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. 2019 Nov 14;9(1):16768.
doi: 10.1038/s41598-019-53391-z.

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

Christine Lin Chin  1 , Justin Bryan Goh  1 , Harini Srinivasan  2   3 , Kaiwen Ivy Liu  3 , Ali Gowher  3 , Raghuvaran Shanmugam  3 , Hsueh Lee Lim  1 , Matthew Choo  1 , Wen Qin Tang  1 , Andy Hee-Meng Tan  1 , Terry Nguyen-Khuong  1 , Meng How Tan  4   5 , Say Kong Ng  6
Affiliations

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

Christine Lin Chin et al. Sci Rep. .

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.

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Conflict of interest statement

The authors are funded by A*STAR, and access to materials described in the work may be subject to terms stipulated by the agency.

Figures

Figure 1
Figure 1
Generation of HEK293 GLUL knockout (KO) cells. (a) Schematic of the three GLUL isoforms. HEK293 wildtype (WT) cells were transfected with vectors encoding Cas9 and two gRNAs targeting the first constitutive protein-coding exon of the GLUL gene. The target site is indicated with an asterisk. (b) Immunoblots showing the presence of GLUL protein in wildtype cells, but absence of protein in four isolated KO clones, cultivated as adherent cultures. (c) GLUL sequence at the target site. The spacer sequences of the gRNAs are indicated in bold, while the protospacer adjacent motifs (PAMs) of Cas9 from Streptococcus pyogenes (SpCas9) are underlined. The two gRNAs target opposite strands of the genomic DNA. (d) Relative expression of GLUL in WT and KO cells, as assayed by qPCR. Values represent mean ± S.E.M. (*P < 0.05, **P < 0.01 ***P < 0.001; Student’s t-test) (e) Sensitivity of WT and KO cells to glutamine-deficient media. WT cells are indicated by a dotted line, while the four KO clones are indicated by solid colored lines. The cells were grown in adherent culture conditions. Values represent mean ± S.E.M. (f) Immunoblots showing the presence of GLUL protein in wildtype cells, but absence of protein in four isolated KO clones cultivated in suspension culture conditions. (g) Sensitivity of WT and KO cells to glutamine-deficient media. WT is represented in a broken line, while GLUL-KO #7 (square), #20 (circle), #24 (diamond), and #29 (triangle) are depicted in solid lines and symbols. The cells were grown in suspension culture conditions. Values represent mean ± SD.
Figure 2
Figure 2
Transcriptome analysis of HEK293 WT and GLUL-KO cells. (a) PCA of gene expression levels. Four biological replicates were generated for the original WT cells grown under each culture condition (adherent or suspension). (b) Hierarchical clustering of our gene expression data. The heatmap showed that the various samples first separated based on GLUL gene status (i.e. GLUL+/+ or GLUL−/−) and then by culture condition (adherent or suspension). The values of the heatmap are Euclidean distances. (c,d) GO analysis of the genes that were significantly (c) up-regulated or (d) down-regulated in our KO clones when compared to the unmodified WT cells (adjusted P < 0.0001). (e,f) GO analysis of the genes that were significantly (e) up-regulated or (f) down-regulated in suspension cells when compared to adherent cells (adjusted P < 0.0001).
Figure 3
Figure 3
Production and stability of EPO of HEK293 producer cells. GLUL-KO clones were transfected with bicistronic vector expressing human GLUL and human EPO cultured in glutamine-deficient media. Post-methionine sulfoximine (MSX) selection, nine cell pools were generated and characterized for (a) viable cell density and (b) EPO production. Values represent mean ± SD (c) MSX selection was subsequently removed from culture for stability testing over 12 weeks with specific productivity measured at day 4 cultures. (n = 2).
Figure 4
Figure 4
Characterization of EPO expression in GLUL-MSX-mediated HEK293 producer cell pool. (a) Endogenous (light gray bars) and exogenous (dark gray bars) GLUL and EPO genomic DNA copy count analyzed via droplet digital PCR (ddPCR). Values represent mean ± SD (b) Endogenous (light gray bars) and exogenous (dark gray bars) GLUL and EPO mRNA copy count analyzed via ddPCR. Values represent mean ± SD (c) Immunoblot of GLUL and EPO protein in HEK293 wildtype (WT), GLUL-KO (KO #24) and cell pool #8 (CP#8). Actin was used as a loading control.
Figure 5
Figure 5
Productivity of HEK293 EPO producer cell pool in a 2 L fed-batch culture. Cell pool #8 was cultured in 2 L stirred-tank bioreactors over 10 days and characterized daily for (a) viable cell density (VCD, diamonds) and viability (%, squares). (b) EPO production was measured by ELISA over period of culture together with (c) glucose (g/L), (d) glutamine (mM), (e) glutamate (mM), (f) lactate (g/L), (g) ammonia (mM), and (h) osmolality (mOsm/kg).
Figure 6
Figure 6
HEK293-derived EPO site-specific N-glycosylation. Glycopeptides from all three N-glycan sites were acquired with LCMS/MS Orbitrap HCD (normalized collisional energy 30%) and identified using the Byonic software. Label-free quantitation was done using OpenMS/KNIME.
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