The recombinant human interferon α-2a (rhIFN α-2a) is a cytokine with antiviral, antiproliferative and immunomodulatory properties, indicated for the treatment of hepatitis B and C and leukemias. The rhIFN α-2a consists of a 165166 amino acids with molecular mass of 19.5 kDa. The aim of this work was to develop and validate the reversed-phase (RP-LC) and size-exclusion (SE-LC) liquid chromatography methods for the physico-chemical characterization of rhIFNα-2a in pharmaceutical formulations. The RP-LC method was carried out on a Jupiter C4 column (250×4.6 mm I.D.) with detection at 214 nm. The mobile phase A consisted of water with 0.1% TFA, and the mobile phase B was acetonitrile with 0.1% TFA. The SE-LC method was performed on a BioSep-SEC-S 2000 column (300×7.8 mm I.D.), using the mobile phase with 0.001 M monobasic potassium phosphate, 0.008 M dibasic sodium phosphate and 0.2 M sodium chloride buffer pH 7.4, with detection at 214 nm. The procedure was applied for the potency evaluation of rhIFN α-2a, dimers and higher molecular mass substances. The separation by the RP-LC method was obtained with the retention time of 32.6 min and the method was linear in the range of 0.550 MIU/ml (r2=0.9999). The SE-LC method yielded results with quantitation limit of 0.5 MIU/ml and detection limit of 0.19 MIU/ml. Eight batches of pharmaceutical formulations were analyzed in parallel by RP-LC and SE-LC methods giving results respectively, within 91.53100.75%, with sulphoxides and deamidates content (<0.78%), and within 91.53104.56% of the stated potency, with dimers and aggregates lower than 0.21%. The results were correlated to the anti-proliferative cell-based assay demonstrating the validity of the methods which can contribute to improve the quality control of rhIFN α-2a in pharmaceutical formulations, and to assure the therapeutic efficacy and safety of the biotherapeutic.
25 - 29 Apr 2009
European Society of Endocrinology