Method Modernization for Routine Analysis of Biotherapeutics as Part of Lifecycle Management

Significance of the Topic

This application note addresses the need to modernize routine analytical methods for small biotherapeutics, using insulin analogues as a model. Traditional USP monographs rely on dated HPLC techniques that are time-consuming and resource intensive. By updating these methods, laboratories can improve throughput, reduce solvent consumption and waste, and maintain compliance with regulatory requirements for product quality and safety.

Objectives and Study Overview

The main goal of the study was to demonstrate how a USP monograph–based identity test for insulin and its analogues can be modernized. Insulin human, insulin lispro, and insulin glargine were selected to compare the original HPLC methods with UPLC-based approaches. The work aimed to develop a unified, high-throughput “platform” method applicable to multiple insulin analogues, while preserving the resolution and selectivity required for reliable identity testing.

Methodology and Instrumentation

The study followed USP monograph digestion protocols using endoproteinase Glu-C to generate peptide fragments. Two method development paths were compared:

• Scaled USP method: Sub-2-µm column in a UPLC system with geometrically scaled gradient.
• Developed method: UPLC column with 1.7 µm CSH C18 packing and 0.1% TFA as ion-pairing reagent for a 10 min gradient.

The following equipment was employed:

• ACQUITY UPLC H-Class Bio System
• ACQUITY TUV Detector (λ = 215 nm, 5 mm flow cell)
• ACQUITY UPLC CSH C18 1.7 µm, 2.1 × 100 mm Column
• Empower 3 Software

Key Results and Discussion

Using the scaled USP method reduced insulin human analysis time from 60 to 30 min with similar selectivity and resolution. The developed UPLC method further shortened the gradient to 10 min, cutting run time by 75% while maintaining baseline separation of four diagnostic peptide fragments. When applied to insulin lispro and glargine, the platform method consistently resolved fragment peaks despite minor sequence variations, confirming its robustness and specificity.

Benefits and Practical Applications

The modernized UPLC approach offers:

• Up to 75% reduction in analysis time
• Reduced solvent consumption and waste
• Consistent performance across multiple insulin analogues
• Maintained compliance with USP and ICH guidelines

This platform method can streamline identity testing in quality control labs and support lifecycle management of biotherapeutics.

Future Trends and Potential Applications

Further enhancements may include integrating mass spectrometry for enhanced specificity, automating sample preparation workflows, and extending the platform to larger protein therapeutics. Continuous advancement in UPLC column technology and detectors will drive even faster, more sensitive assays for biopharmaceutical analytics.

Conclusion

The study successfully modernized USP monograph identity tests for insulin analogues by adopting a sub-2-µm UPLC method with TFA ion-pairing, achieving significant time savings and consistent resolution. The approach establishes a versatile platform for routine QC analysis of small biotherapeutics.

Reference

• Guidance for Industry Q10 Pharmaceutical Quality System. ICH, 2009.
• Human Insulin Market. Zion Market Research, 2016.
• USP Monograph: Insulin Human [11061-68-0], USP 39–NF 34, 2015 Revision Bulletin.
• USP Monograph: Insulin Lispro, USP 39–NF 34, 2015 Revision Bulletin.
• USP Monograph: Insulin Glargine [160337-95-1], USP 39–NF 34, 2016 Revision Bulletin.
• Hsu J. et al. Identification of Recombinant Insulin Analogues by Peptide Mapping Method. J. Food Drug Anal., 2012;20(4):957–962.
• <621> Chromatography, US Pharmacopeia, USP 38–NF33 S1.