Assessment of recombinant human erythropoietin by alternative bioassay and its correlation with liquid chromatography methods

Abstract

Biotechnology-derived erythropoietin is a sialoglycoprotein, which stimulates erythropoiesis, and it is clinically used for the treatment of anaemia related to chronic renal failure. N-Acetylneuraminic acid content was quantified by a reversed-phase liquid chromatography method with fluorescence detection giving values higher than 108.74 ng μg⁻¹. An alternative in vitro TF-1 cell proliferation bioassay was studied showing a lower mean difference of the estimated potency of 2.67%, compared to the normocythaemic mice bioassay, with non-significant differences (p > 0.05). Biopharmaceutical products were also analyzed by validated reversed-phase and size-exclusion liquid chromatography methods and compared to the in vivo bioassay, showing lower mean differences of the content/potencies of 2.11 and 1.21%, respectively. Higher molecular mass forms and deamidated/sulphoxide forms showed mean bioactivities reduced to about 10%, for both. The TF-1 cell culture assay in conjunction with the determination of sialic acids represents an advance that can be correlated with the normocythaemic mice bioassay and the physicochemical methods, allowing for the establishment of alternative methods, which can be applied to monitor the stability, quality control, and thereby ensuring the therapeutic efficacy of the biological medicine.