Overexpression of transcription factor Foxa1 and target genes remediate therapeutic protein production bottlenecks in Chinese hamster ovary cells

Abstract

Despite extensive research conducted to increase protein production from Chinese hamster ovary (CHO) cells, cellular bottlenecks often remain, hindering high yields. In this study, a transcriptomic analysis led to the identification of 32 genes that are consistently upregulated in high producer clones and thus might mediate high productivity. Candidate genes were associated with functions such as signaling, protein folding, cytoskeleton organization, and cell survival. We focused on two engineering targets, Erp27, which binds unfolded proteins and the Erp57 disulfide isomerase in the endoplasmic reticulum, and Foxa1, a pioneering transcription factor involved in organ development. Erp27 moderate overexpression increased production of an easy-to-express antibody, whereas Erp27 and Erp57 co-overexpression increased cell density, viability, and the yield of difficult-to-express proteins. Foxa1 overexpression increased cell density, cell viability, and easy- and difficult-to-express protein yields, whereas it decreased reactive oxygen species late in fed-batch cultures. Foxa1 overexpression upregulated two other candidate genes that increased the production of difficult- and/or easy-to-express proteins, namely Ca3, involved in protecting cells from oxidative stress, and Tagap, involved in signaling and cytoskeleton remodeling. Overall, several genes allowing to overcome CHO cell bottlenecks were identified, including Foxa1, which mediated multiple favorable metabolic changes that improve therapeutic protein yields.

Keywords: CHO cells; Foxa1 transcription factor; cell viability; protein folding; recombinant protein production.

Figure 1

Transcriptomic analysis of genes associated with CHO cell high productivity. (a) Identification of upregulated transcripts in trastuzumab (Tras) high producer clones when compared to parental CHO cells and to the Tras polyclonal cell pool using RNA‐seq. Fifty‐one mRNAs, corresponding to 32 genes, were found to be upregulated, relative to the two control cell populations, with a log2 fold change of >0.5 and a p value less than .05. Three biological replicates were used for the Tras high producer clones, whereas three technical replicates (i.e., independent cultures) were used for parental CHO cells and for the Tras polyclonal cell pool. (b) Ontological analysis of the functional classes of the 32 genes upregulated in Tras high producer clones. Functions of noncoding RNAs (ncRNAs) are unknown. (c) Heatmap showing the expression profiles of the candidate genes identified as depicted in Panel a. Expression levels of the candidate genes were also assessed in cells producing at a high level the easy‐to‐express bevacizumab antibody (n = 1) or the difficult‐to‐express interferon β therapeutic protein (n = 2). Expression levels are shown as the natural logarithm of the fold change, relative to the parental CHO cells. ncRNAs were named according to Table 1. Arhgap42 expression was only detected in the Tras high producer clones. CHO, Chinese hamster ovary; mRNA, messenger RNA

Figure 2

Effect of Erp27 and/or Erp57 overexpression on the production of therapeutic proteins. Clones producing easy‐ or difficult‐to‐express therapeutic proteins were stably transfected with Erp27 or Erp57 expression vectors, or cotransfected with both Erp27 and Erp57 expression vectors. Gene expression, cell growth, cell viability, and protein production were evaluated in fed‐batch cultures in stable polyclonal populations (Panels b–e) or in clones (Panels f–h) of the Erp27‐ and/or Erp57‐overexpressing cells or of the control cells. (a) Quantification of Erp27 and Erp57 mRNA levels in the parental trastuzumab (Tras) clone, represented as fold change relative to their levels in the nontransfected parental CHO cells at Days 0 and 8 of fed‐batch cultures, as assessed by qRT‐PCR. Error bars are shown as SD, n = 3. (b) The parental Tras clone was stably transfected with the Erp27 and/or Erp57 expression vectors, and the titers of the secreted Tras antibody were determined from cell culture supernatants at the end of fed‐batch cultures. Cells transfected with a GFP expression vector were used as control. Error bars are shown as SD, n = 3. (c) The parental Tras clone was stably transfected with decreasing amounts of the Erp27 expression vector (1,800, 600, 200, and 66 ng) together with an empty vector to keep the total amount of plasmid constant. Cells transfected with an empty vector plasmid were used as control. Tras titers were determined at the end of fed‐batch cultures. Error bars are shown as SD, n = 3. (d) An infliximab producer clone was characterized in terms of the secreted monoclonal antibody titers obtained during fed‐batch cultures using either the parental clone or derived cell populations obtained after transfection with the Erp27 and/or Erp57, or with the GFP expression vector, as indicated. Titers are illustrated as Tukey box‐and‐whisker diagram with median values (middle bar) and 25–50% and 50–75% quartiles (box). Whiskers extend to the lowest and highest values still within the 1.5‐fold interquartile range. (e) Viable cell density of the fed‐batch cultures analyzed in Panel d. Error bars are shown as SD. n ≥ 4 for Panels d and e. (f) An etanercept producer clone was stably transfected with the Erp27 and Erp57 expression vectors, or with an empty vector as control. Cell colonies were isolated using a ClonePix device, and the clones with the highest etanercept secretion halos were isolated and characterized for the etanercept titer at the end of fed‐batch cultures. The titer fold change relative to control cells is illustrated as Tukey box‐and‐whisker diagram as for Panel d. (g and h) Viable cell density and cell viability of the fed‐batch cultures analyzed in Panel f. The error bars represent the SEM. n ≥ 8 for Panels f–h. mRNA, messenger RNA; qRT‐PCR, quantitative reverse transcription‐polymerase chain reaction; SD, standard deviation; SEM, standard error of mean

