Rapid Size Variant Analysis of Monoclonal Antibodies Using UPLC™ and HPLC Compatible MaxPeak™ Premier Protein SEC Columns

Importance of the Topic

Size-exclusion chromatography is essential for detecting and quantifying protein size variants, including aggregates and fragments that can compromise the safety and efficacy of biotherapeutics. Rapid, high-throughput SEC methods accelerate process development, formulation studies, and enable real-time purity monitoring during production.

Objectives and Study Overview

This study evaluated short-column (150 mm) SEC separations of four monoclonal antibodies—three biosimilars (bevacizumab, infliximab, rituximab) and one originator (trastuzumab)—using Waters MaxPeak Premier columns on UPLC and HPLC systems. Over 500 injections, columns were tested at near-maximum pressures to compare analysis times, separation quality, impurity quantification consistency, and column longevity.

Methodology and Instrumentation

Sample Preparation:

• Biosimilar mAbs analyzed neat after freeze–thaw cycles at 10–25 mg/mL concentrations.

Chromatographic System and Detectors:

• ACQUITY UPLC H-Class Bio with column heater.
• ACQUITY UPLC Tunable UV detector (280 nm, 5 mm titanium cell).
• Empower 3 data system.

Columns:

• ACQUITY Premier Protein SEC (250 Å, 1.7 µm, 4.6×150 mm).
• XBridge Premier Protein SEC (250 Å, 2.5 µm) in 4.6×150 mm and 7.8×150 mm formats.

Chromatographic Conditions:

• Mobile phase: sterile-filtered 1× PBS.
• Flow rates: 0.75–2.0 mL/min (analysis times 2.1–3.0 min).
• Column ambient, sample at 6 °C, injection volumes 1–6 µL based on mAb and column format.

Results and Discussion

All columns produced comparable profiles for high molecular weight species (HMWS) and low molecular weight species (LMWS). Key observations included:

• Analysis durations: 2.8 min (ACQUITY 4.6×150 mm), 2.1 min (XBridge 4.6×150 mm), and 3.0 min (XBridge 7.8×150 mm).
• Smaller particle UPLC column exhibited superior HMWS1–monomer resolution; larger 7.8 mm HPLC format reduced system dispersion, enhancing LMWS2 detection.
• Over 500 injections, relative HMWS and LMWS2 levels and HMWS1 resolution remained stable, confirming robust column performance under aggressive flow conditions.
• LMWS1 appeared only as a minor shoulder on trastuzumab and was otherwise negligible.

Benefits and Practical Applications

• Enables rapid purity assessments of mAbs to streamline development and QC workflows.
• Supports real-time monitoring of aggregation and fragmentation during manufacturing.
• Demonstrates column durability for high-throughput testing with minimal performance loss.

Future Trends and Potential Applications

• Reducing system dispersion on HPLC platforms to match UPLC resolution.
• Incorporating inline cleanup or guard columns for early-stage formulations containing particulates.
• Extending rapid SEC approaches to complex biotherapeutics such as bispecifics and fusion proteins.
• Automating SEC assays for real-time release testing in biomanufacturing.

Conclusion

Waters MaxPeak Premier SEC columns in UPLC and HPLC formats deliver fast, reproducible size-variant analysis of monoclonal antibodies with sustained performance over hundreds of high-pressure injections, facilitating efficient biopharmaceutical development and quality control.

Reference

1. Koza SM, Yu YQ. Rapid Size Variant Analysis of Monoclonal Antibodies Using UPLC and HPLC Compatible MaxPeak Premier Protein SEC Columns. Waters Application Note, 2022.
2. Mou X, Yang X, Li H, Ambrogelly A, Pollard DJ. A High Throughput Ultra-Performance Size Exclusion Chromatography Assay for Aggregates and Fragments of Monoclonal Antibodies. Pharm Bioprocess 2014,2(2):141–156.
3. Moussa EM, Panchal JP, et al. Immunogenicity of Therapeutic Protein Aggregates. J Pharm Sci 2016;105(2):417–430.
4. Eon-Duval A, Broly H, Gleixner R. Quality Attributes of Recombinant Therapeutic Proteins. Biotechnol Prog 2012;28(3):608–622.