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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

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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).

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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).

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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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Authors and Affiliations

  1. Department of Biotechnology, GIS, GITAM University, Gandhi Nagar, Rushikonda, Visakhapatnam, Andhra Pradesh, 530045, India

    Manoj Kumar Metta & Srinivasan Tantravahi

  2. Usha Biotech, 62/A, 1st Floor, Sundari Reddy Bhavan, Beside Heritage Fresh, Vengal Rao Nagar, Hyderabad, Telangana, 500038, India

    Rajkumar Kunaparaju

  3. Department of Chemistry, University College of Science Saifabad, Osmania University, Saifabad, Hyderabad, Telangana, 500004, India

    Vasavi Malkhed

  4. Department of Chemistry, University College of Science, Osmania University, Tarnaka, Hyderabad, Telangana, 500007, India

    Uma Vuruputuri

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  1. Manoj Kumar Metta
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Correspondence to Srinivasan Tantravahi.

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Metta, M., Malkhed, V., Tantravahi, S. et al. 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. Protein J 36, 112–122 (2017). https://doi.org/10.1007/s10930-017-9704-3

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