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The Protein Journal

, Volume 36, Issue 2, pp 112–122 | Cite as

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

  • Manoj Kumar Metta
  • Vasavi Malkhed
  • Srinivasan Tantravahi
  • Uma Vuruputuri
  • Rajkumar Kunaparaju
Article
  • 244 Downloads

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).

Keywords

UT-7 cell line Biological activity In vitro bioassay Recombinant human erythropoietin Procheck and ProSA 

Abbreviations

rHuEPO

Recombinant Human Erythropoietin

EPO

Erythropoietin

G-CSF

Granulocyte Colony Stimulating Factor

GM-CSF

Granulocyte Macrophage Colony Stimulating Factor

IL-3

Interleukin 3

IL-9

Interleukin 9

IGF-1

Insulin Like Growth Factor 1

FBS

Fetal Bovine Serum

BFU-E

Burst Forming Unit-Erythroid

CFU-E

Colony Forming Unit-Erythroid

CFU-GEMM

Colony Forming Unit-Granulocyte, Erythroid, Macrophage, Megakaryocyte

Erythropoietin Receptor

EPO-R

Chinese Hamster Overy

CHO

MTS

(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium).

Notes

Acknowledgements

I am grateful to Usha Biotech and GITAM University for their support in carrying out this research study. I would also like to acknowledge the Head, Department of Chemistry and the Principal, University College of Science, Osmania University for providing the facilities to carry out this bioinformatics study.

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Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  1. 1.Department of Biotechnology, GISGITAM UniversityVisakhapatnamIndia
  2. 2.Usha BiotechHyderabadIndia
  3. 3.Department of Chemistry, University College of Science SaifabadOsmania UniversityHyderabadIndia
  4. 4.Department of Chemistry, University College of ScienceOsmania UniversityHyderabadIndia

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