Results

Delayed impact of BSO on growth

In order to study effect of glutathione on recombinant mAb production, GSH was depleted using BSO. A sterile BSO solution was spiked on day 3 of the fed batch culture to a final BSO concentration of 0.5mM. BSO-treated bioreactors and control bioreactors were monitored by daily measurement of viable cell concentration (VCC), intracellular GSH and product titer. Surprisingly, despite a depletion of GSH already observed on day 4, i.e. 24 hours after BSO addition, the growth and production profiles only started to differ from day 6 (Figure 1). The average cell diameter started to increase from day 6 in the BSO condition instead of day 8 in control condition (Figure S1). Furthermore, the decrease in viability was only observed from day 9 in BSO conditions despite a treatment on day 3 (Figure S1). These results suggest that the CHO cell line phenotype was unaffected by glutathione depletion during the three first days after BSO addition. From day 6, their growth rate was gradually reduced and their cellular volume is increasing until day 10. The viable cell concentration decreased after day 10 due to cell death until the end of the culture.

Cysteine supply modulates intracellular GSH and product titer

Since cysteine is a precursor of glutathione, we also investigated if this lower concentration of cysteine influenced the content of intracellular glutathione during the cultivation using a feed (Process 2) containing lower levels of cysteine (Figure 1C). A correlation between the increase of total glutathione and the increase in specific productivity was observed over time.
Except for product titer, no other significant differences between the two feeding strategies were observed in growth profile, proteomic data and metabolites uptake and production rate. As a result, data from the two feeding strategies (processes 1 and 2) were combined to study BSO effect in the next analysis.

BSO treatment affects metabolite secretion and uptake rates

To further characterize the impact of glutathione depletion on CHO cell line metabolism, daily extracellular concentrations of selected metabolites were measured and associated specific uptake and production rate were calculated. Uptake/secretion rates of glucose, lactate and amino acids are shown in Figure 2 and Figure S2. Glucose uptake rates were similar between control and BSO conditions until day 10. Glucose uptake was slightly faster in the BSO condition compared to the control when viability and cell diameter started to decrease. Similar profiles were also observed for histidine, asparagine, and tyrosine uptake rate from day 10. On the contrary, hydroxyproline and aspartic acid were produced/released from this point. These metabolic changes seem to be more related to cell death than to the BSO stress itself. The production of cystine observed from day 8 suggests a cysteine secretion, but high variability was observed for this amino acid in the BSO conditions.
Lactate uptake/secretion rate already started to differ from day 6. Indeed lactate is produced by BSO-treated cells and consumed by non-treated cells. Regarding amino acid uptake/secretion rates, alanine was the only amino acid that displayed a similar profile to lactate in response to the BSO treatment. Since lactate and alanine can be produced from pyruvate, these profiles suggest a failure or slowdown of the TCA cycle.

Proteome related to TCA cycle, GSH metabolism and cholesterol biosynthesis are modulated by BSO treatment

As glutathione metabolism is seemingly linked to the clone productivity, we evaluated the impact of glutathione depletion on host cell protein expression in the studied cell line. For this purpose, we sampled 50x106 cells on day 6 and 10 to perform proteomics analysis using TMT labelling (see Materials and Methods). Across all samples, 3,281 proteins were identified with the identification criteria defined in the material and method section.
Differentially expressed proteins in the BSO-treated culture have been identified using an empirical Bayes moderated t -test (adj. p. value <0.05, LogFC threshold : 0.5). This analysis was done on data from day 6 and day 10, i.e. 3 and 7 days after treatment (Supplementary table S1). 63 proteins were differentially expressed in response to BSO; 47 proteins were down-regulated and 16 proteins were up-regulated. A heatmap of the differentially expressed proteins is shown in Figure 3. No pattern can be observed between data from process 1 versus process 2. The global protein expression was not impacted by a low cysteine supply in contrast to the product titer and intracellular glutathione content. Overall, except for a few proteins, the BSO impact on proteins levels observed on day 6 was amplified on day 10.
In order to identify the cellular functions most impacted by the BSO treatment, enrichment analysis on differentially expressed proteins was performed in Metacore software based on GO annotations and Pathway maps (Supplementary table S2). Based on this results, each cluster identified on the heatmap has been associated with overrepresented functions (Figure 3). In the first cluster, protein expression increased significantly between day 6 and day 10 for the control condition, while proteins expression remained low at day 10 for the BSO condition. This cluster primarily represented cell matrix adhesion proteins. This observation can be associated to cell growth differences observed on day 10 between the two conditions. Likewise, the opposite response observed for CYR61 protein can also be linked to cell growth response as this protein is a known regulator of apoptosis (Lau, 2011).
The largest cluster of proteins were down-regulated after BSO treatment on both days 6 and 10 (Figure 3, Table 1). These included three main functions: cholesterol biosynthesis, carboxylic acid metabolism, and aminoacyl-t-RNA biosynthesis in mitochondria. These cellular processes were interpreted to be at least partially down-regulated in the BSO conditions relative to the control. All detected enzymes involved in cholesterol biosynthesis were down-regulated with an average logFC between -0.34 to -1.21 (Table 1). Interestingly the related transcription factor SREBP2 was also down-regulated but to a lesser extent (logFC: -0.17 / adj. p. value <0.05).
When we specifically considered mitochondrial proteins related to the TCA cycle, a global down-regulation was observed where 12 mitochondrial proteins were observed to have logFC in the interval [-1.09; -0.13] and adj. p. value <0.05 (Table 1). However, glycolysis-related proteins were not differentially expressed (Supplementary table 1).
In the last cluster (Figure 3), the main response to oxidative stress was observed, as this contains the majority of BSO up-regulated proteins, including three proteins were related to heme metabolism. Heme oxygenase had the strongest signal with a logFC of 1.50 and has also been associated with oxidative stress (Hedblom et al., 2019).
Proteins specifically involved in glutathione metabolism can be found in the two last described clusters (Figure 3). More details about proteins detected related to glutathione are presented in Figure 4. Glutathione synthesis-related proteins were overall up-regulated, especially the glutamate cysteine ligase regulatory subunit and the S-formylglutathione hydrolase. In contrast, some proteins involved in the consumption of glutathione, e.g. for the detoxification or catabolism of glutathione, were overall down-regulated. A good example of this was the down-regulation of glutathione S-transferases from the Mu family. However, GSTs from other families (omega, alpha and pi) were not down-regulated (Figure 4).