In the previous study, the culture medium was treated with nicotinamide adenine dinucleotide (NAD⁺) under the hypothesis that NAD⁺ regeneration is a major factor causing excessive lactate accumulation in Chinese hamster ovary (CHO) cells. The NAD⁺ treatment improved metabolism by not only reducing the Warburg effect but also enhancing oxidative phosphorylation, leading to enhanced antibody production. Building on this, four NAD⁺ precursors – nicotinamide mononucleotide (NMN), nicotinic acid (NA), nicotinamide riboside (NR), and nicotinamide (NAM) – were tested to elevate intracellular NAD+ levels more economically. First, the ability of CHO cells to utilize both the salvage and Preiss‐Handler pathways for NAD⁺ biosynthesis was verified, and then the effect of NAD⁺ precursors on CHO cell cultures was evaluated. These precursors increased intracellular NAD⁺ levels by up to 70.6% compared to the non‐treated group. Culture analysis confirmed that all the precursors induced metabolic changes and that NMN, NA, and NR improved productivity akin to NAD⁺ treatment, with comparable integral viable cell density. Despite the positive effects such as the increase in the specific productivity and changes in cellular glucose metabolism, none of the precursors surpassed direct NAD⁺ treatment in antibody titer, presumably due to the reduction in nucleoside availability, as evidenced by the decrease in ATP levels in the NAD⁺ precursor‐treated groups. These results underscore the complexity of cellular metabolism as well as the necessity for further investigation to optimize NAD⁺ precursor treatment strategies, potentially with the supplementation of nucleoside precursors. Our findings suggest a feasible approach for improving CHO cell culture performances by using NAD⁺ precursors as medium and feed components for the biopharmaceutical production.

Methotrexate (MTX)-mediated gene amplification has been widely used in Chinese hamster ovary (CHO) cells for the biomanufacturing of therapeutic proteins. Although many studies have reported chromosomal instability and extensive chromosomal rearrangements in MTX-mediated gene-amplified cells, which may be associated with cell line instability issues, the mechanisms of chromosomal rearrangement formation remain poorly understood. We tested the impact of DNA double-strand breaks (DSBs) on chromosomal rearrangements using bleomycin, a DSB-inducing reagent. Bleomycin-treated CHO-DUK cells, which are one of the host cell lines deficient in dihydrofolate reductase (Dhfr) activity, exhibited a substantial number of cells containing radial formations or non-radial formations with chromosomal rearrangements, suggesting that DSBs may be associated with chromosomal rearrangements. To confirm the causes of DSBs during gene amplification, we tested the effects of MTX treatment and the removal of nucleotide base precursors on DSB formation in Dhfr-deficient (i.e., CHO-DUK) and Dhfr-expressing (i.e., CHO-K1) cells. Immunocytochemistry demonstrated that MTX treatment did not induce DSBs per se, but a nucleotide shortage caused by the MTX-mediated inhibition of Dhfr activity resulted in DSBs. Our data suggest that a nucleotide shortage caused by MTX-mediated Dhfr inhibition in production cell lines is the primary cause of a marked increase in DSBs, resulting in extensive chromosomal rearrangements after gene amplification processes.