Expression of Human Erythropoietin Containing 2 Additional N-Link in CHO-K1 Cells under Different Culture Conditions

Adi Santoso, Larasati Larasati, Arizah Kusumawati, Popi Hadi Wisnuwardhani, Ratih Asma Ningrum, Endah Puji Septisetyani

Abstract


Human erythropoietin (hEPO) is a glycoprotein that regulates the formation of erythrocytes and mainly used in anemia patients. Previously, we have reported the expression of modified human EPO with 2 additional N-linked in mammalian cell CHO-K1. The aim of this current research was to study the optimum condition for modified recombinant hEPO (rhEPO) production in CHO-K1. To do this, several parameters of culture conditions were applied including antibiotic concentrations, seeding densities, time of incubations, fetal bovine serum (FBS) concentrations and cell culture media. The result showed that the presence of antibiotic G418 improved the expression level with the highest was at 1% of concentration. Meanwhile, seeding density of 2–3x105 cells/6 cm dish and seven day of incubation time were the best condition for rhEPO protein expression. From five different combination media used, F12 medium with 10% FBS gave the highest expression of rhEPO protein. From this study was also found that at passage 16 the expression level was still increasing proving that the clone expressing the protein of our interest is promisingly stable.

Keywords : EPO, erythropoietin, protein expression, CHO-K1, optimation


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References


Blackstone, A.E. and Fuhr Jr, J.P., 2013, The Economics of Biosimilar, Am. Health Drug. Benefits, 6(8), 469-478. Link

Bussow, K., 2015, Stable Mammalian Producer Cell Lines for Structural Biology, Curr. Opinion in Struct. Biol, 32, 81-90. CrossRef

Carrillo, C.L., Genel Rey, M.T., Hernandez, D.A., Pacheco, F.L., Meza, J.L., Pizana, M.R.R., et al., 2015, Amino Acid Consumption in Naıve and Recombinant CHO Cell Cultures: Producers of a Monoclonal Antibody, Cytotechnology, 67(5), 809-820. CrossRef

Chung, I.J., Dai, C. and Krantz, S.B., 2003, Stem Cell Factor Increases The Expression of FLIP That Inhibits IFN Gamma Induced Apoptosis in Human Erythroid Progenitor Cells, Blood, 101, 1324 1328. CrossRef

D'Andrea, D.A., Lodish, H.F. and Wong, G.G., 1989, Expression Cloning of the Murine Erythropoietin Receptor, Cell, 57(2), 277-285. CrossRef

Declerck, P., Danesi, R., Petersel, D. and Jacobs, I., 2017, The Language of Biosimilars: Clarification, Definitions, and Regulatory Aspects, Drugs, 77, 671–677. CrossRef

Egrie, J.C. and Browne, J.K., 2001, Development and Characterization of Novel Erythropoiesis Stimulating Protein (NESP), Br. J. Cancer, 84(S1), 3-10. CrossRef

Egrie, J.C., Strickland, T.W., Lane, J., Aoki, K., Cohen, A.M., Smalling, R., et al., 1986, Characterization and Biological Effects of Recombinant Human Erythropoietin, Immunobiology, 172, 213–224. CrossRef

Feng, L., Vijayasankaran, N., Shen, A., Kiss, R. and Amanullah, A., 2010, Cell Culture Processes for Monoclonal Antibody Production, mAbs, 2(5), 466-477. CrossRef

Fontes, J., Paschoal, B., Patiño, S.S., Bernardino, T., Rezende, A., Lemos, M., et al., 2014, Adaptation to Serum free Culture of HEK 293T and Huh7.0 Cells, BMC Proceeding, 8(S4), P259. CrossRef

Freshney, R.I., 2010, Serum-Free Media. In: Freshney R.I. ed., Culture of Animal Cells. Hoboken, NJ: John Wiley & Sons, Inc. pp. 115-132. CrossRef

Fried, W., 1972, The Liver as a Source of Extrarenal Erythropoietin Production, Blood, 40, 671-677. Link

Kasper, C., Terhaar, A., Fossa, A., Welt, A., Seeber, S. and Nowrousian, M.R., 1997, Recombinant Human Erythropoietin in The Treatment of Cancer-related Anaemia, Eur. J. Haematol., 58, 251-216. PubMed

Katherine, H., Tkaczuka, R. and Jacobsb, I.A., 2014, Biosimilars in Oncology: From Development to Clinical Practice, Semin. Oncol., 41(2), S3-S12. CrossRef

Krantz, S.B., 1991, Eryhropoietin, Blood, 77(43), 419-434. Link

Kunert, R. and Reinhart, D., 2016, Advances in Recombinant Antibody Manufacturing, Appl. Microbiol. Biotechnol., 100, 3451-3461. CrossRef

