Continuous production and recovery of recombinant Ca2+ binding receptor from HEK 293 cells using perfusion through a...

The extracellular domain of human parathyroid Ca(2+) receptor was needed in order to study itsstructure and clinical application. The Ca(2+)receptor is a unique member of the G protein-coupledreceptor super-family, expressed in parathyroid andkidney cells where it has been shown to play acritical role in extracellular calcium homeostasis.The desired protein was produced by immobilizing thetransformed HEK 293 cells in a packed-bedconfiguration using a 1.6 l (working volume)bioreactor equipped with a vertical mixing impellerassembly and an internal basket. The process includeda propagation phase followed by a production phase. Inthe propagation phase, lasting approximately 160 h, the bed was perfused with a serum-containingmedium, allowing the cells to grow at a constantgrowth rate to approximately 3 x 10(10). At this point the production phase was begun, replacing themedium with serum-free medium and continuing theperfusion process for additional 350 h. Duringthis phase, the medium was pumped through the packedbed at a rate of 4-6 l per day, keeping theresidual glucose concentration around 1 g l(-1) andcollecting and processing approximately 80 l ofspent medium. This continuous perfusion method of thepacked-bed bioreactor was compared to a repeated batchmethod in which existing medium was replenished whenthe glucose concentration was down to 1 g l(-1). Using this method, serum-free medium was replaced withserum containing medium a few times when a decline inthe glucose consumption was observed. Though mediumconsumption and protein yield are similar in bothmethods (roughly 10 mg l(-1)), there aredifferences related to the ease of operation andprocessing of the produced protein. The continuousperfusion operation was found to be preferable and waschosen as the production strategy.