Introduction

CHO cells are currently the workhorse for therapeutic protein production of various therapeutic modalities including monoclonal antibodies, antibody drug conjugates, enzymes, vaccines, etc. Over the last decade, fed-batch has been the predominant culture mode for protein production using CHO cells. While titers for mAb production in fed-batch cultures have significantly increased over this time span, currently commonly reaching 5 - 10 g/L, that productivity progress may have reached a ceiling. To add to this, the advent of the biosimilars has created an additional driver for cost of goods reduction and an increase in process productivities.
To push the productivities higher, perfusion or hybrids of perfusion and fed-batch processes are currently being explored (Hiller et al., 2017). The underlying rational for such a move towards perfusion processes is the building consensus that productivity is principally being limited due to the accumulation of growth-inhibitory metabolic byproducts (Pereira et al., 2018; Mulukutla et al., 2017). Flushing such inhibitors out of the culture using perfusion is a straightforward way of improving the culture environment and boosting cellular proliferation (Hiller et al., 2017). However, the introduction of perfusion does present manufacturing challenges in the form of operational and facility fit complexities. Fed-batch processes are still a preferred way of manufacturing biologics due to manufacturing ease they present. Therefore, improving titers and productivities in fed-batch cultures continues to be very desirable.
Lactate and ammonia are the classical metabolic byproducts that are known to accumulate in fed-batch cultures. It has been extensively documented in the published literature that the aforementioned byproducts inhibit growth and productivities to varying degrees at high concentrations (Lao and Toth, 1997; Cruz et al., 2000; Hansen and Emborg, 1994; Kurano et al., 1990; Ozturk et al., 1992) and significant research efforts have been devoted towards their control in culture (O’Brien et al., 2020). In the case of lactate, process and genetic engineering methods have been employed to reduce its formation, some of which have been reviewed recently (Hartley et al., 2018; Kim and Lee, 2007; Zhou et al., 2011; Toussaint et al., 2016). The use of HiPDOG technology to control lactate accumulation by limiting residual glucose levels in culture has shown successful results (Gagnon et al., 2011). HiPDOG technology uses culture pH as an indirect measure of glucose to maintain residual culture glucose levels in a low concentration range. This results in net reduced lactate accumulation in culture, thereby allowing cells to grow to higher densities which yield higher titers. However, even in such cultures cells eventually stop proliferating due to inhibitory action of other byproducts which accumulate throughout the culture (Mulukutla et al., 2017).
More recently, the cell culture community has been investing in understanding metabolic byproducts beyond lactate and ammonia that accumulate in CHO cell fed-batch cultures (Pereira et al., 2018). We and others have shown that CHO cells produce metabolic byproducts from amino acid catabolism and glucose metabolism (Mulukutla et al., 2017; Alden et al., 2020). These byproducts can accumulate to growth-inhibitory levels in CHO cell fed-batch cultures. We have reported that reducing accumulations of these inhibitory byproducts by maintaining levels of the corresponding amino acids within a low concentration range, under conditions of reduced lactate accumulation (using HiPDOG control), results in higher peak cell densities and productivities (Mulukutla et al., 2017). Alternatively, we have shown that genetic engineering approaches can be employed to overexpress certain enzymes, as in the Phe-Tyr pathway, to direct flux away from byproduct production. Additionally, knockout (KO) of enzymes, as in case of the branched-chain amino acid (BCAA) catabolic pathway, can be used to eliminate overflow metabolism and byproduct formation (Mulukutla et al., 2019). In the BCAA pathway, we have shown that KO of the BCAT1 gene, encoding for branched chain amino acid aminotransferase 1 enzyme, eliminates production of three growth-inhibitory byproducts: isovalerate, 2-methylbutyrate and isobutyrate. These inhibitors are catabolic byproducts of the amino acids, leucine, isoleucine and valine, respectively. The KO clones when grown in conditions with lactate control yield higher peak cell densities and productivities. Other groups have replicated this work, subsequently (Pereira et al., 2019). However, the impact of lactate control on such metabolically engineered cells hasn’t been carefully evaluated yet.
In the present study, the benefit of controlling lactate production by employing the HiPDOG glucose limitation strategy on growth and productivity of BCAT1 KO cells was evaluated. In fed-batch cultures without control of lactate due, BCAT1 KO clones achieved, to a smaller degree, higher peak viable cell densities and titers when compared with their wild-type counterparts. However, in glucose-limited HiPDOG cultures BCAT1 KO cells grew to significantly higher cell densities and produced higher titers than the wild-type cells.