Product titers from Chinese hamster ovary (CHO) processes have increased steadily over the past decade, with reports from fed-batch processes now exceeding 5 g/L. Several approaches are being implemented for titer increases based mostly on volumetric productivity by supporting high viable cell densities (VCD). Another variable to focus on is increasing cell-specific productivity (CSP). CSP factors in the process titer with cell density and cell-specific perfusion rate. It is a variable that is gaining attention for intensified continuous bioprocesses like perfusion processes. The market for continuous bioprocesses is expected to grow at greater than 10% per year through 2025, as supported by new commercial facilities utilizing the approach.

A recent report from a collaboration between the University of Stuttgart, Xell AG, Evonik, and Boehringer Ingelheim looked at how media additives improved CSP in CHO cell cultures. Of particular interest was  S-adenosylmethionine (SAM), a methyl donor cosubstrate integral to connecting cell metabolism to gene regulation and epigenetics. Metabolically, SAM is involved in polyamine synthesis, methylation, and sulfuration. Therefore, the team was interested in learning its potential to boost CSP as it could affect cell growth and gene expression. The CHO-DP12 cell line was grown in disposable shake flasks and fed varying levels of SAM and the degradation of metabolite methylthioadenosine (MTA). The concentrations of both additives were monitored by LC-MS/MS. VCD, viability, average cell diameters were recorded on a Cedex XS cell counter, glucose and lactate were monitored on a Labotrace analyzer, titer was measure by ELISA, and cell cycle analysis was measured using propidium iodide staining with a flow cytometer.

Initially, adding SAM 48 hr after the cultures’ start lowered VCD by 10% and 18% (duplicate cultures) compared to control cultures. However, the titers in both SAM treated cultures were higher by more than 50%. Also, these cultures had a CSP that was 57% higher compared to the controls. Analysis of the cell media throughout the process showed no evidence of SAM’s cellular uptake since the amounts in solution were the same between cultures with and without cells. Also, the degradation product of SAM, MTA, was monitored. MTA was detected at increasing levels in the flasks without the cells indicating gradual degradation of SAM. In contrast, when cells were present, the MTA concentration steadily decreased throughout the culture. When MTA was directly added to cultures, it also displayed similarly increased titers and CSPs to SAM compared to the control without additives.

Cell cycle analysis of the SAM and MTA supplemented cells 12 hrs after addition showed differences in the cultures. SAM supplemented cultures had a higher number of G2-phase cells, while the MTA cultures had more S-phase cells. Both cultures had low amounts of cells in the G1-phase. 12 hrs later, G1-phase cells increased to more than G2-phase cells in the SAM cultures. Conversely, the relative populations in the MTA-treated cultures remained the same. After another 12 hrs of culture, the differences in the proportion of cells in the S-phase leveled off between the two treated cultures. Both treatments had higher amounts of cells in the G1-phase and less in the G2-phase than the control. Finally, both SAM and MTA cultures had average cell diameters that were approximately 10% larger than the control cultures 36 hrs after treatment. This difference was gradually removed towards the end of the culture when cell diameters converged to about 12.5 µm.

It was clear that both the addition of SAM and MTA significantly increased titers and CSPs compared to controls. There was evidence of cell cycle arrest and an early increase in cell volume after treatment. This could indicate a more efficient use of cell resources for protein production instead of biomass formation. That said, the exact mechanism was not determined in this study. Nevertheless, both compounds could be explored in more detail since they are commercially available and can be added to existing cell media formulations easily. With 50% increases in both titer and CSP, likely, other groups have already begun to use these supplements with different CHO cell lines and processes. Combining new media supplements, innovative bioreactors, and process improvements, intensified processes have a bright future.

 

By, Glenn A. Harris