Enhanced metabolism and negative regulation of ER stress support higher erythropoietin production in HEK293 cells

Rasool Saghaleyni; Magdalena Malm; Noah Moruzzi; Jan Zrimec; Ronia Razavi; Num Wistbacka; Hannes Thorell; Anton Pintar; Andreas Hober; Fredrik Edfors; Veronique Chotteau; Per-Olof Berggren; Luigi Grassi; Aleksej Zelezniak; Thomas Svensson; Diane Hatton; Jens Nielsen; Jonathan L Robinson; Johan Rockberg

doi:10.1016/j.celrep.2022.110936

Enhanced metabolism and negative regulation of ER stress support higher erythropoietin production in HEK293 cells

Rasool Saghaleyni, Magdalena Malm, Noah Moruzzi, Jan Zrimec, Ronia Razavi, Num Wistbacka, Hannes Thorell, Anton Pintar, Andreas Hober, Fredrik Edfors, Veronique Chotteau, Per-Olof Berggren, Luigi Grassi, Aleksej Zelezniak, Thomas Svensson, Diane Hatton, Jens Nielsen, Jonathan L Robinson, Johan Rockberg

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Abstract

Recombinant protein production can cause severe stress on cellular metabolism, resulting in limited titer and product quality. To investigate cellular and metabolic characteristics associated with these limitations, we compare HEK293 clones producing either erythropoietin (EPO) (secretory) or GFP (non-secretory) protein at different rates. Transcriptomic and functional analyses indicate significantly higher metabolism and oxidative phosphorylation in EPO producers compared with parental and GFP cells. In addition, ribosomal genes exhibit specific expression patterns depending on the recombinant protein and the production rate. In a clone displaying a dramatically increased EPO secretion, we detect higher gene expression related to negative regulation of endoplasmic reticulum (ER) stress, including upregulation of ATF6B, which aids EPO production in a subset of clones by overexpression or small interfering RNA (siRNA) knockdown. Our results offer potential target pathways and genes for further development of the secretory power in mammalian cell factories.

Original language	English
Article number	110936
Pages (from-to)	1-14
Number of pages	14
Journal	Cell Reports
Volume	39
Issue number	11
DOIs	https://doi.org/10.1016/j.celrep.2022.110936
Publication status	Published (in print/issue) - 14 Jun 2022

Bibliographical note

Funding Information:
This work was supported by the Knut and Alice Wallenberg Foundation ; AstraZeneca ; Swedish Foundation for Strategic Research (SSF); Swedish innovation agency Vinnova through AAVNova , CellNova , and AdBIOPRO ; and the Novo Nordisk Foundation (grant no. NNF10CC1016517 ). The computations/data handling was enabled by resources provided by the Swedish National Infrastructure for Computing (SNIC), partially funded by the Swedish Research Council through grant agreement no. 2018-05973 .

Funding Information:
This work was supported by the Knut and Alice Wallenberg Foundation; AstraZeneca; Swedish Foundation for Strategic Research (SSF); Swedish innovation agency Vinnova through AAVNova, CellNova, and AdBIOPRO; and the Novo Nordisk Foundation (grant no. NNF10CC1016517). The computations/data handling was enabled by resources provided by the Swedish National Infrastructure for Computing (SNIC), partially funded by the Swedish Research Council through grant agreement no. 2018-05973. Conceptualization, J.N. J.L.R. and J.R.; methodology, R.S. J.Z. N.M. M.M. J.L.R. and J.R.; formal analysis, R.S. J.Z. and M.M.; investigation, R.S. M.M. J.Z. V.C. R.R. N.M. P.-O.B. N.W. H.T. A.P. A.H. F.E. J.L.R. and J.R.; writing – original draft, R.S. J.Z. M.M. J.L.R. and J.R.; writing – review & editing, R.S. J.Z. M.M. N.M. P.-O.B. D.H. J.N. L.G. V.C. J.L.R. and J.R.; visualization, R.S. J.Z. L.G. and J.L.R.; supervision, D.H. T.S. J.N. J.L.R. and J.R. funding acquisition, J.N. and J.R. P.-O.B. is cofounder and CEO of Biocrine AB. L.G. and D.H. are employees of AstraZeneca and may own AstraZeneca stock or stock options.

