Results and
Discussion
CHO cells produce the growth-inhibitory metabolites isovalerate,
isobutyrate and 2-methylbutyrate through catabolism of leucine,
isoleucine and valine. Previously, we have reported the generation of
CHO cell clones with KO of the BCAT1 gene from an antibody producing
wildtype (WT) cell line, Cell Line B (Mulukutla et al., 2019). The names
of these clones are BCAT1 KO Clone 83, 86 and 90. To probe the impact of
lactate control on performance of BCAT1 KO clones, Clone 83, Clone 90
and WT cells were cultivated in fed-batch cultures and HiPDOG cultures.
All process parameters were kept the same between fed-batch and HiPDOG
conditions, except for residual glucose levels (see Materials and
Methods). Nutrient feed was delivered such that amount of feed added on
a per cell basis was relatively constant across all conditions.
In non-HiPDOG fed-batch cultures, residual glucose levels were
maintained above 2 g/L throughout the culture with the exception of day
3, where glucose levels fell to 1 g/L in WT conditions (Figure 1). The
BCAT1 KO clones (83 and 90) had comparable growth rates to the WT in the
initial few days of the culture but achieved 3 - 7 x
106 cells/mL higher peak cell densities than the WT
cells. Viability profiles were similar across all conditions. Lactate
profiles were also similar across the conditions, with WT cultures
peaking slightly higher than BCAT1 KO cultures. The lactate peak across
all conditions was within a relatively high concentration range (6.5 - 8
g/L), but a good metabolic shift to lactate consumption was observed
across all conditions. Notably, the BCAT1 KO cultures had higher titers
than the WT, attributable to higher specific productivity
(qP ) in the BCAT1 KO cultures when compared with
the WT cultures.
For HiPDOG cultures, the HiPDOG control strategy was operational from
day 2 to 6, during which residual glucose levels were maintained in the
0.01 - 0.2 g/L range. After ending the HiPDOG control strategy, residual
glucose levels were maintained above 2 g/L (Figure 2). BCAT1 KO clones
(83 and 90) and WT cells had very similar growth rates for the initial
four days of the culture. Subsequently WT growth rates slowed, and these
cultures peaked at roughly 21 × 106 cells/mL on day 9.
However, BCAT1 KO clones continued to grow at high growth rates,
reaching peak VCD on day 10 of 44 × 106 and 50 ×
106 cells/mL; about 23 – 30 x 106cells/mL higher peak VCD than WT counterparts. Severe foaming in BCAT1
KO cultures may have limited the peak cell densities. Lactate was
well-controlled across all conditions, with peak concentrations around 3
g/L and good metabolic shift to lactate consumption. BCAT1 KO cultures
also had significantly higher titers than the WT counter parts, which
can be attributed to higher peak VCDs as the qPwas similar across all the conditions.
Levels of the metabolic byproducts isovalerate, isobutyrate and
2-methylbutyrate are negligible across fed-batch and HiPDOG conditions
for BCAT1 KO clones (Figure 1, Figure 2), which is expected given the
encoded enzyme’s role as the entry point for BCAAs into the catabolic
pathways responsible for production of these compounds (Mulukutla et
al., 2019). Accumulation of these byproducts was observed in all the
wild-type cultures, albeit to different levels across fed-batch and
HiPDOG conditions. The cause of differences in byproduct accumulation
between fed-batch and HIPDOG cultures will be investigated in a future
study.
The levels of other previously reported growth-inhibitory metabolic
byproducts were probed in the culture milieu of HiPDOG and fed-batch
cultures of BCAT1 KO and WT clones on days 6, 8 and 10 (Figure 3)
(Mulukutla et al., 2017). 3-phenyllactate and 4-hydroxyphenyllactate
levels were similar between HiPDOG and fed-batch cultures until day 6
but increased over time in the HiPDOG cultures. Indole-3-lactate levels
were higher in HiPDOG cultures across all the time points probed.
Butyrate levels were similar between HiPDOG and fed-batch cultures
across all the time points probed. Formate and glycerol levels were
similar between HiPDOG and fed-batch cultures on day 6 but accumulated
to lower levels in HiPDOG cultures thereafter. Concentrations of all
tested metabolic byproducts were very similar between HiPDOG and
fed-batch cultures on day 6, thus the reduced growth observed in
fed-batch BCAT1 KO cultures by day 6 can be concluded not to be due to
accumulation of these other metabolic byproducts in the fed-batch
culture milieu.
Previously, it has been reported that fed-batch cultures with robust
metabolic shifts perform better than those without (Le et al., 2012).
The above data suggests even though the BCAT1 KO clone can undergo a
strong metabolic shift in fed-batch cultures, controlling peak lactate
levels, as in the case of HiPDOG cultures, appears to greatly increase
peak cell densities. The higher peak lactate and corresponding higher
osmolality observed in standard fed-batch cultures could be negatively
impacting the proliferative capability of BCAT1 KO cells. This
hypothesis was put to test in a subsequent experiment. The BCAT1 KO
Clone 83 was inoculated in shake flask cultures at 0.1 ×
106 cells/mL and treated with different levels of
lactate spanning the levels observed in the fed-batch and HiPDOG
conditions (Figure 4). Growth characteristics of all conditions were
probed over the course of 6 days. An osmolality control was also
included, targeting the 8 g/L lactate condition. BCAT1 KO clone growth
was similar across 0 and 2.5 g/L conditions between days 0 and 3, with
slightly reduced growth rate for the remainder of the batch. Substantial
growth inhibition was observed at and above the 5 g/L condition. The
osmolality control also had significantly lower growth when compared
with the 0 g/L condition. When WT cells in shake-flask cultures were
subjected to similar lactate concentrations, similar growth inhibition
to that of BCAT1 KO clone cultures was observed. These data suggest that
higher peak lactate levels and the associated osmolality could have a
negative effect on the growth of CHO cells in fed-batch cultures even if
they undergo a robust metabolic shift. The above result that lactate
suppresses growth rate is not novel. This has been reported widely in
the past across multiple mammalian cell lines used for therapeutic
production, including CHO, hybridoma, baby hamster kidney (BHK) cells
etc. (Cruz et al., 2000; Lao and Toth, 1997; Kurano et al., 1990; Ozturk
et al., 1992).
Control of residual glucose at low concentrations could also have a
positive effect on growth of the BCAT1 KO cells, independent of lactate
accumulation in fed-batch cultures. Previous studies with hybridoma
cells have shown that exposure to lower residual glucose levels for
prolonged amount of time in batch or fed-batch mode before transition
into continuous culture mode can result in a lower ratio of lactate
produced to glucose consumed during the continuous phase of the culture
(Europa et al., 2000). Expression of certain key glycolytic genes were
downregulated in culture with exposure to lower glucose concentration
(Korke et al., 2004). Whether any such changes at the transcriptome
level due to reduced exposure to glucose in HiPDOG cultures have a role
to play in improved performance of BCAT1 KO cells remains to be
ascertained.
In summary, metabolic engineering towards reduction of novel growth
inhibitors can help increase cell densities and titers of fed-batch
cultures. However, close attention needs to be paid to the level of
lactate accumulation as lactate is the major growth-inhibitory metabolic
byproduct produced by CHO cells in culture. Accumulation of lactate to
very high levels can reduce the proliferative advantage offered by
control of other inhibitory metabolic byproducts in fed-batch cultures.
Future metabolic engineering attempts that can simultaneously eliminate
production of lactate and BCAA metabolic products including isovalerate,
isobutyrate and 2-methylbutyrate can result in fed-batch culture cell
growth rivaling that of perfusion cultures.