2.4 Shaken microwell cultures
The microwell format used for this study was the 24 deep square well (24-DSW) microtiter plate (Adolf Kuhner AG, Switzerland) with a square cross section of side = 17 mm, height = 40 mm and maximum well volume = 11 mL. The microwells were inoculated using a diluted mid-exponential phase shake-flask culture. Once the target induction cell density was reached, the cultures were induced to initiate the production of LVV. Induction was carried out by the addition of Doxycycline (Sigma-Aldrich, St. Louis, Missouri, United States) and Sodium Butyrate (Sigma-Aldrich, St. Louis, Missouri, United States). Similar to the STR, the induction agents were diluted in complete culture medium prior to being added in the microwell. The plates were sealed with a ‘sandwich lid system’ (W. A. Duetz et al., 2000) in conjunction with a metal clamp (EnzyScreen BV, AL Leiden, Netherlands) to minimize evaporation over extended periods of culture (Figure 1). The plates were incubated in an orbital shaker (shaking diameter = 25 mm) (Adolf Kuhner AG, Switzerland) with shaking speed set at 250 rpm, temperature at 37°C and CO2 at 5%. The well fill volume varied between 5 mL at the start of the culture and 2.5 mL at the harvest time point. The reduction in volume was due to sampling of cell culture each day for analysis. For sampling, 0.5 mL of cell culture was removed from triplicate wells. For this work, sacrificial well sampling was not considered since the 24-DSW plate is not limiting in terms of working volume, as is the case with the 24-SRW plate. In addition, sacrificial well sampling also leads to a reduction in experimental throughput. To validate the aforementioned well sampling approach, the cell culture performance using sacrificial well sampling was compared against the same well sampling method used in this work.