Monitoring of intracellular ribonucleotide pools is a powerful tool in the development 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 features of a particular culture which are amenable to improvement. Current on- and off-line monitoring parameters, however useful, provide only late indications (cell concentration, viability) and circumstantial evidence (lactate, ammonia, etc.) with regard to the physiologic status of cells at the time of sampling. The relative intracellular content of purine to pyrimidine nucleotide triphosphates as well as the ratio of UTP to UDP-N-acetylhexosamines have been previously described as sensitive indicators of a cell's metabolic status, growth potential, and overall physiological condition. The sensitivity of such nucleotide ratios and their usefulness in commercially relevant 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. Monitoring of these intracellular parameters allows a timely and reliable assessment of cell state and growth potential, which is possible neither by classical cell number and viability measurements nor by a variety of fermentation data typically monitored. The view inside the cell afforded by nucleotide monitoring enables prediction of the behavior of a culture up to 2 days before any hint of physiological changes is given by cell number and viability estimation. In this paper, data relating the growth behavior of CHO and hybridoma cell lines to their nucleotide pools are shown. Two very different processes for the production of recombinant tPA in 10,000-litre bioreactors 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 discussed.

Intracellular UDP-N-acetylhexosamine pool affects N-glycan complexity: a mechanism of ammonium action on protein glycosylation.

Understanding the mechanisms by which ammonium ions affect glycosylation may suggest strategies for producing glycoproteins with homogeneous biological activity in the presence of undesirable byproducts of cellular energy metabolism. We have previously shown that ammonium ions cause an increase in the intracellular UDP-N-acetylhexosamine (UDPGNAc) pool, which may be responsible for the ammonium-induced increase in complexity and decrease in sialylation state of the N-linked oligosaccharide. To investigate this novel hypothesis, we induced an artificial increase in UDPGNAc pool by treating recombinant BHK cells expressing an IL-2 variant that features an artificial site for N-glycosylation, with glucosamine (1:2 molar ratio to glucose) and uridine (2 mmol L-1) in the absence of ammonium ions or glutamine. The product fractions collected during this treatment showed increased antennarity compared to product collected under control conditions. When this pool was returned to normal levels, the glycosylation pattern regained its original (control) features. However, the sialylation state remained unaffected, suggesting that the decreased sialylation observed under ammonium treatment is due to a different mechanism of action, possibly involving changes in intracellular pH. By pretreating the cells with 0.5 mmol L-1 adenosine, and exposing them continuously to NH4Cl and adenosine we were able to prevent the ammonium-induced increase in UDPGNAc. Product fractions collected during this treatment showed unchanged antennarity but decreased sialylation of the N-linked oligosaccharide, thus conclusively demonstrating that ammonium ions act on protein glycosylation by at least two independent mechanisms, one of which involves an increase in the UDPGNAc pool.

Diversity in the ability of cultured cells to elongate and desaturate essential (n-6 and n-3) fatty acids.

Essential fatty acids (EFAs) cannot be synthesized by mammalian cells. Once taken in with the diet, they can undergo desaturations/saturations and chain elongations/shortenings to yield a variety of polyunsaturated fatty acids of the same family. Cells in vitro from a variety of tissues are capable of processing EFAs to varying extents. Conversion of the parent EFAs, linoleic (LA, n-6) and alpha-linolenic (LNA, n-3) acids, to the 20-carbon polyunsaturated fatty acids, arachidonic (AA, n-6) and eicosapentanoic (EPA, n-3), requires chain elongation and delta 6 and delta 5 desaturations. AA and EPA are required by many tissues for optimal biological function and are precursors of biologically active eicosanoid hormones. All cultured cells are able to elongate exogenous LA and LNA, and most can perform delta 5 desaturation, so delta 6 desaturation is the limiting step in AA and EPA production. Longer fatty acids that have more double bonds than AA or EPA are less frequently produced due to a deficiency in delta 4 desaturating ability. The process of retroconversion (chain shortening) is less extensively studied, but evidence from a variety of cells suggests that this type of metabolic conversion is normally active. The example of MCF-7 (human breast cancer cell line) and MCF-10A cells (human noncancerous breast cell line) is discussed in order to emphasize the diversity in EFA processing ability of cultured cells. Under identical culture conditions, MCF-10A cells perform extensive desaturations, elongations, and retroconversions, whereas MCF-7 cells can only elongate and retroconvert exogenous EFAs. Given the great diversity in the ability of cultured cells to process EFAs, no conclusions can be drawn regarding the mechanisms responsible for the effects of exogenous EFAs on a particular cell until that cell's EFA processing patterns have been evaluated.

