A FLOW-INJECTION (FI) MEDIATED BIOSENSOR FOR ONLINE MONITORING OF LACTATE IN MAMMALIAN-CELL CULTURE

A flow-injection (FT) biosensor system has been developed for on-line monitoring of lactate formation during mammalian cell cultivation. The culture sample was peristaltically withdrawn from the bioreactor and, after cell separation by a steam sterilizable ceramic microfilter, th e cell-free filtrate was fed to the Fl system. Lactate oxidase was cov alently immobilized onto a preactivated nylon membrane and attached to the sensing area of a platinum working electrode. The enzyme reaction was coupled with a water-soluble mediator 1,1'-dimethylferricinium (D MFe+)-cyclodextrin inclusion complex to recycle the reduced lactate ox idase to its original active state. 1,1'-Dimethylferrocene (DMFe) was then reoxidized to DMFe+ at the surface of the platinum electrode pois ed at +0.15 V vs. silver/silver chloride. The FI biosensor was linear up to 3 mM lactate with a detection limit of 0.05 mM, and possessed +/ -1.5% reproducibility over 154 repeated analyses during a 77 h continu ous operation. Due to a significant accumulation of lactate during fed -batch cultivation of 293S mammalian cells, a dialysis membrane with 1 000 molecular weight cut-off was placed over the lactate oxidase membr ane to extend the linear detection range up to 40 mM. Excellent reprod ucibility (+/-1.3%) was observed for 96 repeated hourly analyses with culture medium containing 15 mM lactate. When applied to (5-10)-day fe d-batch cultivation of 293S mammalian cells, the results obtained from the biosensor system compared well with HPLC and spectrophotometric a ssay data. (C) 1997 Elsevier Science B.V.