Understanding Oxidation Propensity in GCSF and Assessment of its Safety and Efficacy.

To understand the impact of methionine oxidation in GCSF on efficacy (neutrophil production/activation) and safety (biochemical and histopathological changes). Nine GCSF biosimilars were analyzed for the levels of residual iron and copper content. Oxidation in GCSF was induced by H2O2 treatment and four samples were prepared: wtGCSF (no oxidation), MetO (1138), MetO (1,138,127) and MetO (1138,127,122). These samples were used to evaluate binding affinity with the GCSF receptor (GCSFR) using biolayer interferometry, thermal stability using circular dichroism and in vitro potency using a relevant cell-based assay. In vivo pharmacodynamics examined changes in neutrophil production upon GCSF methionine oxidation, with the outcome correlated with the differential expression of genes implicated in the GCSF mediated neutrophil activation/ maturation. Pre-clinical safety studies including biochemical and histopathological changes were also performed. Met 122 and Met 127 have the most deleterious effect on the potency. Lower binding affinity with GCSFR was identified as the underlying cause for lower efficacy and potency. Role of Asp 110 in GCSF as the critical residue having adverse impact on efficacy in context of methionine oxidation has been elucidated. Impairment of in vitro binding affinity with GCSF manifests as in vivo pharmacodynamic differences via differential expression of downstream genes required for neutrophil maturation. The data from the present study suggests that methionine oxidation in GCSF is a critical quality attribute that needs careful monitoring and control during commercial manufacturing and subsequent supply chain stages.