Figure 3

Effect of Foxa1 overexpression on trastuzumab (Tras) production. The parental Tras clone was stably transfected with the Foxa1 or GFP expression vector. (a) The Tras titers of the resulting polyclonal populations were determined after 10 days of fed‐batch cultures. Viable cell density (b) and cell viability (c) were evaluated throughout fed‐batch cultures. n = 5 for Panels a–c. Titers are illustrated as a Tukey box‐and‐whisker diagram as described for Figure 2, whereas error bars are shown as SD (Panels b and c). (d) An RT‐qPCR analysis of the mRNA levels of Foxa1 target genes and other relevant genes identified in Figure 1 was performed on Foxa1‐overexpressing cells, GFP‐expressing cells, or the parental Tras clone at Day 8 of the fed‐batch culture. Error bars are shown as SD, n = 3. Arhgap42 expression could not be detected in these samples. (e) RT‐qPCR quantification of Foxa1, Ca3, Rassf9, and Tagap mRNA levels in Foxa1‐overexpressing cells, GFP‐expressing cells, or in the parental Tras clone at Day 0 of the fed‐batch. Error bars are shown as SD, n = 3. (f) Evaluation of intracellular ROS levels using carboxy‐H₂DCFDA in Foxa1‐overexpressing cells and in parental Tras clone at Days 0, 3, 6, 8, and 9 of the fed‐batch cultures. Error bars are shown as SD, n = 3. mRNA, messenger RNA; qRT‐PCR, quantitative reverse transcription‐polymerase chain reaction; ROS, reactive oxygen species; SD, standard deviation

Figure 4

Effect of Ca3, Rassf9, and Tagap overexpression on trastuzumab (Tras) production. The parental Tras clone was stably transfected with the Ca3, Rassf9, Tagap, or GFP expression vector. (a) The Tras titers of the resulting polyclonal populations were determined after 10 days of fed‐batch cultures. Viable cell density (b) and cell viability (c) were evaluated throughout fed‐batch cultures. Error bars are shown as SD. n ≥ 3 for Panels a–c. (d) Quantification of the mRNA levels of candidate genes by RT‐qPCR analyses in Ca3‐, Rassf9‐ or Tagap‐expressing stable polyclonal populations. Data are presented relative to the mRNA levels in control GFP‐expressing cells. Error bars are shown as SD, n = 3. (e) The Tras clone was stably transfected with various amounts of the Ca3 expression vector (1,800, 600, 200, and 66 ng) together with an empty vector to keep the total amount of plasmid constant. The Tras titers obtained from these polyclonal populations were assessed at the end of fed‐batch cultures. Error bars are shown as SD, n = 3. mRNA, messenger RNA; qRT‐PCR, quantitative reverse transcription‐polymerase chain reaction; SD, standard deviation

Figure 5

Effect of Foxa1 overexpression on infliximab production. (a) The parental infliximab clone was stably transfected with the Foxa1 or GFP expression vector, and the infliximab titers of the resulting polyclonal populations were determined after 9 days of fed‐batch cultures. Viable cell density (b) and cell viability (c) were evaluated throughout fed‐batch cultures. n = 5 for Panels a–c. Titers are depicted as described for Figure 2 (Panel a), whereas error bars are shown as SD (Panels b and c). (d) Evaluation of intracellular ROS levels using carboxy‐H₂DCFDA for Foxa1‐overexpressing cells and for the parental infliximab‐producing clone at Days 3, 6, 7, and 8 of the fed‐batch cultures. Error bars are shown as SD, n = 3. (e) RT‐qPCR quantification of Foxa1, Ca3, Rassf9, and Tagap mRNA levels in Foxa1‐overexpressing cells, GFP‐expressing cells or in the parental infliximab clone at Day 6 of the fed batch. Error bars are shown as SD, n = 3. mRNA, messenger RNA; qRT‐PCR, quantitative reverse transcription‐polymerase chain reaction; SD, standard deviation

Figure 6

Effect of Tagap overexpression on infliximab production. (a) The parental infliximab clone was stably transfected with the Tagap or GFP expression vector, and the infliximab titers of the resulting polyclonal populations were determined after 9 days of fed‐batch cultures. Viable cell density (b) and cell viability (c) were evaluated throughout fed‐batch cultures. n = 4 for Panels a–c. Titers are illustrated as described in Figure 2. Error bars are shown as SD for Panels b and c. (d) RT‐qPCR quantification of Foxa1, Ca3, Rassf9, and Tagap mRNA levels in Tagap‐overexpressing cells, GFP‐expressing cells or in the parental clone at Day 6 of the fed batch by RT‐qPCR. Error bars are shown as SD, n = 3. mRNA, messenger RNA; qRT‐PCR, quantitative reverse transcription‐polymerase chain reaction; SD, standard deviation