title: Development of an in vitro Bioassay for Recombinant Human Erythropoietin (rHuEPO) Based on Proliferative Stimulation of an Erythroid Cell Line and Analysis of Sialic Acid Dependent Microheterogeneity: UT-7 Cell Bioassay.
abstract: 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).
authors.affiliation: Department of Biotechnology, GIS, GITAM University, Gandhi Nagar, Rushikonda, Visakhapatnam, Andhra Pradesh, 530045, India.
authors.forename: Manoj Kumar
authors.initials: MK
authors.lastname: Metta
authors.affiliation: Department of Chemistry, University College of Science Saifabad, Osmania University, Saifabad, Hyderabad, Telangana, 500004, India.
authors.forename: Vasavi
authors.initials: V
authors.lastname: Malkhed
authors.affiliation: Department of Biotechnology, GIS, GITAM University, Gandhi Nagar, Rushikonda, Visakhapatnam, Andhra Pradesh, 530045, India. saveplants@gmail.com.
authors.forename: Srinivasan
authors.identifier: 0000-0002-3966-8281
authors.initials: S
authors.lastname: Tantravahi
authors.affiliation: Department of Chemistry, University College of Science, Osmania University, Tarnaka, Hyderabad, Telangana, 500007, India.
authors.forename: Uma
authors.initials: U
authors.lastname: Vuruputuri
authors.affiliation: Usha Biotech, 62/A, 1st Floor, Sundari Reddy Bhavan, Beside Heritage Fresh, Vengal Rao Nagar, Hyderabad, Telangana, 500038, India.
authors.forename: Rajkumar
authors.initials: R
authors.lastname: Kunaparaju
mesh_terms: D001681:Biological Assay / Q000379:methods*; D049109:Cell Proliferation / Q000187:drug effects*; D002470:Cell Survival; D041905:Erythroid Cells; D004921:Erythropoietin / Q000737:chemistry* / Q000494:pharmacology*; D006023:Glycoproteins; D006031:Glycosylation; D006801:Humans; D008958:Models, Molecular; D019158:N-Acetylneuraminic Acid / Q000737:chemistry*; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry* / Q000494:pharmacology
chemical_list: C554270:EPO protein, human; D006023:Glycoproteins; D011994:Recombinant Proteins; D004921:Erythropoietin; D019158:N-Acetylneuraminic Acid
keywords: Biological activity; In vitro bioassay; Procheck and ProSA; Recombinant human erythropoietin; UT-7 cell line