×100 mm, 2.1×50 mm).
• Mobile phases: 100 mM MES monohydrate/sodium salt, 1 M NaCl, water.
• Gradient delivery: AutoBlend Plus with typical salt ramp from 0 to 700 mM NaCl over defined times.
• Detection: UV at 280 nm; injection volumes 0.2–2 µL; flow rate 0.8 mL/min unless varied; sample at 10 °C, column at 30 °C.

Main results and discussion

• Mobile phase pH: Lower pH enhanced acidic variant resolution but compromised basic peaks, requiring an optimal compromise.
• Salt concentration slope: Narrowing initial/final NaCl concentrations yielded shallower gradients and higher peak-to-valley ratios.
• Gradient time: Extending run time improved resolution until a plateau was reached beyond which no further gains occurred.
• Flow rate: At constant gradient volume, higher flow slightly reduced resolution due to dispersion; at constant time, increased flow produced shallower slopes and improved separation.
• Temperature: Affected retention times marginally with minimal impact on selectivity, highlighting the importance of temperature control for reproducibility.
• Organic modifiers: Addition of isopropanol caused minor retention shifts but did not alter selectivity, indicating low hydrophobic interaction with the stationary phase.
• Column geometry: Longer columns significantly boosted resolution compared with longer gradients on shorter columns; narrow-bore columns required delay volume correction to match performance.

Benefits and practical applications

• Enables precise characterization of mAb charge heterogeneity for release testing and stability studies.
• Supports method transfer and routine monitoring in QA/QC laboratories.
• Provides parameter guidelines for robust, reproducible separations adaptable to various mAbs.

Future trends and potential applications

• Integration of pH and ionic-strength gradients to further enhance resolution.
• Automation and high-throughput method optimization using design-of-experiments or AI-driven approaches.
• Development of novel stationary phases to minimize non-specific interactions.
• Extension to other protein therapeutics and profiling during process development.

Conclusion

Optimizing ionic-strength gradient CEX parameters—pH, gradient slope, flow rate and column configuration—is crucial for resolving monoclonal antibody charge variants. Controlled temperature and minimal hydrophobic interactions contribute to robust, reproducible analytical methods.

References

• Khawli LA et al. Charge variants in IgG1. mAbs 2(6):613–624 (2010).
• Lauber MA et al. Designing a new particle technology for robust charge variant analysis of mAbs. Waters Application Note 720006475EN (2018).