Metta Manoj Kumar, Malkhed Vasavi, Tantravahi Srinivasan, Vuruputuri Uma, Kunaparaju Rajkumar
Department of Biotechnology, GIS, GITAM University, Gandhi Nagar, Rushikonda, Visakhapatnam, Andhra Pradesh, 530045, India.
Department of Chemistry, University College of Science Saifabad, Osmania University, Saifabad, Hyderabad, Telangana, 500004, India.
Protein J. 2017 Apr;36(2):112-122. doi: 10.1007/s10930-017-9704-3.
Determination of biological activity and its comparison with clinical behavior is important in the quality assessment of therapeutic glycoproteins. In vivo studies are usually employed for evaluating bioactivity of these glycomolecules. However, alternative methods are required to simplify the bioassay and avoid ethical issues associated with in vivo studies. Negatively charged sialic acid residues are known to be critical for in vivo bioactivity of rHuEPO. To address this need, we employed the human acute myeloid leukemia cell line UT-7 for the determination of proliferative stimulation induced by rHuEPO. Relative potencies of various intact and sugar-trimmed rHuEPO preparations were estimated using the International Standard for Human r-DNA derived EPO (87/684) as a reference for bioactivity. The cellular response was measured with a multi-channel photometer using a colorimetric microassay, based on the metabolism of the Resazurin sodium by cell viability. For a resourceful probing of physiological features of rHuEPO with significance, we obtained partly or completely desialylated rHuEPO digested by the neuraminidase enzyme without degradation of carbohydrates. Two-fold higher specific activity was shown by asialoerythropoietin in in vitro analysis compared with the sialoerythropoietin. Further, computational studies were also carried out to construct the 3D model of the erythropoietin (EPO) protein structure using standard comparative modeling methods. The quality of the model was validated using Procheck and protein structure analysis (ProSA) server tools. N-glycan units were constructed; moreover, EPO protein was glycosylated at potential glycosylation amino acid residue sites. The method described should be suitable for potency assessments of pharmaceutical formulations of rHuEPO (European Pharmacopeia, 2016).
在治疗性糖蛋白的质量评估中，确定其生物活性并将其与临床行为进行比较非常重要。体内研究通常用于评估这些糖分子的生物活性。然而，需要替代方法来简化生物测定并避免与体内研究相关的伦理问题。已知带负电荷的唾液酸残基对rHuEPO的体内生物活性至关重要。为满足这一需求，我们使用人急性髓系白血病细胞系UT-7来测定rHuEPO诱导的增殖刺激。使用人r-DNA衍生的EPO国际标准品（87/684）作为生物活性参考，估计各种完整和糖修饰的rHuEPO制剂的相对效价。基于刃天青钠的细胞活力代谢，使用比色微量测定法通过多通道光度计测量细胞反应。为了对rHuEPO的生理特征进行有意义的深入探究，我们获得了经神经氨酸酶消化且碳水化合物未降解的部分或完全去唾液酸化的rHuEPO。与唾液酸促红细胞生成素相比，去唾液酸促红细胞生成素在体外分析中显示出两倍高的比活性。此外，还使用标准的比较建模方法进行了计算研究，以构建促红细胞生成素（EPO）蛋白质结构的三维模型。使用Procheck和蛋白质结构分析（ProSA）服务器工具验证了模型的质量。构建了N-聚糖单元；此外，EPO蛋白在潜在的糖基化氨基酸残基位点进行了糖基化。所描述的方法应适用于rHuEPO药物制剂的效价评估（欧洲药典，2016年）。
