Modernizing Compendial SEC Methods for Biotherapeutics Using the Alliance™ iS Bio HPLC System

Importance of the Topic

Size-exclusion chromatography (SEC) remains a cornerstone in the quality control of biotherapeutics, providing critical insights into protein aggregation and size variants. As regulatory bodies place growing emphasis on method lifecycle management, modernizing legacy compendial methods can deliver enhanced resolution, faster runtimes, and reduced solvent consumption without requiring full revalidation.

Objectives and Study Overview

This study evaluated the migration and modernization of the United States Pharmacopeia General Chapter 129 SEC method for monoclonal antibodies using the Alliance iS Bio HPLC System. A direct method transfer was performed alongside two scaled adaptations following USP General Chapter 621 guidance. Performance metrics were compared against a traditional HPLC platform to assess improvements in throughput, resolution, and operational robustness.

Methodology and Instrumentation

The investigation employed both a legacy HPLC system and the next-generation Alliance iS Bio HPLC System. Key features and consumables included

• BioSuite Diol SEC column (250 Å, 5 μm, 7.8 × 300 mm) for the original compendial protocol
• XBridge Premier Protein SEC columns (250 Å, 2.5 μm) in two formats: 7.8 × 150 mm and 4.6 × 150 mm for modernized methods
• Bio-inert flow path materials (titanium, PEEK, MP35N) and MaxPeak High Performance Surfaces to minimize non-specific adsorption
• Mobile phase composed of 0.20 M potassium phosphate and 0.25 M potassium chloride at pH 6.2
• Detection at 280 nm via tunable UV detector
• Empower Chromatography Data System with Intelligent Method Translator App for error-reduced method migration

Main Results and Discussion

Direct migration using the Intelligent Method Translator yielded comparable system suitability on both instruments, with the Alliance iS Bio HPLC System exhibiting increased resolution of monomer and low molecular weight species, facilitating more accurate impurity quantitation. Method modernization with the 7.8 × 150 mm column reduced runtime fourfold (30 to 7.5 minutes) while preserving selectivity and revealing previously undetected impurity peaks. The 4.6 × 150 mm column delivered a sixfold reduction in solvent and sample usage, demonstrating significant cost and waste reduction potential.

Benefits and Practical Applications

• Substantial reduction in analysis time and mobile phase consumption
• Enhanced resolution and sensitivity toward size variants
• Bio-inert flow path ensures reproducible analysis of metal-sensitive proteins
• Pre-run checks and touchscreen control minimize transcription errors and operational downtime

Future Trends and Opportunities

Ongoing advancements in column chemistry and system automation will further streamline SEC workflows. The integration of artificial intelligence for predictive maintenance and real-time method adjustment is anticipated. Continued harmonization of pharmacopeial guidance will support flexible method scaling and expedite adoption of high-throughput bioanalytical platforms.

Conclusion

The Alliance iS Bio HPLC System effectively modernizes compendial SEC methods, delivering up to 75 percent faster analysis, over 80 percent less solvent use, and superior resolution of protein size variants. Enhanced software tools and bio-inert design collectively empower laboratories to meet stringent regulatory expectations while maximizing operational efficiency.

References

1. USP. Chromatography <621>. In: USP-NF. Rockville, MD: USP; Dec 1, 2022.
2. Analytical Procedures for Recombinant Therapeutic Monoclonal Antibodies. USP General Chapter <129>. 2022.
3. Aggregation Analysis Using SE-HPLC and SE-UHPLC Methods in USP Chapter <129>. USP Application Note. 2023.
4. Kizekai L, Shiner SJ, Lauber MA. Waters ACQUITY and XBridge Premier Protein SEC 250 Å Columns: A New Benchmark in Inert SEC Column Design. Waters Application Note. 2022.