Quantification of a pegylated interferon-alpha2a product by a customised and validated reverse phase-high performance...

Area of Science:

• Biochemistry
• Analytical Chemistry
• Pharmacology

Background:

• Pegylated proteins are increasingly used as therapeutics.
• Accurate quantification of these complex molecules is challenging.
• Existing calibrants may not be suitable for heterogeneous pegylated products.

Purpose of the Study:

• To develop and validate a customized RP-HPLC method for quantifying a specific pegylated interferon alfa-2a product (PEG20-IFN-α2a).
• To assess the suitability of a non-pegylated interferon alfa-2a chemical reference substance (CRS) as a primary calibrant for PEG20-IFN-α2a.
• To ensure accurate mass determination for therapeutic pegylated protein products.

Main Methods:

• Development and validation of a customized RP-HPLC method.
• Analysis of PEG20-IFN-α2a (Reiferon Retard®) using the developed method.
• Quantification of base IFN-α2a molecules by UV absorbance at 210nm.
• Correlation analysis between PEG20-IFN-α2a and IFN-α2a CRS or internal reference substance (IRS).

Main Results:

• The customized RP-HPLC method successfully quantified PEG20-IFN-α2a.
• A high correlation (r² ≥ 0.995) was observed between PEG20-IFN-α2a and IFN-α2a CRS.
• The IFN-α2a CRS was confirmed as a suitable primary calibrant for PEG20-IFN-α2a mass determination.
• The method provided accurate and consistent results for multiple batches of Reiferon Retard®.

Conclusions:

• The customized RP-HPLC method is suitable for the accurate mass determination of PEG20-IFN-α2a.
• A non-pegylated interferon alfa-2a CRS can be effectively used as a primary calibrant for this pegylated therapeutic.
• This validated method supports quality control for current and future batches of Reiferon Retard®.