A human expression system based on HEK293 for the stable production of recombinant erythropoietin

A human expression system based on HEK293 for the stable production of recombinant erythropoietin
Title:
A human expression system based on HEK293 for the stable production of recombinant erythropoietin
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Scientific Reports
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Publication Date:
14 November 2019
Citation:
Chin, C.L., Goh, J.B., Srinivasan, H. et al. A human expression system based on HEK293 for the stable production of recombinant erythropoietin. Sci Rep 9, 16768 (2019). https://doi.org/10.1038/s41598-019-53391-z
Abstract:
Mammalian host cell lines are the preferred expression systems for the manufacture of complex therapeutics and recombinant proteins. However, the most utilized mammalian host systems, namely Chinese hamster ovary (CHO), Sp2/0 and NS0 mouse myeloma cells, can produce glycoproteins with non-human glycans that may potentially illicit immunogenic responses. Hence, we developed a fully human expression system based on HEK293 cells for the stable and high titer production of recombinant proteins by first knocking out GLUL (encoding glutamine synthetase) using CRISPR-Cas9 system. Expression vectors using human GLUL as selection marker were then generated, with recombinant human erythropoietin (EPO) as our model protein. Selection was performed using methionine sulfoximine (MSX) to select for high EPO expression cells. 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. Mass spectrometry analysis revealed that N-glycosylation of the produced EPO was similar to endogenous human proteins and non-human glycan epitopes were not detected. Collectively, our results highlight the use of a human cellular expression system for the high titer and xenogeneic-free production of EPO and possibly other complex recombinant proteins.
License type:
http://creativecommons.org/licenses/by/4.0/
Funding Info:
Tis work was supported by the Biomedical Research Council (BMRC) of the Agency for Science, Technology and Research (A*STAR), Singapore, and Biological Design Tools and Applications Funding scheme funded by the National Research Foundation (NRF2013-THE001-093), Singapore. Glycan analysis work was supported by the Strategic Positioning Fund (SPF) (“GlycoSing”) from the BMRC of A*STAR and A*STAR’s Joint Council Ofce Visiting Investigator Programme (“HighGlycoART”).
Description:
ISSN:
2045-2322
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