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 mg À1 . 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.

Evaluation of an In Vitro Cell Culture Assay for the Potency Assessment of Recombinant Human Erythropoietin

“…The assay was based on the growth-promoting activity of the factor-dependent human cell line, TF-1 (ATCC CRL-2003), with a modification of the method described elsewhere (8,10). The cells were maintained in RPMI 1640 culture medium, supplemented with 10% (v/v) fetal bovine serum (Sigma-Aldrich), in 75cm 2 flasks.…”

Reversed-phase high performance liquid chromatography as an alternative to animal bioassay for human thyrotropin potency determination

“…9 RP-HPLC and HPSEC were also shown to be useful alternatives to the normocythaemic mice bioassay for erythropoietin potency assessment, the estimated content/ potency determined by these methods being respectively 2% and 1% lower than those determined by in vivo bioassay, a difference that is statistically non-signicant. 10 Given the current interest in reducing and/or rening the currently required animal bioassays, the present work reports an RP-HPLC methodology that represents a potential alternative to the in vivo bioassay for testing the potency of human thyrotropin (hTSH). This hormone is a heterodimeric glycoprotein formed by two non-covalently linked subunits whose association is mandatory for expression of the biological activity.…”

Determinação de potência de diferentes preparações de foliculotrofina, luteotrofina e tireotrofina: comparação entre a quantificação por cromatografia líquida em fase reversa e por bioensaio in vivo

“…Da mesma forma, um método de cromatografia líquida de alta eficiência em fase reversa (RP-HPLC) em combinação com HPSEC mostrou correlação significativa com os bioensaio in vivo para avaliar a potência do fator estimulador de colônia de granulócito humano recombinante (rhG-CSF) (Dalmora, 2006) e do hormônio recombinante da paratireóide (rhPTH) (Stamm, 2013).…”

“…A substituição de bioensaios por métodos físico-químicos no caso de glicoproteínas é particularmente difícil, principalmente levando em conta a complexidade destas moléculas e a heterogeneidade da sua glicosilação. RP-HPLC, bem como HPSEC, foram propostos como uma possível alternativa para o bioensaio em camundongos normocitêmicos para a determinação da potência de eritropoietina humana recombinante (rhEPO) (Barth, 2007;Ferreto, 2009;Schutkoski, 2013 (Driebergen, 2003;Bassett, 2005). (Hartree, 1992;Galet, 2004).…”