Publisher Copyright:
© 2022 The Authors

Keywords

ATF6B
CP: Cell biology
CP: Molecular biology
Cell engineering
GFP
HEK293
erythropoietin
protein production
ribosome heterogeneity
secretory pathways

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

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Saghaleyni, R., Malm, M., Moruzzi, N., Zrimec, J., Razavi, R., Wistbacka, N., Thorell, H., Pintar, A., Hober, A., Edfors, F., Chotteau, V., Berggren, P.-O., Grassi, L., Zelezniak, A., Svensson, T., Hatton, D., Nielsen, J., Robinson, J. L., & Rockberg, J. (2022). Enhanced metabolism and negative regulation of ER stress support higher erythropoietin production in HEK293 cells. Cell Reports, 39(11), 1-14. Article 110936. https://doi.org/10.1016/j.celrep.2022.110936

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title = "Enhanced metabolism and negative regulation of ER stress support higher erythropoietin production in HEK293 cells",

abstract = "Recombinant protein production can cause severe stress on cellular metabolism, resulting in limited titer and product quality. To investigate cellular and metabolic characteristics associated with these limitations, we compare HEK293 clones producing either erythropoietin (EPO) (secretory) or GFP (non-secretory) protein at different rates. Transcriptomic and functional analyses indicate significantly higher metabolism and oxidative phosphorylation in EPO producers compared with parental and GFP cells. In addition, ribosomal genes exhibit specific expression patterns depending on the recombinant protein and the production rate. In a clone displaying a dramatically increased EPO secretion, we detect higher gene expression related to negative regulation of endoplasmic reticulum (ER) stress, including upregulation of ATF6B, which aids EPO production in a subset of clones by overexpression or small interfering RNA (siRNA) knockdown. Our results offer potential target pathways and genes for further development of the secretory power in mammalian cell factories.",

keywords = "ATF6B, CP: Cell biology, CP: Molecular biology, Cell engineering, GFP, HEK293, erythropoietin, protein production, ribosome heterogeneity, secretory pathways",

author = "Rasool Saghaleyni and Magdalena Malm and Noah Moruzzi and Jan Zrimec and Ronia Razavi and Num Wistbacka and Hannes Thorell and Anton Pintar and Andreas Hober and Fredrik Edfors and Veronique Chotteau and Per-Olof Berggren and Luigi Grassi and Aleksej Zelezniak and Thomas Svensson and Diane Hatton and Jens Nielsen and Robinson, {Jonathan L} and Johan Rockberg",

note = "Funding Information: This work was supported by the Knut and Alice Wallenberg Foundation ; AstraZeneca ; Swedish Foundation for Strategic Research (SSF); Swedish innovation agency Vinnova through AAVNova , CellNova , and AdBIOPRO ; and the Novo Nordisk Foundation (grant no. NNF10CC1016517 ). The computations/data handling was enabled by resources provided by the Swedish National Infrastructure for Computing (SNIC), partially funded by the Swedish Research Council through grant agreement no. 2018-05973 . Funding Information: This work was supported by the Knut and Alice Wallenberg Foundation; AstraZeneca; Swedish Foundation for Strategic Research (SSF); Swedish innovation agency Vinnova through AAVNova, CellNova, and AdBIOPRO; and the Novo Nordisk Foundation (grant no. NNF10CC1016517). The computations/data handling was enabled by resources provided by the Swedish National Infrastructure for Computing (SNIC), partially funded by the Swedish Research Council through grant agreement no. 2018-05973. Conceptualization, J.N. J.L.R. and J.R.; methodology, R.S. J.Z. N.M. M.M. J.L.R. and J.R.; formal analysis, R.S. J.Z. and M.M.; investigation, R.S. M.M. J.Z. V.C. R.R. N.M. P.-O.B. N.W. H.T. A.P. A.H. F.E. J.L.R. and J.R.; writing – original draft, R.S. J.Z. M.M. J.L.R. and J.R.; writing – review & editing, R.S. J.Z. M.M. N.M. P.-O.B. D.H. J.N. L.G. V.C. J.L.R. and J.R.; visualization, R.S. J.Z. L.G. and J.L.R.; supervision, D.H. T.S. J.N. J.L.R. and J.R. funding acquisition, J.N. and J.R. P.-O.B. is cofounder and CEO of Biocrine AB. L.G. and D.H. are employees of AstraZeneca and may own AstraZeneca stock or stock options. Publisher Copyright: {\textcopyright} 2022 The Authors",

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doi = "10.1016/j.celrep.2022.110936",

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Saghaleyni, R, Malm, M, Moruzzi, N, Zrimec, J, Razavi, R, Wistbacka, N, Thorell, H, Pintar, A, Hober, A, Edfors, F, Chotteau, V, Berggren, P-O, Grassi, L, Zelezniak, A, Svensson, T, Hatton, D, Nielsen, J, Robinson, JL & Rockberg, J 2022, 'Enhanced metabolism and negative regulation of ER stress support higher erythropoietin production in HEK293 cells', Cell Reports, vol. 39, no. 11, 110936, pp. 1-14. https://doi.org/10.1016/j.celrep.2022.110936

TY - JOUR

T1 - Enhanced metabolism and negative regulation of ER stress support higher erythropoietin production in HEK293 cells

