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

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.

Rapid and cost-effective development of stable clones for the production of anti-Ebola monoclonal antibodies in HEK293T cells

“…39,[44][45][46] The specific cell productivities observed in our four stable clone cultures (5.33 +/-0.7 pg cell -1 day -1 ) were above the range of values obtained in previous studies that used similar or more complex cell engineering strategies on HEK cells. 35,47,48 These titer, productivity, and specific productivity values are competitive for experimental cultures but still distant (although in the same order of magnitude) from those expected for commercial scale production (approximately 20-40 pg cell -1 day -1 ). 43 We anticipate that the application of standard optimization protocols (i.e., fed-batch feeding protocols) could enhance productivity by at least one order of magnitude.…”

“…43 Further optimization is expected from adaptation of the clones to suspended culture conditions and development of perfusion feeding protocols. These two optimization stages frequently result in a titer enhancement of at least two orders of magnitude 48 . The methodologies presented here can be directly generalized to any other mAb and easily adapted to any other glycosylated protein.…”

“…43 We anticipate that the application of standard optimization protocols (i.e., fed-batch feeding protocols) could enhance productivity by at least one order of magnitude. 49,50 Further enhancement is possible by translating to suspended culture in instrumented bioreactors 48,51 or culturing by continuous perfusion in cell adherent culture systems.…”

Effects of Decomplexation Rates on Ternary Gene Complex Transfection with α-Poly(l-Lysine) or ε-Poly(l-Lysine) as a Decomplexation Controller in An Easy-To-Transfect Cell or A Hard-To-Transfect Cell

“…This fact indicates that the gene release rates of gene complexes could differentially affect cells to express transgenes depending on the cell. HEK293 cells, a human embryonic kidney cell line, have often been used to transfect genes of interest in biological experiments and to produce recombinant proteins due to their high and stable gene expressing capability [ 29 , 30 ]. On the other hand, although HepG2 cells (a human hepatocellular carcinoma cell line) have been used as a well-known and suitable in vitro model of human hepatocytes to understand cellular functions and disease mechanisms [ 31 , 32 ], their gene of interest expression levels are poor.…”

Transcriptome analysis of EPO and GFP HEK293 Cell-lines Reveal Shifts in Energy and ER Capacity Support Improved Erythropoietin Production in HEK293F Cells

“…Although human derived cell-lines benefit from the ability to generate human PTMs, challenges still remain, such as increasing the protein production titer (Chin et al, 2019;Dietmair et al, 2012;Mori et al, 2020) and creating a genetic engineering toolbox with specialized tools for human cells (Xu and Qi, 2019). Recent publications have pursued some of these challenges, including the aim to increase the protein production and secretion power either by cell-line development approaches (Chin et al, 2019;Rahimpour et al, 2013) or cell culture process optimization (Schwarz et al, 2019), as well as to increase the quality of the secreted proteins by engineering folding and PTM pathways (Behrouz et al, 2020;Del Val et al, 2016;Liang et al, 2020;Meuris et al, 2014). However, despite the current knowledge of protein production and secretion in mammalian cells, there is still notable ambiguity in understanding and predicting the production and secretion rates and product quality under different conditions.…”