Each cell line, except CHO line 4, was cultured in its optimal basal chemically defined (CD) medium for maintenance and batch and fed batch studies. Venetoclax clinical trial Cell line 4 was maintained in a peptone containing medium, while batch and fed batch studies were performed in CD medium. Cell culture media utilized were: BD Select CHO and BD Select CD1000 (BD Advanced Bioprocessing), CDM4CHO (Thermo Fisher Scientific), and EX CELL CD CHO3 (SAFC). Feeds and media supplements utilized were: TC Yeastolate (TCY) and Proteose Peptone 3 (PP3) (BD Advanced Bioprocessing) and CD Cell Boost 6 (Thermo Fisher). The Duetz
sandwich-cover system and 24DW plates were obtained from Enzyscreen BV (Haarlem, Netherlands). The system includes 24DW plates (40 mm deep, pyramid bottom, volume 11 mL/well, transparent polystyrene plates), sandwich covers (CR1224a) and clamps (CR1700). Bioassay cultures were seeded at identical seeding density in 24DW plates and shake flasks. Culture volume was 3 mL and 50 mL in 24DW plates and shake flasks, respectively. Plate and shake flask cultures were incubated on a shaking platform in 5% CO2 at 37 °C. The shaking speed for plates was 300 rpm, while Dabrafenib manufacturer the shaking speed for flasks was 125 rpm.
The orbital diameter of the shaking platform was 25 mm for both plates and shake flasks. For 24DW plates, 300 μl samples were collected from the same well on various days of culture. These samples were diluted 2–3 times with PBS (Cellgro®) for assessment of cell growth (Viable cell density; VCD), viability and protein production. VCD and viability were determined using a Vi-CELL® (Beckman Coulter) and protein production was measured using a ForteBio Octet®
(Pall Life Sciences). For batch culture studies, peptones were added to basal media on Day 0. Fed batch cultures were fed with CD feeds on alternate days starting on Day PJ34 HCl 0 of the culture. Peptone titration studies were performed to test the effect of various concentrations of peptones on growth and production of CHO cells in a batch culture. Minitab 16 software (Minitab Inc) was used to generate multivariate charts and to perform other statistical analyses. Correlation analysis was performed to determine growth and production performance relationship between shake flask and 24DW plates. The Pearson correlation coefficient is a measure of linear association between two variables. Values of the correlation coefficient are always between −1 and +1. A correlation coefficient of +1 indicates that two variables are perfectly related in a positive linear sense. Two-Way ANOVA was used to assess effect of plates and peptone titration in plate-to-plate variation study. Four CHO lines were grown concurrently in their respective optimal base media in 24DW plates with Duetz sandwich-covers and in conventional shake flasks.