Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of...
Reexamination Certificate
2001-09-17
2004-03-16
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
C435S183000, C435S189000, C435S190000, C435S252300, C435S320100, C435S325000, C435S383000, C435S440000, C435S455000, C536S023200, C536S023600, C536S023700, C536S023740, C536S024100
Reexamination Certificate
active
06706524
ABSTRACT:
The invention relates to a method of improving the primary energy metabolism of mammalian cell lines, to expression vectors for use in the method and to recombinant mammalian cells having improved primary energy metabolism obtainable in accordance with the method.
Only a very small proportion of glucose, which is one of the main sources of energy, can be fully oxidised to CO
2
. The majority is released as lactate and alanine. Since the amount of energy gained in aerobic glycolysis is only very small, the energy requirement is met by glutaminolysis, in which ammonium is formed as a toxic by-product.
Various studies have shown that the crucial enzymes that transfer the end product of glycolysis, pyruvate, into the citric acid cycle (TCA) have only weak activity in cell lines (Fitzpatrick et al., 1993; Petch and Butler, 1994; Neermann and Wagner, 1996) (a list of the quoted literature references will be found at the end of this description). If there is a connection between glycolysis and the citric acid cycle, it will increase the contribution of glucose to energy metabolism and reduce the glutamine requirement.
Overview of Energy Metabolism in Mammalian Cell Lines
For culturing of permanent mammalian cell lines, glucose and glutamine occupy a special position amongst the numerous essential components of the relatively complex nutrient medium because, unlike in the case of bacteria or yeasts, both substrates are necessary as suppliers of energy, glutamine serving as a primary source of cellular energy (ATP). The degree of cellular energy supplied by glutamine depends on the individual cell line and on the presence and concentration of glucose.
The primary energy metabolism of mammalian cells is therefore composed of glucose and glutamine oxidation and includes the metabolic pathways of glycolysis, glutaminolysis and the citric acid cycle. A simplified overview of those primary metabolic pathways with branches and crucial enzyme functions is given in FIG.
1
.
Glucose Metabolism
In mammalian cell lines, between 80% and 97% of the glucose is converted by means of glycolysis. In contrast to insect or primary cells, however, in transformed mammalian cell lines almost all the glucose converted in glycolysis is processed to lactate, and only a very small proportion (about 0.2 to 5%) of the glucose carbon, i.e. the glycolytic intermediates, passes into the energy-supplying citric acid cycle.
It is largely unexplained why glucose is converted almost completely into lactate and the transition of glycolytic intermediates into the citric acid cycle is blocked in the case of almost all mammalian cell lines.
An initial supposition is that the activity of the mediating enzyme pyruvate dehydrogenase is very low as a result of low expression rates or permanent inhibition by irreversible phosphorylation. As a result of that false regulation, large amounts of lactate are secreted into the nutrient medium where they lead to uncontrolled acidification of the culture. Those factors result in a low degree of efficiency in the utilisation of the nutrients, high glucose consumption and a low energy yield.
Glutamine Metabolism
Unlike glycolysis, glutaminolysis is not a single, complete metabolic pathway, but forms a network of up to eight, partly interconnected, alternative metabolic pathways by which glutamine can be oxidised to different degrees and which therefore result in different energy yields and product combinations.
A large proportion of the glutamine is deaminated and introduced via the intermediate a-ketoglutarate into the citric acid cycle where it can be fully oxidised to CO
2
. In addition, glutamine may also be converted partly into the amino acids aspartate and alanine, and also into lactate. These may either be secreted into the culture medium or, in the case of aspartate, introduced into the citric acid cycle via oxaloacetate.
The choice of the pathways to the complete or only partial oxidation of the glutamine decides the contribution of glutamine to the energy balance of the cell. Investigations carried out on various mammalian cell lines have shown that glutamine in the presence of glucose meets from 30% to 65%, and possibly even up to 98%, of the cell's energy requirement. In general, the contribution of glutamine to the energy balance of the mammalian cell is higher, the lower the concentration of glucose in the medium.
A crucial by-product of glutamine breakdown is ammonium. Depending upon the glutaminolytic metabolic branch, one or two moles of ammonium may be released per mole of glutamine, this having a growth-inhibiting to toxic effect on the cells. On the one hand, the intracellular pH value is lowered, while on the other hand large amounts of ammonium result in changes to the nucleotide and sugar-nucleotide pool (Ryll et al., 1994; Valley et al., 1999), which has a significant effect on the expression of the N-glycosidically bonded carbohydrate side chains of glycoproteins and thus alters the product quality of therapeutic agents (Gawlitzek et al., 1998; Grammatikos et al., 1998).
Metabolic Engineering in Mammalian Cell Lines
Attempts to achieve a significant improvement in the growth and productivity of cellular systems probably require the radical modification of certain substrate streams of the energy metabolism. For that purpose, very accurate information relating to key points of the metabolism are necessary.
Since, however, it is precisely in sensitive mammalian cells that an irreversible intervention in metabolism is associated with considerable difficulties, only very few attempts to obtain a rational design of the metabolism have been carried out successfully in mammalian cells, as compared with bacteria and yeasts.
In fact, according to present knowledge only six successful attempts at metabolic engineering in the so-called primary metabolism of mammalian cells have taken place.
The first approach in mammalian cell lines was disclosed by Pendse and Bailey (1994). On the assumption that an increase in the innercellular ATP or energy level would raise productivity, the gene for the bacterial Vitreoscilla haemoglobin (Vhb) was cloned into a tPA-producing CHO cell line. In comparison with a non-transfected control cell line, the resulting Vhb-expressing cell line exhibited a reduction in growth, but a 40% to 100% increase in specific tPA production. It is clearly possible to influence both growth and productivity by way of increased cellular energy states.
Renner et al. (1995) established a connection between cell cycle phases, growth and cyclin-E expression through experiments with growth factors such as bFGF (basic fibroblast growth factor). Where cyclin-E expression is high, in certain CHO cells there is a cell cycle having a relatively long G
1
-phase and a short S-phase, and the cell growth is relatively high. The authors then cloned a cyclin-E expression vector into CHO cells. As a result of the then increased amount of cyclin-E, the transfected cell line exhibited higher proliferation rates than the non-transfected control cells, especially in protein-free basal media. In addition, the cell morphology and the cell cycle phase distribution were similar to the bFGF-stimulated CHO cells. Cell growth and morphology can therefore be influenced by this metabolic design of the cell cycle.
Bell et al. (1995) cloned a vector having a glutamine synthetase gene into a hybridoma cell line. Unlike the non-transfected control cell line, the transfected hybridoma cell line was able, as a result of its glutamine synthetase activity, to grow in a glutamine-free nutrient medium.
That transfection represented a crucial intervention in glutamine metabolism and therefore in the primary and energy metabolism of the mammalian cell. At the same time, the problem of toxicity caused by the ammonium formed from glutamine was significantly reduced by the possibility of glutamine-free culture.
Rees and Hay (1995) cloned the complete bacterial threonine metabolic pathway into a mammalian cell line, so that the cell line was able to grow in a threonine-free nutrient medium. The cell
Irani Noushin
Van Der Heuvel Joop
Wagner Roland
Wirth Manfred
Gesellschaft fuer Biotechnologiesche Forschung mbH (GBF)
Marshall & Gerstein & Borun LLP
Prouty Rebecca E.
Rao Manjunath N.
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