Cell Line Development
CHO-K1 host cells that were originally purchased from ATCC and suspension-adapted by WuXi Biologics were maintained as a suspension culture in CD CHO medium (ThermoFisher Scientific) supplemented with 4 mM L-glutamine, and incubated at 36.5 ℃ in a humidified 6% CO2 in air (v/v) orbital shaking incubator at 150 rpm with an orbital diameter of 50 mm. The recombinant biologics-expressing cell lines were generated by transfecting vectors encoding recombinant genes by electroporation using a Bio-Rad Gene PulserTM (Bio-Rad) in the cGMP facilities of WuXi Biologics. The transfectants were resuspended with CD CHO medium and returned to the shaking incubator. Cells were then sub-cultured and expanded accordingly every 3 days. A few weeks of sub-culturing are needed before compatible with fed-batch culturing for toxicology and GMP production.
Single-cell cloning was done by depositing single cells into microplates by FACSAria IITM after transfected cells recovered. Time-lapse images of each well were taken daily to monitor the growth of clones from the day of FACS sorting till three days after. Clonal colonies were expanded to 24 deep well plates or spin tubes, in which the cells were sub-cultured every three days, and seeded in AMBR 250 bioreactors for clone fed-batch culture screening.