Assessment of the Effects of Glycosylation on the Pattern and Kinetics of Degradation of Lenograstim in Comparison to Filgrastim Using a Stability-Indicating Orthogonal Testing Protocol

In the biopharmaceutical industry, protein aggregation and/or degradation has profound pathological implications and is encountered routinely during production, shipping, storage and administration. Lenograstim (glycosylated recombinant human granulocyte colony-stimulating factor) was subjected to stress conditions, namely, oxidation, pH, temperature, agitation and repeated freeze–thaw to generate all possible degradation products. An orthogonal stability-indicating testing protocol (RP-HPLC, SE-HPLC, ELISA and SDS-PAGE) was developed and validated for assessment of the pattern and kinetics of aggregation/degradation, under the studied experimental conditions. Results indicated clearly that Lenograstim is susceptible to degradation induced by the studied stress conditions. However, Lenograstim was found relatively more stable than Filgrastim (non-glycosylated recombinant human granulocyte colony-stimulating factor) which was attributed to the effect of glycosylation. Oxidized forms and high molecular weight aggregates of Lenograstim and Filgrastim were detected in all samples subjected to stress conditions to different degrees. ELISA assay and SDS-PAGE results were generally in agreement to those obtained using SE-HPLC assay which confirmed its selectivity to the intact drug. However, formation of soluble aggregates of both drugs was found to occur via physical adsorption and formation of intermolecular disulfide bonds. Results confirmed the need for an orthogonal testing protocol since it was impossible to reveal all types of degradation products using a single technique. Results raised a concern about the efficacy and safety of such sensitive products and highlighted the need for simple tools to inspect biologics for soluble aggregates and sub-visible particles before administration.