## 4 Discussion
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 (q\({}_{\text{P}}\)) and volumetric productivity (V\({}_{\text{P}}\)). 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 Tables 1-3) 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]. [PERSON] 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; 47]. At present, the most accepted hypothesis