Size Exclusion Chromatography of Biosimilar and Innovator Insulin

Importance of the Topic

This summary addresses the critical role of size exclusion chromatography (SEC) in the analysis of biopharmaceutical insulin products. Accurate separation and quantification of monomeric insulin, its aggregates, and related impurities ensure product safety, efficacy, and regulatory compliance in both innovator and biosimilar formulations.

Objectives and Study Overview

The primary objective was to develop and validate a robust SEC-UV method for distinguishing innovator and biosimilar insulin analogs. Emphasis was placed on:

• Establishing system suitability and method precision
• Determining sensitivity (LOD/LOQ) and linear response range
• Detecting high-molecular-weight impurities and preservative peaks
• Demonstrating column lot-to-lot consistency and extended lifetime

Materials and Methods

Samples consisted of commercial innovator and biosimilar insulins stored under recommended conditions. The mobile phase combined anhydrous acetic acid, acetonitrile, and water (200:300:400 mL), adjusted to pH 3.0 with ammonia, and diluted to 1,000 mL. A 10 µL injection volume, isocratic flow of 0.5 mL/min, and UV detection at 276 nm were employed. System suitability and calibration were evaluated using triplicate injections across nine concentration levels (10.6–3,400 µg/mL). Aggregates were generated by heating insulin at 60 °C for 6 hours before analysis.

Instrumentation Used

• Agilent 1260 Infinity Bio-inert Quaternary LC Pump (G5611A)
• Agilent 1260 Infinity Bio-inert HPLC Autosampler (G5667A)
• Agilent 1200 Infinity Thermostat (G1330B)
• Agilent 1260 Infinity Thermostatted Column Compartment with bio-inert heating elements (G1316C, option 19)
• Agilent 1260 Infinity DAD VL detector (G1315D)
• Agilent AdvanceBio SEC column, 130 Å, 7.8×300 mm, 2.7 µm (p/n PL1180-5350)

Main Results and Discussion

Separation and Detection

• Monomeric insulin eluted at ~16.45 min with a symmetric peak and RSD < 0.06% (RT) and < 0.7% (area).
• m-Cresol preservative appeared at ~49.54 min, confirming its clear baseline resolution.

System Suitability

• Symmetry factor for insulin peaks was ≤ 1.72 (limit ≤ 2.0).
• Peak-to-valley ratio and total impurities (< 0.3% of total area) met draft pharmacopeial criteria.

Method Sensitivity

• LOD and LOQ for innovator insulin were determined at 11.3 and 28 µg/mL, respectively, with S/N > 3 and > 10.

Linearity

• Calibration across 10.6–3,400 µg/mL yielded R² = 0.9999, indicating excellent dose-response correlation.

Aggregate Analysis

• Heat-induced insulin aggregates eluted at 11.18 and 13.88 min, demonstrating the column’s ability to resolve high-molecular-weight species.

Column Reproducibility and Lifetime

• Batch-to-batch evaluation using protein markers across four lots showed consistent retention times and peak shapes.
• Over 250 injections of 3 mg/mL insulin exhibited negligible drift in RT, area, tailing, and plate count, highlighting long column lifetime.

Benefits and Practical Applications of the Method

• Reliable differentiation of innovator vs. biosimilar insulin for QA/QC and regulatory submissions.
• Sensitive impurity and aggregate detection ensures patient safety.
• Bio-inert construction minimizes sample adsorption and metal chelation artifacts.
• Extended column lifetime reduces operational costs and downtime.
• High reproducibility supports routine batch release and stability studies.

Future Trends and Perspectives

Advancements in SEC media and bio-inert system design will further improve the detection of subvisible aggregates and trace impurities. Integration with high-resolution mass spectrometry can enable simultaneous molecular weight confirmation. Automation and rapid-screening formats may accelerate throughput for biopharma process development and real-time QC testing.

Conclusion

The validated SEC-UV method using the Agilent AdvanceBio SEC 130 Å column provides a robust, sensitive, and reproducible approach to characterize monomeric insulin, aggregates, and preservatives in both innovator and biosimilar products. Its demonstrated system suitability, linearity, and extended column performance make it well suited for routine quality control, method transfer, and regulatory support in biopharmaceutical development.

References

1. Kannan V; Narayanaswamy P; Gadamsetty D; Hazra P; Khedkar A; Iyer H. A tandem mass spectrometric approach to the identification of O-glycosylated glargine glycoforms in active pharmaceutical ingredient expressed in Pichia pastoris. Rapid Commun. Mass Spectrom. 2009, 23(7), 1035–1042.
2. European Pharmacopoeia. Pharmeuropa Vol. 23, No. 2, April 2011.