Optimizing Aggregates Analysis – Mobile Phase

Significance of the Topic

Monoclonal antibodies are key therapeutic agents but are susceptible to aggregation, which can impair potency and safety.
Size exclusion chromatography (SEC) provides a critical tool for quantifying monomer and aggregate species, supporting quality control and method development.

Objectives and Overview

This study aimed to optimize mobile phase ionic strength and pH for SEC separation of the anti-HER2 monoclonal antibody trastuzumab, using a Shim-pack Bio Diol column on a Nexera Bio UHPLC system.

Methodology

Trastuzumab was prepared at 1 mg/mL in water and analyzed under isocratic conditions with 50 mM sodium phosphate buffer supplemented with 100–250 mM NaCl at pH 6.0–7.5.
Chromatographic parameters included a flow rate of 0.4 mL/min, column temperature of 25 °C, a 300 mm × 4.6 mm I.D., 3 μm column, 5 μL injection, and UV detection at 280 nm.

Instrumentation

• Nexera Bio UHPLC system
• Shim-pack Bio Diol-300 SEC column (300 mm × 4.6 mm, 3 μm)
• UV detector set at 280 nm
• Standard consumables (amber vials, solvent bottles, fittings)

Main Results and Discussion

Increasing NaCl concentration reduced electrostatic interactions between trastuzumab and the stationary phase, yielding improved monomer peak symmetry and resolution of high-molecular-weight species.
Optimal separation occurred at 200 mM NaCl and pH 7.0, showing minimal tailing (USP tailing factor ~1.30) and clear aggregate resolution.
Salt concentrations above 200 mM induced hydrophobic interactions, resulting in increased retention time and peak tailing across tested pH values.
pH variation influenced protein conformation and non-ideal interactions, with pH 7.0 balancing ionic suppression and native size distribution.

Benefits and Practical Applications

This optimized SEC method ensures reliable quantitation of trastuzumab monomer and aggregates, supporting process development and quality control in biopharmaceutical workflows.
Its robustness and reproducibility make it suitable for routine aggregate monitoring and formulation screening.

Future Trends and Opportunities

Advancements may include integration of SEC with mass spectrometry for detailed aggregate profiling, development of low-interaction stationary phases, and application of automated high-throughput platforms.
Data-driven optimization using machine learning could further streamline method development and enhance sensitivity.

Conclusion

Mobile phase composition, notably ionic strength and pH, critically affects SEC performance for monoclonal antibody analysis.
The identified optimal conditions on a Shim-pack Bio Diol column provide a reliable approach for aggregate characterization of trastuzumab.

References

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3. Golovchenko N.P., Kataeva I.A., Akimenko V.K. J. Chromatogr., 1992, 591 (1-2), pp 121-128.