Lacombe, C. and Mayeux, P., 1998, Biology of Erythropoietin, Haematologica, 83(8),724-732. Link

Leng, H.M., Albrecht, C.F., Kidson, S.H. and Folb, P.I., 1999, Erythropoietin Production in Anemia Associated with Experimental Cancer, Exp. Hematol., 27, 806-810. CrossRef

Matasci, M., Hacker, D.L., Baldi, L., Wurm, F.M., 2008, Drug Discovery Today, Technologies, 5, 37-42. CrossRef

Reinhart, D., Kaisermayer, C., Damjanovic, L. and Kunert, R., 2013, Benchmarking of Commercially Available CHO Cell Culture Media for Antibody Production, BMC Proceedings, 7(S6), pp. 13. CrossRef

Rice, L., Alfrey, C.P., Driscoll, T., Whitley, C.E., Hachey, D.L. and Suki, W., 1999, Neocytolysis Contributes to The Anemia of Renal Disease, Am. J. Kidney Dis., 33, 59-62. CrossRef

Rolfe, M.D., Rice, C.J., Lucchini, S., Pin, C., Thompson, A., Cameron, A.D.S., et al., 2012, Lag Phase Is a Distinct Growth Phase That Prepares Bacteria for Exponential Growth and Involves Transient Metal Accumulation, J. Bacteriol., 194(3), 686-701. CrossRef

Santoso, A., Septisetyani, E.P., Meiyanto, E., Dyaningtyas, D.P.P. and Ningrum, R.A., 2014, Expression of Modified Recombinant Human Erythropoietin in CHO-K1 Cells and Its In-vitro Proliferation Assay in TF-1 Cells, Indonesian J. Pharm., 25(1), 9-16. CrossRef

Schroder, M., Matischak, K. and Friedl, P., 2004, Serum and Protein Free Media Formulations for The Chinese Hamster Ovary Cell Line DUKXB11, J. Biotechnol., 108, 279-292. CrossRef

Septisetyani, E.P., Rubiyana, Y., Wisnuwardhani, P.H., Wardiana, A. and Santoso, A., 2012, Expression of Recombinant Human Erythropoietin with Glycosylation Modification in HEK293T cells, Indonesian J. Pharm., 3, 177-182.

">CrossRef

Shahdadfar, A., Frønsdal, K., Haug, T., Reinholt, F.P. and Brinchmanna, J.E., 2005, In Vitro Expansion of Human Mesenchymal Stem Cells: Choice of Serum Is a Determinant of Cell Proliferation, Differentiation, Gene Expression, and Transcriptome Stability, Stem Cells, 23, 1357-1366. CrossRef

Tatsuma, Y. and Asayama, Y., 2017, Animal-Cell Culture Media: History, Characteristics, and Current Issues, Reprod. Med. Biol., 16, 99-117. CrossRef

Valk, J.V.D., Bieback, K., Buta, C., Cochrane, B., Dirks, W.G., Fu, J., et al., 2017, Fetal Bovine Serum (FBS): Past – Present – Future, Altex, 35(1), 99-118. CrossRef

Wu, H., Liu, X., Jaenisch, R. and Lodish, H.F., 1995, Generation of Committed Erythroid BFU-E and CFU-E Progenitors Does Not Require Erythropoietin or The Erythropoietin Receptor, Cell, 83(1), 59-67. CrossRef

Yin, H. and Blanchard, K.L., 2000, DNA Methylation Represses The Expression of The Human Erythropoietin Gene by Two Different Mechanisms, Blood, 95, 111-119. Link

Zanjani, E.D., Ascensao, J.L., McGlave, P.M., Banisadre, M., Ash, R.C., 1981, Studies on The Liver to Kidney Switch of Erythropoietin Poduction, J. Clin. Invest., 67, 1183-1188. CrossRef

Zhang, H., Wang, H., Liu, M., Zhang, T., Zhang, J., Wang, X. and Xiang, W., 2013, Rational Development of a Serum-Free Medium and Fed-Batch Process for a GS-CHO Cell Line Expressing Recombinant Antibody, Cytotechnology, 65, 363-378. CrossRef

Zhao, F., Wang, Y., Liu, L. and Bian M., 2017, Erythropoietin for Cancer-Associated Malignant Anemia: A Meta-Analysis, Mol. Clin. Oncol., 6, 925-930. CrossRef

Zhou, H., Weir, M.D. and Xu, H.K., 2011, Effect of Cell Seeding Density on Proliferation and Osteo Differentiation of Umbilical Cord Stem Cells on Calcium Phosphate Cement-Fiber Scaffold, Tissue Eng., 17(21-22), 2603-2613. CrossRef




DOI: http://dx.doi.org/10.14499/indonesianjcanchemoprev10iss1pp7-15

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