The demand for monoclonal antibodies has significantly increased in recent years, mainly due to new applications in therapy, but also for clinical diagnoses and highly specific purification processes [1
]. In this regard, the capacity of mammalian cells to perform complex post-translational modifications to yield biologically active proteins has led to their preferential use for biopharmaceuticals production. About 70–80% of all biopharmaceuticals, including monoclonal antibodies, viral vaccines and gene therapy vectors, are produced in mammalian cells. Among the top ten selling protein biopharmaceuticals in 2014, six were antibodies or antibody-derived proteins. It is therefore not surprising that in 2016, monoclonal antibody-based drug production using mammalian cell-based systems almost doubled that of 2010 [1
]. In recent years, monoclonal antibodies industrial manufacturing has been based on mammalian cell lines such as hybridoma, among others [3
Mammalian cell-based processes present important limitations regarding apoptosis: the accumulation of metabolic byproducts (i.e., lactate and ammonia) up to cytotoxic concentration, and the depletion of essential nutrients, which triggers apoptosis (programmed cell death) [7
]. The prevention of apoptosis during cell growth has a critical effect on the productivity of the final process, as an increase of cell life-span results in an increase in the synthesis and accumulation of the product of interest, since cells remain productive for a longer time, even after the exponential cell growth phase [8
]. Moreover, more robust cell lines which are less sensitive to apoptosis make it possible to design high cell density culture strategies based on consistently low nutrient concentrations in narrower ranges [9
In recent years, strategies to generate stress-resistant cell lines preventing apoptosis have been focused on blocking the apoptotic transduction pathways [10
]. Although different pathways control the activation of signaling cascades of cell apoptosis activation, many of the apoptosis signals converge on the mitochondria, which stores numerous molecules that activate apoptosis [7
]. The most widely used strategy to prevent apoptosis has been the overexpression of bcl-2 or bcl-xL genes, which inhibits the release of pro-apoptotic molecules from the mitochondria [10
]. This strategy has been successfully applied in different mammalian cell lines, such as CHO or hybridoma, showing higher viabilities and improved robustness in cell culture [13
]. The expression of mcl-1, another antiapoptotic gene with mechanism similar to that of bcl-2/bcl-xL, has also shown good results [17
]. Another approach has been to directly target the caspase cascade, expressing X-linked inhibitor of apoptosis (XIAP) or cytokine response modifier CrmA, which both act to directly inhibit the caspases [18
]. The expression of different viral proteins has also been reported to have antiapoptotic effects in hybridoma cell cultures, such as ksblc-2 from Karposi’s sarcoma-associated herpesvirus [19
] and bhrf-1 from Epstein-Barr virus [20
]. Furthermore, the downregulation of the pro-apoptotic genes Bak, Bax and Casp3 has been shown to reduce apoptosis in CHO cells [21
Additionally, antiapoptotic genes have been shown to have an effect on metabolism, although this is not yet fully understood [22
]. This is remarkable, as cell-based processes present an important limitation regarding metabolism: deregulation of the uptake of substrates (i.e., high consumption rates of mainly glucose and glutamine), linked to the secretion and accumulation of lactate and ammonia as byproducts of metabolism [24
The reduction of the secretion and accumulation of lactate remains a hot topic in the biomanufacturing industry. Many approaches have been explored to reduce or delay lactate generation in cell culture, including media design by the substitution of glucose for alternative sources of carbon, like fructose or galactose [25
], different fed-batch strategies limiting glucose concentration [9
] and several cell engineering approaches such as the expression of pyruvate carboxylase [27
] and downregulation of lactate dehydrogenase [28
]. In all of the described scenarios, a reduction, but never a total depletion, in lactate accumulation was observed.
In the present work, a murine hybridoma cell line (KB26.5) was engineered to overexpress the BHRF-1 gene (KB26.5-BHRF1). Besides the observed protective effect against apoptosis, the overexpression of BHRF-1 had an unexpected direct effect on cell physiology and metabolism. Mainly higher cell growth rate and more efficient nutrient usage were observed in batch cultures, significantly reducing the production of lactate. Therefore, metabolic flux balancing techniques were applied to better understand the interactions and effects of BHRF-1 expression with cell metabolism. Analysis of the obtained intracellular fluxes provided us with a better understanding of the effects of BHRF-1 expression on the improved cell metabolism, and gave rise to some hypotheses about possible BHRF-1 interactions with the metabolic pathways. Nowadays, the prevention of apoptosis continues to be a relevant research topic, e.g., in a review by Henry et al. (2020) [11
], the effects of the various antiapoptotic strategies which have been applied in recent years were presented and discussed.
