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

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.

Evaluation and use of disaccharides as energy source in protein-free mammalian cell cultures.

Mammalian cells are generally considered to be unable to utilize polysaccharides for cell growth because the phospholipid bilayer in the cell membrane has very low permeability to sugars. With the recent discovery of the only known animal disaccharide transporter, a sucrose transporter, we considered the potential use of polysaccharides as energy source, because that can impact biopharmaceutical manufacturing by potentially increasing carbohydrate loading in the culture medium and decreasing lactate accumulation. In this study, we found that mammalian cells can utilize maltose for growth in the absence of glucose and successfully adapted CHO-K1, CHO-DG44 and HEK293 cells to grow in glucose-free, maltose-containing serum-free protein-free media. We then cultivated a non-adapted CHO-K1 producer cell line in media containing both glucose and maltose to show that the cells can utilize maltose in a biphasic manner, that maltose enters the cells, and that maltose utilization only took place in the presence of the cells. This is the first report of a protein-free mammalian cell culture using a disaccharide as energy source.

Engineering selection stringency on expression vector for the production of recombinant human alpha1-antitrypsin using Chinese Hamster ovary cells.

BACKGROUND: Expression vector engineering technology is one of the most convenient and timely method for cell line development to meet the rising demand of novel production cell line with high productivity. Destabilization of dihydrofolate reductase (dhfr) selection marker by addition of AU-rich elements and murine ornithine decarboxylase PEST region was previously shown to improve the specific productivities of recombinant human interferon gamma in CHO-DG44 cells. In this study, we evaluated novel combinations of engineered motifs for further selection marker attenuation to improve recombinant human alpha-1-antitrypsin (rhA1AT) production. Motifs tested include tandem PEST elements to promote protein degradation, internal ribosome entry site (IRES) mutations to impede translation initiation, and codon-deoptimized dhfr selection marker to reduce translation efficiency. RESULTS: After a 2-step methotrexate (MTX) amplification to 50 nM that took less than 3 months, the expression vector with IRES point mutation and dhfr-PEST gave a maximum titer of 1.05 g/l with the top producer cell pool. Further MTX amplification to 300 nM MTX gave a maximum titer of 1.15 g/l. Relative transcript copy numbers and dhfr protein expression in the cell pools were also analysed to demonstrate that the transcription of rhA1AT and dhfr genes were correlated due to the IRES linkage, and that the strategies of further attenuating dhfr protein expression with the use of a mutated IRES and tandem PEST, but not codon deoptimization, were effective in reducing dhfr protein levels in suspension serum free culture. CONCLUSIONS: Novel combinations of engineered motifs for further selection marker attenuation were studied to result in the highest reported recombinant protein titer to our knowledge in shake flask batch culture of stable mammalian cell pools at 1.15 g/l, highlighting applicability of expression vector optimization in generating high producing stable cells essential for recombinant protein therapeutics production. Our results also suggest that codon usage of the selection marker should be considered for applications that may involve gene amplification and serum free suspension culture, since the overall codon usage and thus the general expression and regulation of host cell proteins may be affected in the surviving cells.