Collaborative study for the validation of an improved HPLC assay for recombinant IFN-alfa-2

Area of Science:

• Pharmaceutical Sciences
• Analytical Chemistry
• Biotechnology

Background:

• Current European Pharmacopoeia (Ph. Eur.) methods for Interferon (IFN)-alfa-2 rely on nonspecific protein assays and variable cell-based potency tests.
• Official Medicines Control Laboratories (OMCLs) require improved methods for robust batch control and market surveillance of recombinant IFN-alfa-2, including formulations with Human Serum Albumin (HSA).

Purpose of the Study:

• To transfer and validate an improved High-Performance Liquid Chromatography (HPLC) method for the quantitative analysis of IFN-alfa-2 products.
• To enhance the accuracy, reproducibility, and robustness of IFN-alfa-2 batch control and market surveillance testing.

Main Methods:

• A previously developed HPLC method was modified and collaboratively studied (BSP071) by ten laboratories.
• The study utilized marketed IFN-alfa-2 preparations, Ph. Eur. Chemical Reference Substances (CRSs), and in-house standards for quantitative assay.
• Method transferability and performance were assessed across different laboratory equipment and sample types, including HSA-containing products.

Main Results:

• The improved HPLC method demonstrated successful transferability across all participating laboratories.
• Enhanced resolution of IFN-alfa-2 forms (including oxidized) and acceptable peak symmetry were achieved.
• Excellent linearity (R² > 0.9995) was observed for calibration curves using Ph. Eur. CRSs, with good assay repeatability, intermediate precision, and reproducibility.
• Accurate quantification was achieved, with results comparable to manufacturer-declared contents, indicating good test accuracy.

Conclusions:

• The validated HPLC method is suitable, reproducible, and transferable for the quality control of IFN-alfa-2 products.
• The findings support proposals for revising the current Ph. Eur. texts for IFN-alfa-2 testing.
• The method provides a more reliable alternative to existing assays for batch release and market surveillance.