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).