Modernizing the Insulin USP Monograph HPLC Method for Assay and Related Compounds

Importance of the Topic

The modernization of USP monograph methods for insulin assay and related compound analysis is critical to reduce analysis time, minimize solvent consumption, and enhance laboratory throughput. Implementing new stationary phase technologies under revised USP <621> guidelines supports sustainable and efficient quality control in biopharmaceutical manufacturing.

Objectives and Study Overview

This study aimed to transfer and modernize the original USP HPLC methods for insulin and human insulin assay (isocratic) and related compounds analysis (gradient) from conventional 5 µm totally porous particle (TPP) C18 columns (4.6×150 mm and 4.6×250 mm) to 2.7 µm superficially porous particle (SPP) Agilent InfinityLab Poroshell 120 SB-C18 and EC-C18 columns (4.6×50 mm and 4.6×100 mm), in compliance with the December 2022 USP <621> guidelines, without revalidation.

Methodology and Instrumentation

All experiments were conducted on an Agilent 1260 Infinity II LC system equipped with a binary pump, multisampler, multicolumn thermostat, and diode array detector using HPLC-grade reagents. Assay mobile phase comprised 0.2 M sodium sulfate buffer (pH 2.3) and acetonitrile (74:26). Gradient analysis used two acetonitrile mixtures (82:18 and 50:50) with the same buffer. Injection volume and flow rate adjustments followed USP <621> equations to maintain plate count or (tR/Wh)² within −25% to +50% of the original method. System suitability criteria included tailing factor ≤1.8, resolution ≥2.0, and peak‐area RSD ≤1.6%.

Main Results and Discussion

• Assay method: Transferred from 4.6×150 mm, 5 µm columns to 4.6×50 mm, 2.7 µm Poroshell columns reduced run time from 15 to 6 min (60% saving) and solvent use proportionally, with injection volume adjusted from 20 to 6.7 µL and flow maintained at 1 mL/min. Plate counts remained within allowable ranges and system suitability metrics were met.
• Related compounds analysis: Original 4.6×250 mm, 5 µm TPP columns were replaced by 4.6×100 mm, 2.7 µm Poroshell SB-C18 and EC-C18 columns. Insulin analysis time decreased from 97 to 39 min and human insulin from 73 to 29 min (60% reduction). Both column chemistries delivered symmetrical peaks with resolution >5, exceeding USP requirements. SB-C18 provided alternative selectivity for impurity separation.

Benefits and Practical Applications

• Significant reduction in analysis time and solvent consumption enhances lab productivity and reduces operating costs.
• Compliance with USP <621> modernization guidelines without additional method validation simplifies method transfer.
• SPP column technology can be implemented on existing HPLC/UHPLC systems, enabling flexible deployment in quality control and process development laboratories.

Future Trends and Potential Applications

Advances in superficially porous particle columns will continue to drive faster and greener separations. Future work may extend this approach to other protein and peptide monographs, integrate with mass spectrometry detection, and explore novel column chemistries. Automation and ultrafast gradient capabilities could further improve throughput and data quality.

Conclusion

The study successfully modernized USP insulin and human insulin assay and related compounds methods by transferring to Agilent InfinityLab Poroshell 120 SPP columns, achieving 60% reduction in run time and solvent use while meeting all USP system suitability criteria. This approach supports sustainable, high-throughput analysis in biopharma quality control.

References

1. USP Harmonized Standards Home Page. Supplement USP Stage 4 Harmonization, Official, 1 December 2022.
2. Fu R.; Joseph M. The Influence of Silica Pore Size and Particle Size on Insulin—a Small Protein Molecule Separation. Agilent Technologies Application Note, 5990-9028EN, 2011.
3. Fu R. Converting a CHP Method of Insulin to Agilent Poroshell 120 Columns. Agilent Technologies Application Note, 5990-9029EN, 2011.
4. Fu R. Converting a CHP Method of Related Compounds Analysis in Human Insulin to Agilent InfinityLab Poroshell 120 Columns. Agilent Technologies Application Note, 5994-4756EN, 2022.
5. USP NF Monographs. USP Insulin, United States Pharmacopeia 3503.
6. USP NF Monographs. USP Insulin Human, United States Pharmacopeia 3506.