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

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

doi:10.1016/j.celrep.2022.110936

Cited by 8 publications

(3 citation statements)

References 88 publications

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“…5), suggesting these cells cannot keep up with the increased energy and raw material demands of recombinant protein production and secretion. Other studies have reported similar metabolic restructuring when comparing cells producing secreted vs. intracellular proteins, implicating increased energy demand of the secretory pathway during recombinant protein production 50 . The strongest metabolic differences we observed involve the metabolism of FAs, which serve as integral constituents of the secretory pathway endomembrane system and as a cell energy source.…”

Section: Discussionmentioning

confidence: 69%

Deciphering the determinants of recombinant protein yield across the human secretome

Masson

Kuo

Lewis

et al. 2022

Preprint

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Mammalian cells are critical hosts for the production of most therapeutic proteins and many proteins for biomedical research. While cell line engineering and bioprocess optimization have yielded high protein titers of some recombinant proteins, many proteins remain difficult to express. Here, we decipher the factors influencing yields in Chinese hamster ovary (CHO) cells as they produce 2165 different proteins from the human secretome. We demonstrate that variation within our panel of proteins cannot be explained by transgene mRNA abundance. Analyzing the expression of the 2165 human proteins with machine learning, we find that protein features account for only 15% of the variability in recombinant protein yield. Meanwhile, transcriptomic signatures account for 75% of the variability across 95 representative samples. In particular, we observe divergent signatures regarding ER stress and metabolism among the panel of cultures expressing different recombinant proteins. Thus, our study unravels the factors underlying the variation on recombinant protein production in CHO and highlights transcriptomics signatures that could guide the rational design of CHO cell systems tailored to specific proteins.

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Section: Discussionmentioning

confidence: 69%

Deciphering the determinants of recombinant protein yield across the human secretome

Masson

Kuo

Lewis

et al. 2022

Preprint

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“…Notably, our analysis highlighted ATF6B as a key regulator of the high expression of FKBP11 in plasma cells. ATF6B has been associated with endoplasmic reticulum function [10]. Combining this finding with the results of the differential expression analysis, we identified 37 target genes that were up-regulated in FKBP11(+) plasma cells, with a significant enrichment in intrinsic apoptotic signaling and protein processing in the endoplasmic reticulum (Figure 11A and 11B).…”

Section: Identification Of Tf-target Gene Pairs Using Scenic Analysis...mentioning

confidence: 77%

Integrative analysis of multi-omics and machine learning highlighted an m6A-related mRNA signature as a robust AAA progression predictor

He,

Xing,

Wang

et al. 2023

Preprint

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ObjectiveAbdominal aortic aneurysm (AAA) is a life-threatening disease in vascular surgery with significant morbidity and mortality rates upon rupture. Despite surgical interventions, effective targeted drugs for non-surgical candidates are lacking. M6A methylation, a dynamic RNA modification, has been implicated in various diseases, but its role in AAA remains poorly understood. In this study, we aimed to explore the participation of M6A in the progression of AAA progression through multi-omics and machine learning.Approach and Resultswe conducted methylated RNA immunoprecipitation with next-generation sequencing (MeRIP-seq) to profile the m6A methylome in AAA tissues, identifying differentially methylated genes (DMGs). Integrating multi-omics data from RNA-sequencing (RNA-seq) in GEO databases, we developed a machine learning-based AAA m6A-related mRNA signature (AMRMS) to predict AAA dilation risk. The AMRMS demonstrated robust predictive performance in distinguishing AAA patients with large AAA and small AAA. Notably, the AMRMS highlighted FKBP11 as a key gene with a significant impact on the predicted model. Subsequent single-cell RNA sequencing (ScRNA-seq) revealed the pivotal role of FKBP11-positive plasma cells in AAA progression.ConclusionsOur study provides novel insights into the regulatory role of m6A modification in AAA pathogenesis, and further develop a promising AMRMS for risk evaluation in AAA patients. Furthermore, the identification of FKBP11 positive plasma cells as significant contributors to AAA progression opens new avenues for targeted therapeutic interventions.

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“…Different products may require different levels of UPR activation as a result of varying metabolic burden (i.e., easy-to-express versus difficult-to-express). For example, both ATF6β and WFS1 were reported as upregulated in erythropoietin (EPO)-producing HEK293 cells, and WFS1 is upregulated during insulin secretion in pancreatic β-cells 31 , 32 , 87 . Further investigation is required to better understand the benefits, or lack thereof, of ATF6β and WFS1 knockdown on a product-specific basis.…”

Section: Discussionmentioning

confidence: 99%

RNASeq highlights ATF6 pathway regulators for CHO cell engineering with different impacts of ATF6β and WFS1 knockdown on fed-batch production of IgG1

Rives,

Peak,

Blenner

2024

Sci Rep

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Secretion levels required of industrial Chinese hamster ovary (CHO) cell lines can challenge endoplasmic reticulum (ER) homeostasis, and ER stress caused by accumulation of misfolded proteins can be a bottleneck in biomanufacturing. The unfolded protein response (UPR) is initiated to restore homeostasis in response to ER stress, and optimization of the UPR can improve CHO cell production of therapeutic proteins. We compared the fed-batch growth, production characteristics, and transcriptomic response of an immunoglobulin G1 (IgG1) producer to its parental, non-producing host cell line. We conducted differential gene expression analysis using high throughput RNA sequencing (RNASeq) and quantitative polymerase chain reaction (qPCR) to study the ER stress response of each cell line during fed-batch culture. The UPR was activated in the IgG1 producer compared to the host cell line and our analysis of differential expression profiles indicated transient upregulation of ATF6α target mRNAs in the IgG1 producer, suggesting two upstream regulators of the ATF6 arm of the UPR, ATF6β and WFS1, are rational engineering targets. Although both ATF6β and WFS1 have been reported to negatively regulate ATF6α, this study shows knockdown of either target elicits different effects in an IgG1-producing CHO cell line. Stable knockdown of ATF6β decreased cell growth without decreasing titer; however, knockdown of WFS1 decreased titer without affecting growth. Relative expression measured by qPCR indicated no direct relationship between ATF6β and WFS1 expression, but upregulation of WFS1 in one pool was correlated with decreased growth and upregulation of ER chaperone mRNAs. While knockdown of WFS1 had negative impacts on UPR activation and product mRNA expression, knockdown of ATF6β improved the UPR specifically later in fed-batch leading to increased overall productivity.

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

Cited by 8 publications

References 88 publications

Deciphering the determinants of recombinant protein yield across the human secretome

Deciphering the determinants of recombinant protein yield across the human secretome

Integrative analysis of multi-omics and machine learning highlighted an m6A-related mRNA signature as a robust AAA progression predictor

RNASeq highlights ATF6 pathway regulators for CHO cell engineering with different impacts of ATF6β and WFS1 knockdown on fed-batch production of IgG1

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