n-3 and n-6 fatty acid processing and growth effects in neoplastic and non-cancerous human mammary epithelial cell lines.

The type rather than the amount of dietary fat may be more important in breast carcinogenesis. While animal studies support this view, little is known about the effects of essential fatty acids (EFAs) at the cellular level. The MCF-7 breast cancer and the MCF-10A non-cancerous human mammary epithelial cell lines are compared in terms of growth response to EFAs and ability to incorporate and process the EFAs. Eicosapentaenoic (EPA, n-3) and docosahexaenoic (DHA, n-3) acids, presented bound to albumin, inhibited the growth of MCF-7 cells by as much as 50% in a dose-dependent manner (6-30 microM) in medium containing 0.5% serum. alpha-Linolenic (LNA, n-3) and arachidonic (AA, n-6) acids inhibited growth less extensively, while linoleic acid (LA, n-6) had no effect. In contrast, MCF-10A cells were not inhibited by any of the EFAs at levels below 24 microM. The differential effects of AA, EPA and DHA on MCF-7 and MCF-10A cells support a protective role of highly unsaturated essential fatty acids against breast cancer. The EFAs were primarily incorporated into phosphoglycerides. MCF-7 cells showed chain elongations and possibly delta 8 desaturation, but no AA was formed from LA, nor EPA or DHA from LNA. In contrast, MCF-10A cells desaturated and elongated the exogenous EFAs via all the known pathways. These findings suggest defects in the desaturating enzymes of MCF-7 cells. LNA, DHA and AA presented to MCF-7 cells in phospholipid liposomes inhibited growth as extensively as albumin-bound free acids, but were less extensively incorporated, suggesting different mechanisms of inhibition for the two methods.

Diverse effects of essential (n-6 and n-3) fatty acids on cultured cells.

Fatty acids (FAs) have long been recognized for their nutritional value in the absence of glucose, and as necessary components of cell membranes. However, FAs have other effects on cells that may be less familiar. Polyunsaturated FAs of dietary origin (n-6 and n-3) cannot be synthesized by mammals, and are termed 'essential' because they are required for the optimal biologic function of specialized cells and tissues. However, they do not appear to be necessary for normal growth and metabolism of a variety of cells in culture. The essential fatty acids (EFAs) have received increased attention in recent years due to their presumed involvement in cardiovascular disorders and in cancers of the breast, pancreas, colon and prostate. Many in vitro systems have emerged which either examine the role of EFAs in human disease directly, or utilize EFAs to mimic the in vivo cellular environment. The effects of EFAs on cells are both direct and indirect. As components of membrane phospholipids, and due to their varying structural and physical properties, EFAs can alter membrane fluidity, at least in the local environment, and affect any process that is mediated via the membrane. EFAs containing 20 carbons and at least three double bonds can be enzymatically converted to eicosanoid hormones, which play important roles in a variety of physiological and pathological processes. Alternatively, EFAs released into cells from phospholipids can act as second messengers that activate protein kinase C. Furthermore, susceptibility to oxidative damage increases with the degree of unsaturation, a complication that merits consideration because lipid peroxidation can lead to a variety of substances with toxic and mutagenic properties. The effects of EFAs on cultured cells are illustrated using the responses of normal and tumor human mammary epithelial cells. A thorough evaluation of EFA effects on commercially important cells could be used to advantage in the biotechnology industry by identifying EFA supplements that lead to improved cell growth and/or productivity.