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High titer stable production of recombinant erythropoietin in HEK293

Authors

Christine Lin Chin1,4, Justin Bryan Goh1,4, Harini Srinivasan2,3, Kaiwen Ivy Liu3, Ali Gowher3, Raghuvaran Shanmugam3, Hsueh Lee Lim1, Matthew Choo1, Wen Qin Tang1, Andy Hee-Meng Tan1, Terry Nguyen-Khuong1, Meng How Tan2,3 & Say Kong Ng 1

1 Bioprocessing Technology Institute, Agency for Science, Technology and Research (A*STAR), Singapore, Singapore
2 School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore, Singapore
3 Genome Institute of Singapore, Agency for Science, Technology and Research (A*STAR), Singapore, Singapore
4 These authors contributed equally: Christine Lin Chin and Justin Bryan Goh

Published in Scientific Reports 2020 9: 16768 (Online Version)

Abstract

Mammalian cells are typically used to manufacture complex therapeutics and recombinant proteins for the treatment of diseases such as cancer, rheumatoid arthritis, stroke, hemophilia and enzyme deficiencies. The most commonly utilized mammalian cells for this purpose, such as Chinese Hamster Ovary cells, are non-human and can produce glycoproteins with non-human sugar chains attached. These non-human sugar chains can trigger the human immune system to produce undesirable responses against the protein, thereby reducing their therapeutic benefit.

To alleviate this concern, the Human Embryonic Kidney cell line, HEK293, presents a good alternative since it is a human cell line. HEK293 has been approved by regulatory agencies for protein drug manufacture and is better than other mammalian cell lines at increasing the activity of some therapeutic products like Drotrecogin alfa and recombinant factor IX-Fc. HEK293 is also amenable to genetic engineering and has been used to rapidly produce recombinant proteins via transient gene expression. However, stable recombinant protein production in HEK293 has been achieved mainly using non-human genes as selection markers and titers of such a system have been low to moderate. The use of non-human genes will also result in higher expression of non-human proteins. Trace amounts of these proteins can potentially contaminate the final product and trigger allergic and adverse immunogenic reactions to the product.

As such, we developed a fully human expression system based on HEK293 to produce recombinant proteins at high titer. We first engineered the host cell line by knocking out the GLUL (encoding glutamine synthetase) gene using CRISPR-Cas9. Expression vectors using human GLUL as a selection marker were then generated, with recombinant human erythropoietin (EPO) as our model protein. EPO production of up to 92700 U/mL of EPO as analyzed by ELISA or 696 mg/L by densitometry was demonstrated in a 2-L stirred-tank fed-batch bioreactor, reaching one of the highest EPO titers ever reported in literature. We confirmed that the sugar chains on the EPO are fully human and non-human glycan epitopes were not detected. Our xeno-free, high-titer platform may be extended to other complex recombinant proteins beyond EPO.

2020_07 Say Kong Fig 1