Violacein improves recombinant IgG production by controlling the cell cycle of Chinese hamster ovary cells.

Chinese hamster ovary (CHO) cells are used as host cells for industrial monoclonal antibody (mAb) production. Cell cycle control is an effective approach to increase mAb production in the cell culture. Violacein, a purple-colored pigment produced by microorganisms, has diverse bioactive properties and has been proposed for various industrial applications. In this study, we evaluated the potency of violacein for cell cycle control and improvement of recombinant immunoglobulin G (IgG) production in CHO cells. Compared with the control, 0.9 µM violacein in a 14-day fed-batch culture increased the maximum IgG concentration by 37.6% via increasing the specific production rate and cell longevity. Cell cycle analysis showed that violacein induced G1 and G2/M phase arrest. However, the G1 arrest was observed only on day 1, while G2/M arrest lasted more than 3 days, suggesting that G2/M arrest mediated the violacein-induced enhanced IgG production. Moreover, in line with the increased protein expression, the expression levels of IgG mRNA and nutrient metabolic rates were also increased. N-Linked glycosylation and charge variant profiles were barely affected by violacein treatment. Our results indicate that violacein affects the cell cycle of CHO cells and increases IgG production without changing product quality, showing promise as a mAb production enhancer in CHO cells. The study provides insight into violacein utilization in industrial mAb manufacturing and can help develop advanced, effective mAb production technologies using CHO cell cultures.

Low-concentration staurosporine improves recombinant antibody productivity in Chinese hamster ovary cells without inducing cell death.

Chinese hamster ovary (CHO) cells are used as host cells for biopharmaceutical production, including monoclonal antibodies (mAbs). Arresting the cell cycle with chemical compounds is an effective approach to improve biopharmaceutical productivity. In a previous study, potential new cell cycle-arresting compounds were screened from marine-derived microorganism culture extracts, and it was suggested that staurosporine might improve mAb productivity in CHO cells via cell cycle arrest. The purpose of this study was to demonstrate the effectiveness of staurosporine as a cell-cycle arresting compound to improve mAb productivity. The optimal staurosporine concentration range was initially investigated using batch cultures. Thereafter, the effects on the culture profile and mAb productivity were evaluated using fed-batch cultures. Staurosporine at concentrations ≥10 nM induced cell death, but at concentrations ≤5 nM did not. In the range of 2-4 nM, cell growth was inhibited, whereas the specific production rate (Qp) and cell longevity were improved in a dose-dependent manner. The Qp and maximum mAb concentration with 4 nM staurosporine improved by 36.3 and 5.2%, respectively, compared to those with control conditions. Cell viability post-culture without staurosporine was 40.0 ± 0.3%, whereas with 4 nM staurosporine, it was 90.1 ± 1.0%. Flow cytometric analysis indicated cell-cycle arrest at the G1/G0 phase with 4 nM staurosporine addition. The present study highlighted the efficacy of staurosporine in improving mAb production by causing cell-cycle arrest. Further research into staurosporine analogs and how to use them will lead to development of more effective industrial production technologies of biopharmaceuticals.

Screening of new cell cycle suppressive compounds from marine-derived microorganisms in Chinese hamster ovary cells.

Monoclonal antibodies (mAbs) are active pharmaceutical ingredients in antibody drugs, produced mainly using recombinant Chinese hamster ovary (CHO) cells. The regulation of recombinant CHO cell proliferation can improve the productivity of heterologous proteins. Chemical compound approaches for cell cycle regulation have the advantages of simplicity and ease of use in industrial processes. However, CHO cells have genetic and phenotypic diversity, and the effects of such compounds might depend on cell line and culture conditions. Increasing the variety of cell cycle inhibitors is a promising strategy to overcome the dependency. Marine microorganisms are a vast and largely undeveloped source of secondary metabolites with physiological activity. In this study, we focused on secondary metabolites of marine microorganisms and evaluated their effectiveness as cell cycle inhibitory compounds. Of 720 extracts from microorganisms (400 actinomycetes and 320 filamentous fungi) collected from the Okinawan Sea, we identified nine extracts that decreased the specific growth rate and increased the specific production rate without reducing cell viability. After fractionating the extracts, the components of active fractions were estimated using time-of-flight mass spectrometry analysis. Then, four compounds, including staurosporine and undecylprodigiosin were deduced to be active compounds. These compounds have been reported to exert a cell cycle inhibitory effect on mammalian cells. These compounds might serve as additives to improve mAb production in CHO cells. This study indicates that secondary metabolites of marine microorganisms are a useful source for new cell cycle inhibitory compounds that can increase mAb production in CHO cells.