Minimización de residuos

Based on the Plackett-Burman analysis the initial medium composition was modified to

improve cell growth and IFN-y production. In the previous Chapter, the positive group of

variables was also further subdivided into BSA and amino acids plus sodium pyruvate. It was

shown that the stimulating growth effect of sodium pyruvate plus amino acids was independent of BSA. Although BSA proved to be a major component per se for both CHO cell growth and IFN-y production, it was not initially included in the positive group due to the

variability it introduces into the culture medium. There are other reasons not to increase its concentration in the culture medium; it was further analysed as a separate component (see

Chapter 5).

The conditions of the culture medium deteriorate during a batch culture and cell growth

usually becomes limited by nutrient depletion or by the accumulation of toxic components, which may become the bottleneck of the process. Analysis of nutrient consumption in the

previous Chapter has shown that glutamine was depleted early in the culture, after

approximately 75 h, while glucose was also used extensively and depleted in the later stages;

both conditions could be related to the onset of the stationary phase. Final concentrations of

ammonia and lactate obtained in batch culture, ca. 1.7 mM and 13 mM respectively, should not be inhibitory to the cells as 2 mM ammonia and 17.5 mM lactate have shown no effect on

the growth of this cell line (Hayter et a i, 1991a). However, cell growth limitation by either glucose and/or glutamine starvation was a possibility to consider. Simply increasing the initial concentration of these nutrients in the culture medium is often unsuccessful due to their

inefficient utilisation and an increase in the accumulation of inhibitory metabolites. This strategy has been investigated for this cell line by increasing initial glutamine and glucose

concentrations to 4 mM and 22 mM respectively (Hayter et ai, 1991a), and no improvements on cell growth or IFN-y production were obtained; nutrient utilisation seemed to be less

effective, as indicated by the higher glucose and glutamine uptake rates. Higher initial

concentration of glutamine also lead to an increase in ammonia production. Glucose and

glutamine were not identified as positive variables by the Plackett-Burman analysis although

they were depleted early in culture; this may be also related to inefficient utilisation of these

nutrients at higher initial concentrations. As discussed in section 1.3.2.1, ammonia and lactate

are major products of glutamine and glucose metabolism, respectively. Controlled feeding of these components in order to reduce ammonia and lactate accumulation has been previously

reported (Glacken e ta i, 1986; Glacken, 1988; Ljunggren and Haggstrom, 1990). A significant

reduction in ammonia production with a simultaneous increase in cell yield of MDCK cultures has been achieved by controlled feeding of glutamine, which was maintained at low levels

throughout the culture (Glacken et a i, 1986).

The nutritional environment of the cells may affect the glycosylation patterns of a recombinant protein. A decrease in the proportion of the 2N-glycoform of IFN-y was previously observed

during batch culture (section 3.4.1). This shift follows the decline in glucose levels and rapid

depletion of glutamine from culture. Glucose-limited chemostat cultures of these cells have shown an increase in the proportion of the 2N form of IFN-y following transient periods of

excess glucose (Hayter et a i, 1992), suggesting that glycosylation could be affected by the availability of this nutrient. However, studies from batch cultures with high initial

concentration of glucose (22 mM) did not have the same effect on protein glycosylation

(Hayter et a i, 1991b; Curling et a i, 1990) indicating that factors other than the availability of

glucose per se could be affecting the glycosylation pathway. It has also been reported that CHO cells utilise an alternate glycosylation pathway when deprived of glucose, and this was

thought to be due to depletion of nucleotide sugar precursors, leading to the synthesis of glycoproteins with truncated oligosaccharides (Rearick et a i, 1981). This is supported by the

early suggestion that the major function of aerobic glycolysis is to maintain high amounts of glycolytic precursors for macromolecule synthesis (Hume et a i, 1978).

The glycosylation biosynthetic pathway has been already described in section 1.4.1. It starts

with the synthesis of a lipid-linked oligosaccharide, containing glucose, mannose and N-

acetylglucosamine. The oligosaccharide processing reactions in the Golgi compartments yield

complex oligosaccharide structures and use uridine-diphosphate (UDP), guanosine

diphosphate (GDP) and cytidine monophosphate (CMP) as co-substrates (e.g. UDP-N-

acetylglucosamine, CMP-sialic acid). It is possible that the supply of glycosylation precursors,

such as aminosugars and nucleotides, are a limiting step for that pathway. Both glucose and glutamine are involved in the biosynthesis of these precursors. Ribose-5-phosphate, primarily

formed by the pentose-phosphate pathway, and the amino group of glutamine are both

necessary for the synthesis of purine and pyrimidine nucleotides. Glucose and/or glutamine depletion from the culture medium may thus constitute a block for the efficient glycosylation

of the protein and it is a subject that needs to be addressed.

In this Chapter, glucose and glutamine fed-batch cultures were used in order to avoid the

depletion of these nutrients from the culture, while keeping them at a concentration which would not allow excessive production of inhibitory metabolites. Cell growth, cell metabolism

and IFN-y production under these conditions were investigated. At the same time, the

possibility that glucose and/or glutamine availability in culture affects the glycosylation of

IFN-y was evaluated.