Monitoring of intracellular ribonucleotide pools is a powerful tool in thedevelopment and characterization of mammalian cell culture processes

Efficient cell culture process development for the industrial production of recombinant therapeutics is characterized by constraints which pertain to issues such as costs, competitiveness and the meeting of project timelines. These constraints require tools which can help the developer learn as much as possible as quickly as possible about the cell at hand and identify fea tures of a particular culture which are amenable to improvement. Current on - and off-line monitoring parameters, however useful, provide only late ind ications (cell concentration, viability) and circumstantial evidence (lacta te, ammonia, etc.) with regard to the physiologic status of cells at the ti me of sampling. The relative intracellular content of purine to pyrimidine nucleotide triphosphates as well as the ratio of UTP to UDP-N-acetylhexosam ines have been previously described as sensitive indicators of a cell's met abolic status, growth potential, and overall physiological condition. The s ensitivity of such nucleotide ratios and their usefulness in commercially r elevant process development and characterization were tested at Boehringer Ingelheim Pharma KG in a large number of fermentations (>80) with a variety of culture modes, cells, and products in scales up to 10,000 litres. Monit oring of these intracellular parameters allows a timely and reliable assess ment of cell state and growth potential, which is possible neither by class ical cell number and viability measurements nor by a variety of fermentatio n data typically monitored. The view inside the cell afforded by nucleotide monitoring enables prediction of the behavior of a culture up to 2 days be fore any hint of physiological changes is given by cell number and viabilit y estimation. In this paper, data relating the growth behavior of CHO and h ybridoma cell lines to their nucleotide pools are shown. Two very different processes for the production of recombinant tPA in 10,000-litre bioreactor s are compared and characterized with respect to their nucleotide profiles. Examples from industrial process development cases in which intracellular nucleotide information is used to advantage are also presented and discusse d. (C) 1999 John Wiley & Sons, Inc.