Scaling analytical RP-HPLC to semi-preparative for fractionation and characterization of pegfilgrastim oxidized variants.

Pegfilgrastim, a 40 kDa PEGylated form of recombinant human granulocyte colony-stimulating factor (rhG-CSF), is a biotherapeutic protein used to treat chemotherapy-induced neutropenia. To ensure the product is safe and effective, stringent monitoring of product-related impurities, particularly those arising from oxidative degradation, is necessary. This study focuses on the isolation and characterization of oxidized variants in pegfilgrastim using a multi-step approach that includes method transfer to semi-preparative High-Performance Liquid Chromatography (HPLC), mass spectrometry, and an in vitro cell-based potency assay (CBPA). The analytical reversed-phase (RP)-HPLC method was successfully scaled up and optimized for isolating oxidized variants in H2O2-treated pegfilgrastim. Mass spectrometry analysis identified the degree and specific sites of oxidation, with Met1 being the most susceptible. CBPA showed that oxidation at Met1 alone had minimal impact on functional activity, while oxidation at both Met127 and Met138 led to significant reductions in activity. The impact of oxidation at all four sites in pegfilgrastim could not be assessed due to significant degradation. These findings highlight the importance of robust analytical strategies in the characterization and control of pegfilgrastim impurities.