AU - Saghaleyni, Rasool

AU - Malm, Magdalena

AU - Moruzzi, Noah

AU - Zrimec, Jan

AU - Razavi, Ronia

AU - Wistbacka, Num

AU - Thorell, Hannes

AU - Pintar, Anton

AU - Hober, Andreas

AU - Edfors, Fredrik

AU - Chotteau, Veronique

AU - Berggren, Per-Olof

AU - Grassi, Luigi

AU - Zelezniak, Aleksej

AU - Svensson, Thomas

AU - Hatton, Diane

AU - Nielsen, Jens

AU - Robinson, Jonathan L

AU - Rockberg, Johan

N1 - Funding Information: This work was supported by the Knut and Alice Wallenberg Foundation ; AstraZeneca ; Swedish Foundation for Strategic Research (SSF); Swedish innovation agency Vinnova through AAVNova , CellNova , and AdBIOPRO ; and the Novo Nordisk Foundation (grant no. NNF10CC1016517 ). The computations/data handling was enabled by resources provided by the Swedish National Infrastructure for Computing (SNIC), partially funded by the Swedish Research Council through grant agreement no. 2018-05973 . Funding Information: This work was supported by the Knut and Alice Wallenberg Foundation; AstraZeneca; Swedish Foundation for Strategic Research (SSF); Swedish innovation agency Vinnova through AAVNova, CellNova, and AdBIOPRO; and the Novo Nordisk Foundation (grant no. NNF10CC1016517). The computations/data handling was enabled by resources provided by the Swedish National Infrastructure for Computing (SNIC), partially funded by the Swedish Research Council through grant agreement no. 2018-05973. Conceptualization, J.N. J.L.R. and J.R.; methodology, R.S. J.Z. N.M. M.M. J.L.R. and J.R.; formal analysis, R.S. J.Z. and M.M.; investigation, R.S. M.M. J.Z. V.C. R.R. N.M. P.-O.B. N.W. H.T. A.P. A.H. F.E. J.L.R. and J.R.; writing – original draft, R.S. J.Z. M.M. J.L.R. and J.R.; writing – review & editing, R.S. J.Z. M.M. N.M. P.-O.B. D.H. J.N. L.G. V.C. J.L.R. and J.R.; visualization, R.S. J.Z. L.G. and J.L.R.; supervision, D.H. T.S. J.N. J.L.R. and J.R. funding acquisition, J.N. and J.R. P.-O.B. is cofounder and CEO of Biocrine AB. L.G. and D.H. are employees of AstraZeneca and may own AstraZeneca stock or stock options. Publisher Copyright: © 2022 The Authors

PY - 2022/6/14

Y1 - 2022/6/14

N2 - Recombinant protein production can cause severe stress on cellular metabolism, resulting in limited titer and product quality. To investigate cellular and metabolic characteristics associated with these limitations, we compare HEK293 clones producing either erythropoietin (EPO) (secretory) or GFP (non-secretory) protein at different rates. Transcriptomic and functional analyses indicate significantly higher metabolism and oxidative phosphorylation in EPO producers compared with parental and GFP cells. In addition, ribosomal genes exhibit specific expression patterns depending on the recombinant protein and the production rate. In a clone displaying a dramatically increased EPO secretion, we detect higher gene expression related to negative regulation of endoplasmic reticulum (ER) stress, including upregulation of ATF6B, which aids EPO production in a subset of clones by overexpression or small interfering RNA (siRNA) knockdown. Our results offer potential target pathways and genes for further development of the secretory power in mammalian cell factories.

AB - Recombinant protein production can cause severe stress on cellular metabolism, resulting in limited titer and product quality. To investigate cellular and metabolic characteristics associated with these limitations, we compare HEK293 clones producing either erythropoietin (EPO) (secretory) or GFP (non-secretory) protein at different rates. Transcriptomic and functional analyses indicate significantly higher metabolism and oxidative phosphorylation in EPO producers compared with parental and GFP cells. In addition, ribosomal genes exhibit specific expression patterns depending on the recombinant protein and the production rate. In a clone displaying a dramatically increased EPO secretion, we detect higher gene expression related to negative regulation of endoplasmic reticulum (ER) stress, including upregulation of ATF6B, which aids EPO production in a subset of clones by overexpression or small interfering RNA (siRNA) knockdown. Our results offer potential target pathways and genes for further development of the secretory power in mammalian cell factories.

KW - ATF6B

KW - CP: Cell biology

KW - CP: Molecular biology

KW - Cell engineering

KW - GFP

KW - HEK293

KW - erythropoietin

KW - protein production

KW - ribosome heterogeneity

KW - secretory pathways

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U2 - 10.1016/j.celrep.2022.110936

DO - 10.1016/j.celrep.2022.110936

M3 - Article

C2 - 35705050

SN - 2211-1247

VL - 39

SP - 1

EP - 14

JO - Cell Reports

JF - Cell Reports

IS - 11

M1 - 110936

ER -

Enhanced metabolism and negative regulation of ER stress support higher erythropoietin production in HEK293 cells

Abstract

Bibliographical note

Keywords

UN SDGs

Access to Document

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