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

Saghaleyni, R.; Malm, M.; Zrimec, J.; Razavi, R.; Wistbacka, N.; Chotteau, V.; Hatton, D.; Grassi, L.; Zelezniak, A.; Svensson, T.; Nielsen, J.; Robinson, J. L.; Rockberg, J.

doi:10.1101/2020.09.16.299966

bioRxiv · 10.1101/2020.09.16.299966

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

Saghaleyni, R.·Malm, M.·Zrimec, J.·Razavi, R.·Wistbacka, N.·Chotteau, V.·Hatton, D.·Grassi, L.·Zelezniak, A.·Svensson, T.·Nielsen, J.·Robinson, J. L.·Rockberg, J.

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Abstract

Higher eukaryotic cell lines like HEK293 are the preferred hosts for production of therapeutic proteins requiring human post translational processing. However, recombinant protein production can result in severe stress on the cellular machinery, resulting in limited titre and product quality. To investigate the cellular and metabolic characteristics associated with these limitations, we compared erythropoietin (secretory) and GFP (non-secretory) protein producer HEK293 cell-lines using transcriptomics analysis. Despite the high demand for ATP in all protein producer clones, a significantly higher capacity for ATP production was observed with erythropoietin producers as evidenced by the enrichment of upregulated genes in the oxidative phosphorylation pathway. In addition, ribosomal genes exhibited specific patterns of expression depending on the recombinant protein and the production rate. In a clone displaying a dramatically increased erythropoietin secretion, we detected higher ER stress, including upregulation of the ATF6B gene. Our results are significant in recognizing key pathways for recombinant protein production and identifying potential target genes for further development of secretory power in mammalian cell factories. In BriefAlthough the protein secretion process has been widely studied, the complexity of it leaves many questions with regards to defining bottlenecks for successful protein secretion to be answered. By investigating the transcriptomic profiles of different HEK293 clones with varying translational rates producing either the secreted protein erythropoietin or the intracellular GFP, we reveal that high ATP production and improved capacity of specific post-translational pathways are key factors associated with boosting erythropoietin production. HighlightsO_LITranscriptomics analysis of a panel of HEK293 stable cell lines expressing GFP or erythropoietin (EPO) at varying translational rates C_LIO_LIExpression of mitochondrial ribosomal genes is positively correlated with EPO secretion C_LIO_LIExpression of different cytosolic ribosomal genes are correlated with productivity in a recombinant-protein specific manner C_LIO_LIHigh EPO producing clones have significant upregulation of ATF6B, potentially enabling a beneficial ER stress response to cope with high protein secretion C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/299966v1_ufig1.gif" ALT="Figure 1"> View larger version (87K): org.highwire.dtl.DTLVardef@1e35efforg.highwire.dtl.DTLVardef@71cee2org.highwire.dtl.DTLVardef@1c56d33org.highwire.dtl.DTLVardef@843a6f_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Saghaleyni, R., Malm, M., Zrimec, J., Razavi, R., Wistbacka, N., Chotteau, V., Hatton, D., Grassi, L., Zelezniak, A., Svensson, T., Nielsen, J., Robinson, J. L., Rockberg, J.. 2020-09-16. Transcriptome analysis of EPO and GFP HEK293 Cell-lines Reveal Shifts in Energy and ER Capacity Support Improved Erythropoietin Production in HEK293F Cells. https://doi.org/10.1101/2020.09.16.299966

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