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.