Ex cell advanced cho fed batch media

Cells were inoculated at 600,000 cells per mL in Ex-Cell® Advanced CHO Fed-Batch media (MilliporeSigma) and incubated in a humidified (70-80%) shaking incubator with 5% CO 2 and temperature of 37°C (shift to 34°C on day 4). Cultures were fed every other day starting on day 2 with 6.25% (V:V) of a feed blend containing 66% Ex-Cell® Advanced CHO Feed 1 and 33% Cellvento® 4Feed (MilliporeSigma). Glucose was monitored daily and supplemented if below 5 g/L. Cultures were terminated when viabilities were ≤ 70% or at the end of day 20. Cell concentration and viability were measured by a Roche Cedex HiRes® instrument and cell metabolites, glucose, and IgG titer were measured by a Roche Cedex Bio HT® instrument.

Pools or clones were continually passaged in Ex-Cell® Advanced CHO Fed-Batch media (MilliporeSigma) or ActiPro TM (Cytiva) media and frozen weekly. All generations were simultaneously thawed and evaluated for production.

Coding sequences for commercially available biotherapeutics for monoclonal IgG1-kappa antibodies and an Fc fusion protein were cloned into either a single gene or two gene Boat expression construct. 3 million GPEx® Lightning Parental Dock cells were transfected with 2 micrograms (total) of Boat plasmid and recombinase plasmid DNA using ExpiFectamine TM CHO (ThermoFisher Scientific). Cells were allowed to recover in Ex-Cell® Advanced CHO Fed-Batch media (MilliporeSigma) supplemented with 6 mM glutamine and 1% ClonaCell™-CHO ACF Supplement (Stem Cell Technologies) until viability returned to greater than 95% before glutamine was withdrawn. Viability was monitored until the resulting selected cell pools returned to greater than 95% viability and doubling times reached 18-24 hours, typically 16 to 22 days after transfection.