Thesis (M.Sc.) - Cairo University - Faculty of Pharmacy - Department of Analytical Chemistry

Biosimilars are biotechnology-derived products that are biologically similar but not identical to an authorized innovator product. Extensive comparability study for the biosimilar product and the reference product is commonly required. An orthogonal testing protocol was developed and validated to assess the quality of Filgrastim biosimilars. Initial screening was carried out using gel electrophoresis and peptide mapping to confirm the product primary structure. CIEF was used to detect the charged impurities. It was optimized using neutrally coated capillaries over a wide-range pH gradient (pH 3.0{u2013}10.0). Impurities above the acceptable limits were detected in both biosimilar products. CIEF also revealed heterogeneity in the active ingredient that has not been investigated by the manufacturers. RP-HPLC was employed for the determination of Filgrastim in the presence of its oxidative degradation products. The assay was accurate and precise over a linear concentration range of 9.38{u2013}300.00 æg/mL. SE-HPLC was carried out over a molecular weight range of 5{u2013}150 kDa to reveal impurities with high molecular weight. The assay was found accurate and precise over a linear concentration range of 20.00{u2013}400.00 æg/mL.Filgrastim oxidized forms and higher molecular weight impurities were detected in the biosimilars. Immunoassay was carried out to determine the protein content and to predict the tertiary structural confirmation. Antigen- antibody reaction was very low in the two biosimilars. Inability to bind could be due to some degree of protein deformation. Finally; spectrophotometric techniques were used to determine the total protein content. Results confirmed the need for in-house validated orthogonal testing protocols to be developed by local regulatory authorities. This should prevent access of substandard biosimilars to price-sensitive markets

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