A flow-injection (FI) mediated biosensor for on-line monitoring of lactate in mammalian cell culture

Abstract

A flow-injection (FI) 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 The culture sample was peristaltically withdrawn from the bioreactor and, after cell separation by a steam sterilizable ceramic microfilter, the cell-free filtrate was fed to the FI system. Lactate oxidase was covalently 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 (DMFe +)-cyclodextrin inclusion complex to recycle the reduced lactate oxidase to its original active state. 1.1′-Dimethylferrocene (DMFe) was then reoxidized to DMFe + at the surface of the platinum electrode poised 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 continuous operation. Due to a significant accumulation of lactate during fed-batch cultivation of 293S mammalian cells, a dialysis membrane with 1000 molecular weight cut-off was placed over the lactate oxidase membrane to extend the linear detection range up to 40 mM. Excellent reproducibility (±1.3%) was observed for 96 repeated hourly analyses with culture medium containing 15 mM lactate. When applied to (5–10)-day fed-batch cultivation of 293S mammalian cells, the results obtained from the biosensor system compared well with HPLC and spectrophotometric assay data.