MaxPeak Premier Protein SEC 250 Å Column Lifetimes at Physiological pH for Polysorbate (Tween) Formulated Biosimilar Monoclonal Antibodies

Significance of the Topic

Protein aggregation in therapeutic monoclonal antibodies (mAbs) poses a risk of immunogenic responses in patients. Formulation with non-ionic surfactants such as polysorbate (Tween 20 or Tween 80) is a standard approach to prevent self-association and surface adsorption. Evaluating size-exclusion chromatography (SEC) column stability under physiological pH and ionic strength is essential to ensure accurate monitoring of high molecular weight species (HMWS) and low molecular weight species (LMWS) impurities in formulated biosimilars.

Objectives and Study Overview

This study assesses the lifetime performance of three SEC columns—Waters XBridge Premier Protein SEC (250 Å, 2.5 µm), ACQUITY Premier Protein SEC (250 Å, 1.7 µm), and ACQUITY Protein BEH SEC (200 Å, 1.7 µm)—over 500 consecutive analyses. Four biosimilar or originator mAb products (bevacizumab, infliximab, rituximab, trastuzumab) formulated with either Tween 20 or Tween 80 were used to evaluate column robustness at physiological pH (~7.4) and ionic strength (~150–225 mM).

Methods and Instrumentation

The analyses were performed on an ACQUITY UPLC H-Class Bio system equipped with a CH-30A APH column heater and an ACQUITY UPLC TUV detector (280 nm, 214 nm). Mobile phase was Dulbecco’s phosphate-buffered saline (DPBS) at target ionic strength; the BEH column used 1.5× DPBS. Columns and sample handling were designed to minimize particulate fouling, including 0.1 µm sterile filtration of mobile phases and chilled sample storage at 6 °C. Injection volumes and flow rates were optimized per column and mAb to ensure optimal resolution of HMWS and LMWS.

Main Results and Discussion

All three columns maintained consistent retention times, resolution, and quantitative measurements of HMWS and LMWS across 500 injections. Peak-to-valley ratios for the early-eluting LMWS1 fragment remained stable, indicating negligible loss in column efficiency. Minor variations in HMWS abundance for certain early timepoints were attributed to sample handling rather than column degradation. The diol-bonded BEH column also demonstrated robust performance, suggesting that proper filtration and sample preparation mitigate surfactant-induced fouling.

Benefits and Practical Applications

• Enables direct analysis of therapeutic mAbs in physiologically relevant buffer systems without acidic modifiers.
• Supports high-throughput monitoring of aggregate and fragment impurities over extended column usage.
• Provides reproducible quantification of HMWS and LMWS during method development and routine QC.

Future Trends and Opportunities

Further improvements may include integration of guard columns to protect against larger particulates and exploration of novel particle chemistries that enhance surfactant tolerance. Advances in column hardware and heated sample loops could expand the operational pH range and enable faster workflows for next-generation biotherapeutics.

Conclusion

This lifetime study confirms that Waters XBridge Premier, ACQUITY Premier, and BEH Protein SEC columns can reliably separate and quantify protein aggregates and fragments in polysorbate-formulated mAbs under physiological conditions for more than 500 injections. Proper mobile phase filtration and sample handling are key to sustaining long-term column performance.

Reference

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6. Koza SM et al. Impact of LC System Dispersion on SEC Analysis of mAbs. Waters App Note 720006336. 2019.
7. Waters Brochure. Guide to SEC of mAb Aggregates, Monomers, and Fragments. 720006067. 2020.