USP Monograph mAb SEC Method Robustness on the XBridge Premier Protein SEC Column

Significance of the Topic

Size-exclusion chromatography (SEC) is a cornerstone technique for monitoring size-based variants in monoclonal antibody (mAb) development, quality control and regulatory compliance. Reliable quantification of high molecular weight species (HMWS), monomers and low molecular weight species (LMWS) is critical for ensuring product safety, efficacy and batch-to-batch consistency. Demonstrating method robustness across different column technologies and mobile phase conditions enhances confidence in analytical results and supports streamlined method transfer in pharmaceutical laboratories.

Objectives and Study Overview

This study evaluated the United States Pharmacopoeia (USP) monograph SEC method for mAb size variant analysis using a modern column chemistry. Key goals were to:

• Assess robustness of the USP SEC protocol when run on Waters XBridge Premier Protein SEC 250 Å, 2.5 µm columns.
• Compare quantitative results for HMWS and LMWS against the USP-specified L59 column (5 µm, 250 Å).
• Examine effects of mobile phase pH (6.0–6.4) and ionic strength (200–300 mM KCl) on separation performance.
• Evaluate column-to-column reproducibility across multiple production batches.
• Apply the method to four commercially available mAbs (bevacizumab, infliximab, rituximab, trastuzumab) to demonstrate broader applicability.

Methodology

An ACQUITY UPLC H-Class Bio system coupled with a tunable UV detector was used at 280 nm. The USP gradient employed 200 mM potassium phosphate buffer (pH 6.2) with 250 mM potassium chloride. Flow rates ranged from 0.5 to 1.0 mL/min. Monoclonal IgG system suitability standard and biosimilar samples were injected in duplicate. Data were evaluated for percent peak area of HMWS (target 0.4 %–0.67 %) and LMWS (NMT 0.2 %) per USP General Chapter <129>.

Used Instrumentation

• LC system: ACQUITY UPLC H-Class Bio
• Detector: ACQUITY UPLC Tunable UV (280 nm, 5 mm titanium flow cell)
• Primary column: XBridge Premier Protein SEC 250 Å, 2.5 µm, 7.8 × 300 mm
• Reference column: BioSuite Diol (OH) SEC 250 Å, 5 µm, 7.8 × 300 mm (USP L59)
• Autosampler vials: Polypropylene screw neck with PTFE/Silicone septum

Results and Discussion

Robustness tests on the XBridge Premier column demonstrated consistent HMWS elution at 10.7–12.3 min and LMWS at 15.5–16.3 min under pH 6.0–6.4 and KCl 200–300 mM. All conditions met USP criteria (n=2). Comparative runs on the USP L59 column produced similar quantitation but lower resolution of HMWS and LMWS. Column-to-column reproducibility across three XBridge batches showed negligible variation in percent peak area. Analysis of four mAbs confirmed consistent HMWS and LMWS values across columns; a higher flow rate (1 mL/min) yielded similar results, enabling potential throughput gains. In contrast, the USP 5 µm column failed to resolve certain LMWS peaks reliably.

Practical Benefits and Applications

• The XBridge Premier Protein SEC column is USP listed and directly interchangeable with the L59 specification.
• Enhanced resolution of size variants improves confidence in impurity quantitation.
• Robust performance across mobile phase variations reduces method revalidation needs.
• Column interchangeability supports streamlined method transfer in regulated environments.
• Potential two-fold increase in sample throughput via higher flow rates.

Future Trends and Opportunities

Advances in column surface chemistries and particle technologies will further minimize secondary interactions, enhancing accuracy for challenging protein formats. Integration of SEC with native mass spectrometry or multi-angle light scattering will enable simultaneous size and mass characterization. Automated high-throughput platforms and real-time data analytics may streamline in-process control of biologics. Expansion of standardized monographs to novel biotherapeutics will drive demand for universally robust SEC methods.

Conclusion

The USP monograph SEC method for mAb size variant analysis demonstrates excellent robustness and reproducibility when implemented on Waters XBridge Premier Protein SEC columns. Comparable or superior resolution, tolerance to mobile phase variation and compatibility with higher flow rates make this column an effective starting point for method development and quality control across diverse monoclonal antibody products.

Reference

1. USP General Chapter <129> Analytical Procedures for Recombinant Therapeutic Monoclonal Antibodies. United States Pharmacopeia. 2017.
2. Hong P.; Koza S. M.; Fountain K. J. Analysis of Proteins by Size-Exclusion Chromatography Coupled With Mass Spectrometry Under Non-denaturing Conditions. Waters Application Note 720004254EN. 2012.