The growth, viability, glucose and lactate profiles obtained from the KB26.5-BHRF1 cultures were vastly different than those from the parental cell line. Cell culture expansion was much faster, and the maximum cell density reached 3.92 ± 0.03 × 106
cell/mL, representing a two-fold increase. Additionally, the cell viability profile after the cell density peak remained at over 90% for 24 h after the maximum cell density had been achieved, indicating a delay in apoptosis due to the effects of BHRF1, as anticipated by Juanola et al. (2009) [20
]. The glucose and lactate profiles showed an initial phase of glucose consumption and lactate generation. The glucose concentration profile decrease was similar to that obtained in the KB26.5 culture, but lactate production rates were reduced by almost half. However, since the cell density almost doubled, the specific glucose consumption rate was affected by BHRF1 expression and, consequently, by lactate production. In order to better quantify the differences, Table 2
presents the main physiological parameters for both cell lines, i.e., growth rate (µ), doubling time (td
), specific glucose consumption rate (qGlu
), specific lactate production rate (qLac
), glucose yield on biomass (YGlu/X
), lactate yield on biomass (YLac/X
) and maximum VCD (VCDmax
In general terms, the expression of BHRF1 was beneficial for cell physiology and growth. The cell growth rate (µ) increased by 78%, the specific glucose consumption rate (qGlu) decreased by 15% and the specific lactate production rate (qLac) reduced by more than 50%. This resulted in a decrease in both glucose yield on biomass (YGlu/X) and lactate yield on biomass (YLac/X) in KB26.5-BHRF1, meaning that more efficient glucose consumption had been achieved, thereby generating less lactate. Overall, this resulted in a more efficient use of the main carbon source. This fact translated into a more than two-fold increase in total cell density under the same media and culture conditions.
The results obtained in 2 L bioreactors showed similarities to those observed in shake flask cultures. The maximum cell density was slightly improved for KB26.5, reaching 2.34 ± 0.39 × 106 cell/mL, probably due to the pH control. Even though lactate was produced at similar rates, the lack of pH control in the shake flaks might have negatively affected cell growth, a phenomenon which was reversed in the bioreactor cultures. In the case of KB26.5-BHRF1, the maximum cell density observed was significantly increased, reaching 3.93 ± 0.01 × 106 cell/mL. In addition, the viability strongly decayed after 48 h for KB26.5, while it was maintained at above 90% in BHRF1-KB26.5.
Despite the concordance with the results observed in the shake flask cultures, another difference was that the glucose was completely depleted in the KB26.5 culture, indicating the extension of the culture time due to the pH control, which may have had an effect on cell density increase. To further compare the performance of both strains in the bioreactor, the main physiological parameters were calculated and compiled in Table 3
, including maximum titer obtained (IgG3
,max), specific productivity (qP
) and volumetric productivity (VP
Similar to shake flask culture results, cell growth improved by more than 30% in KB26.5-BHRF1, with a reduction of 42% and 54% in specific glucose consumption rate and specific lactate production rate, respectively. Therefore, the differences between strains may have been related to the effects of BHRF1 on cell physiology, rather than a consequence of the culture conditions. The same was observed regarding glucose and lactate yield on biomass, as both were reduced in KB26.5-BHRF1. Due to the extension of the growth phase, KB26.5-BHRF1 increased the final product concentration by 3.7 times. The specific and volumetric productivity were also increased two- and three-fold, respectively.
