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.