IMPROVEMENT OF RECOMBINANT PROTEIN-PRODUCTION WITH THE HUMAN ADENOVIRUS 293S EXPRESSION SYSTEM USING FED-BATCH STRATEGIES/

The human adenovirus/293S cell expression system is used for the produ ction of either recombinant protein or adenovirus vectors for use in g ene therapy. In this work, the production of protein tyrosine phosphat ase (PTP1C) was used as a model for the scale-up of both applications. Maximum specific production of 30 to 45 mu g of active protein/10(6) cells was maintained upon infection with adenovirus vectors at cell de nsities between 2 x 10(6) to 3 x 10(6) cells/ml in a 3.5-L bioreactor. This was achieved by resuspending the culture in fresh medium at infe ction time. The pH was kept at 7.0 throughout the experiment and, at 2 4 h postinfection, glucose and essential amino acids were added. Attem pts to replace the complete change of medium at the time of infection with nutrient supplementation of the used medium led to lower producti on levels, suggesting that protein expression was limited not by the a bsence of a key nutrient but by inhibitory factors. Two potentially in hibitory factors were investigated: lactic acid accumulation and in cr eased osmolarity. Medium acidification such as that which would be bro ught about by lactic acid accumulation was shown to depress PTP1C prod uction. The lactate molecule itself decreased the cell viability when added in concentrations of 20 mM or more. But the specific productivit y was affected at higher lactate concentrations of 40 mM or more. Addi tions of glucose, amino acids, and NaHCO3 used to control pH, led to i ncreases in osmolarity. Osmolarities above 400 mOsm lowered cell densi ty. However, specific production was not significantly affected below 500 mOsm. But, at 500 mOsm, PTP1C production peak was shifted from 48 to 42 hpi. Because of the cell loss, this per cell yield increase did not translate into higher volumetric production. When glucose concentr ations was kept at 5 mM by fed-batch addition, lactate production and increases in osmolarity were reduced. In shake flasks, this method per mitted maximum production with cells resuspended either in fresh or sp ent medium at infection. This fed-batch process was implemented succes sfully at the 3.5-L scale. Fed-batch with glucose may provide a means to increase infected-cell density beyond 3 x 10(6) cells/mL. (C) 1996 John Wiley & Sons, Inc.