Converting a ChP Method of Related Compounds Analysis in Human Insulin to Agilent InfinityLab Poroshell 120 Columns

Importance of the Topic

Quality control of human insulin is critical in biopharmaceutical manufacturing and regulatory compliance. Analysis of related peptide variants ensures product purity and safety in line with pharmacopeial standards.

Objectives and Study Overview

This study adapts the China Pharmacopeia (ChP) method for insulin-related compound analysis, originally using 5 µm C18 columns, to faster and more efficient 2.7 µm superficially porous Agilent InfinityLab Poroshell 120 EC-C18 and SB-C18 columns. The main goals were to improve chromatographic performance, reduce run time from over 70 minutes to approximately 20 minutes, and maintain compliance with ChP system suitability criteria.

Methodology and Instrumentation

The evaluation compared:

• Traditional octadecylsilane (C18) columns: 4.6×250 mm, 5 µm (ZORBAX SB-C18 and Eclipse Plus C18).
• Poroshell 120 columns: 3.0×100 mm, 2.7 µm (EC-C18 and SB-C18).

Mobile phases consisted of (A) 0.2 M sulfate in acetonitrile–water (82:18) adjusted to pH 2.3 with phosphoric acid and ethanolamine, and (B) 50:50 acetonitrile–water. Gradients were modified to achieve a ~25-minute retention for insulin on traditional columns and ~20-minute total run on Poroshell columns. The Agilent 1260 Infinity II LC system was used, comprising a quaternary pump, vialsampler, multicolumn thermostat, and diode array detector at 214 nm.

Results and Discussion

Traditional 5 µm columns yielded low efficiency (plate counts ~2,000–3,100) and marginal tailing and resolution for A21-desamido insulin. Poroshell 120 columns significantly increased efficiency (plate counts ~5,500–6,800), improved peak shapes (tailing factors ~1.0–1.2), and enhanced resolution (Rs >1.6). The EC-C18 variant provided the best separation of minor impurities. Analysis time decreased by over 70%, from 73 minutes to 20 minutes. Repeatability tests on EC-C18 showed RSDs below 0.8% for peak area and retention time.

Benefits and Practical Applications

• Substantial reduction in analysis time increases sample throughput.
• Solvent savings lower operating costs and environmental impact.
• Enhanced sensitivity and resolution improve impurity detection.
• Method meets ChP requirements, supporting routine quality control.

Future Trends and Potential Applications

Advances in superficially porous technologies and UHPLC promise further acceleration of peptide separations. Coupling these columns with mass spectrometry could enable more comprehensive profiling of insulin variants. Similar strategies may be applied to other therapeutic proteins and biopharmaceutical analyses.

Conclusion

Conversion of the ChP insulin related-compound method to Agilent InfinityLab Poroshell 120 columns delivers significant gains in efficiency, resolution, and speed while maintaining compliance with pharmacopeial standards. This approach offers a robust solution for high-throughput quality control of insulin.

References

1. China Pharmacopoeia (2020 edition, III), Human insulin, p. 378, 2020.
2. Converting a ChP Method of Insulin to Poroshell 120 columns, Agilent Technologies Application Note, publication 5990-9029EN, 2011.