The results showed lower specific consumption rates for glucose and almost all amino acids, as well as lower production rates of byproducts, which might indicate efficient cell metabolism mediated by the expression of BHRF1. Additionally, the significant reduction in glutamine consumption, and therefore, in the production of ammonia, well known as a toxic byproduct in mammalian cell cultures [43
], yielded the dual benefits of lactate and ammonia reduction in culture. Alanine generation was also greatly reduced in KB26.5-BHRF1, an amino acid that often accumulates in mammalian cell cultures [40
]. In any case, a metabolic flux balance is required to evaluate and discuss the possible effects of BHRF1 on the general behavior of the engineered line with respect to the parental cell line. Interestingly, the expression of BHRF1 has an effect on metabolism, improving the efficiency of nutrient usage, which is necessary for cell growth (see Table 1
, Table 2
and Table 3
) and the reduction of byproducts.
The engineered KB26.5-BHRF1 hybridoma cell line suffered a modification in its metabolism. BHRF1 is an antiapoptotic protein located in the inner mitochondrial membrane [44
]. For this reason, BHRF1 may have somehow affected carbon and NADH/NAD+ transport between cytoplasm and mitochondria. Most of the antiapoptotic genes reported in the literature are known to be bind at the mitochondrial membrane, regulating apoptosis through modulation of mitochondrial permeability, but also playing an important role in the metabolic processes of mitochondria [45
]. Dorai et al. (2009) [22
] reported the effect of two antiapoptotic genes on the metabolism of CHO, showing an important reduction in the final lactate concentration due to lactate consumption in culture. In addition, engineered cells showed a more efficient nutrient consumption profile and produced fewer byproducts, such as ammonia or alanine, as observed in this study.
Lactate generation in mammalian cell cultures is a well-known challenge that has been extensively studied in recent years [46
]. At present, the most accepted hypothesis for the production of lactate is based on the regeneration of the reducing power (NADH) in the cytoplasm due to the high glycolytic fluxes [46
]. In this regard, there are two ways to regenerate NADH into the cytoplasm: (1) lactate generation and (2) malate-aspartate shuttle [48
]. It should be noted that the malate-aspartate shuttle facilitates the regeneration of NADH and increases the TCA cycle flux (importing malate), thereby generating energy in the form of ATP. However, the generation of lactate provokes a total loss of both carbon sources and ATP generation. Therefore, the mechanism of lactate generation may lie in the flux limit of the Malate-Aspartate Shuttle, leading cells to generate lactate and display this wasteful metabolism.
In the breakdown of glucose into two pyruvate molecules, two molecules of ATP and two NADH are generated. Since the inner mitochondrial membrane is impermeable to NADH, the malate-aspartate shuttle works as an indirect transport system. It has been reported that the flux through this transport occurs at lower rates than glycolysis [49
], and that the increased LDH activity is due to the inability to transport NADH through the shuttle at the same rates at which it is generated. Under conditions of increased cellular energy demand, higher glycolytic fluxes are observed, and consequently, NADH production rates increase proportionally. Such an increase in the need for NAD+
regeneration is compensated for by higher LDH activity in the cytosol, as not much increase is observed in the malate-aspartate shuttle fluxes [50
The results presented in this work provide evidence for a beneficial effect of antiapoptotic gene BHRF1 in hybridoma in terms of cell growth, productivity and metabolism, characterized by a more efficient use of nutrients, doubling the cell density in the culture under identical conditions and using the same culture media. The specific productivity of the cells increased two fold, yielding a three-fold increase in the final concentration of IgG3.
The study and comparison of the intracellular fluxes by means of a flux balance analysis of the KB26.5 and KB26.5-BHRF1 engineered cell lines highlighted the interactions and effects of the BHRF1 protein on the metabolic pathways. In short, BHRF1 primarily affected glucose uptake rate, reducing glycolysis by 50% and, consequently, reducing the generation of lactate by more than 60%. Interestingly, the total ATP generation in the engineered KB26.5-BHRF1 cell line decreased significantly due to the lower energy requirements for maintenance, probably due to the lower energy requirements for maintaining ion gradients (reduction in the generation of lactate), although this hypothesis should be tested in future metabolic flux analysis experiments.
The use of antiapoptotic genes has been extensively explored in recent years, as manifest in a recent review about the attenuation of apoptosis in the CHO cell line [12
]. Many efforts have been made to determine how these genes affect cell apoptosis; however, the impact they have on protein production, and especially on metabolism, has not yet been clarified. To address this, a strategy to obtain isogenic cell lines should be implemented, as this would make it possible to compare different antiapoptotic genes and their effects.