MilliporeSigma
  • Continuous production and recovery of recombinant Ca2+ binding receptor from HEK 293 cells using perfusion through a packed bed bioreactor.

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

Cytotechnology (2008-11-13)
J B Kaufman, G Wang, W Zhang, M A Valle, J Shiloach
ABSTRACT

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.

MATERIALS
Product Number
Brand
Product Description

SAFC
EX-CELL® 293 Serum-Free Medium for HEK 293 Cells, Serum-free, Animal-protein free, without L-glutamine, liquid, suitable for cell culture
SAFC
EX-CELL® 293 Serum-Free Medium for HEK 293 Cells, Animal-protein free, with L-glutamine, without sodium bicarbonate, dry powder, suitable for cell culture