Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Using a micro-organism to make a protein or polypeptide
Reexamination Certificate
2000-09-05
2004-01-06
Lankford, Jr., Leon B. (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Using a micro-organism to make a protein or polypeptide
C435S348000, C435S325000, C435S373000
Reexamination Certificate
active
06673575
ABSTRACT:
The invention concerns a process for the production of a polypeptide with suitable glycosylation by culturing eukaryotic cells and isolating the polypeptide from the culture medium or/and the cells. In this process the desired glycosylated polypeptide can be produced recombinantly with the aid of endogenous gene activation or be produced naturally by the cells.
The production of glycoproteins by culturing eukaryotic cells is generally carried out in commercial culture media. The addition of certain substrates to the culture medium may be necessary in order to achieve a desired glycosylation of the polypeptide. This is described in the Japanese laid-open patent document H6-292 592 for a batch process in small volumes (<1000 ml), a low initial cell density (5×10
4
cells/ml) and a short culture period (48 h) with a human IgM antibody as an example. In this case another sugar such as fructose, mannose, galactose, N-acetylglucosamine, ribose, fucose, N-acetylgalactosamine etc. is used instead of the conventional glucose for the recombinant production of antibodies in mammalian cells e.g. CHO cells. In addition a multi-step culture process is disclosed in which the cells are firstly cultured in a medium containing glucose which is subsequently substituted by a medium containing another sugar.
However, the process described in the Japanese laid-open patent document H6-29592 has serious disadvantages. The concentration of the sugar in the culture medium changes continuously as a result of its consumption during the cell culture so that a constant high degree of glycosylation of the polypeptides is not guaranteed. Furthermore the batch process is unsuitable if a constant substrate concentration is necessary for a desired glycosylation since the initial concentrations of the substrates continuously decrease due to cell metabolism. Moreover the high sugar concentrations required for high cell densities and a constant high degree of glycosylation must already be provided at the start of the fermentation which would, however, inhibit the growth of the cells and hence limit the attainable cell density. Therefore an economical production of highly glycosylated polypeptides is not possible using the process described in the above-mentioned Japanese laid-open patent document.
The culture of eukaryotic cells by a batch process with feeding (fed-batch) in which nutrient solution is added during the culture is known. In this type of process it is possible to achieve a high cell density and a longer culture period by suitable feeding. An example is the continuous and limited feeding of the essential amino acid glutamine which leads to an improved cell growth (Ljunggren et al., Biotech. Lett. 12 (1990), 705-710). The aim of feeding glutamine is to reduce the formation of ammonium since ammonium is toxic for animal cells (Mirabet et al., Biotechnol. Bioeng. 56 (1997), 530-537).
Gawlitzek et al. (Biotechnol. Bioeng. 57 (1998), 518-528) describe that increased concentrations of ammonium ions or glucosamine in the medium of cultured eukaryotic BHK-21 cells lead to an increase of the complexity of N-linked carbohydrate structures in recombinant glycoproteins which are secreted by the cultured cells. However, this finding is inconsistent with the results of Borys et al. (Biotechnol. Bioeng. 43 (1994), 505-514) or Andersen and Goochee (Biotechnol. Bioeng. 47 (1995), 95-105) where an inhibition of glycosylation by elevated ammonium concentrations in the culture medium was found. Hence it becomes clear that the control of ammonium formation in the culture is only an isolated aspect and is therefore not sufficient for the economical production of proteins with suitable glycosylation.
The degree of glycosylation of polypeptides can greatly influence their biological activity. This is elucidated in the following erythropoietin (EPO) example. EPO is a human glycoprotein which stimulates the production of red blood cells. EPO only occurs in the blood plasma of healthy persons in very low concentrations so that it is not possible to provide larger amounts in this manner. EP-B-0 148 605 and EP-B-0 205 564 describe the production of recombinant human EPO in CHO cells. The EPO described in EP-B-0 148 605 has a higher molecular weight than urinary EPO and no O-glycosylation. The EPO described in EP-B-0 205 564 from CHO cells is now available in large amounts and in a pure form.
Furthermore the isolation of human EPO from the urine of patients with aplastic anaemia is known (Miyake et al., J. Biol. Chem. 252 (1977), 5558-5564).
Recombinant and urinary EPO are isolated as a mixture of various isoforms which are known to differ in their degree of sialylation. These EPO isoforms have different isoelectric points and can be separated by isoelectric focussing or capillary electrophoresis (see Tsao et al., Biotech. Bioeng. 40 (1992), 1190-1196; Nieto et al., Anal. Commun. 33 (1996), 425-427; Tran et al., J. Chromatogr. 542 (1991), 459-471; Bietot et al., J. Chromatogr. 759 (1997), 177-184; Watson et al., Anal. Biochem. 210 (1993), 389-393). The isoforms with the highest number of sialic acids have the highest specific activity, whereas those with the lowest number have the lowest activity (see e.g. Imai et al., Eur. J. Biochem. 194 (1990), 457-462; EP-A-0 428 267).
Takeuchi et al., (Proc. Natl. Acad. Sci. USA 86 (1989), 7819-7822) describe a relationship between the biological activity and the sialic acid content and the ratio of biantennary and tetraantennary carbohydrate structures. Takeuchi et al., additionally conclude that the N-acetyl-lactosamine disaccharide units present in the EPO carbohydrate structures do not correlate with the biological activity.
Fukuda et al., (Blood 73 (1989), 84-89) deal with the rate of elimination of EPO from the blood circulation which makes an important contribution to the biological activity and conclude that EPO with a relatively large number of N-acetyl-lactosamine units is more rapidly removed from the circulation than EPO without lactosamine units. Morimoto et al., (Glycoconjugate J. 13 (1996), 1093-1120) describe the separation of EPO isoforms by means of mono-Q chromatography so that the individual fractions are then only composed of a few isoforms. The investigations carried out on these fractions show an equidistribution of all structures in all fractions. No correlation was found between the content of biantennary or triantennary structures or the content of N-acetyl-lactosamine units and the specific activity.
Thus the said prior art shows that there is a general correlation of the biological activity with the sugar structure especially with regard to the content of sialic acids.
Surprisingly it was found that a continuous feeding according to requirements of carbohydrate-containing substrates during a high cell density fermentation or/and use of a mixture of at least 2 carbohydrates during culture enables a high yield of desired protein, such as EPO, with a high degree of glycosylation to be obtained.
Hence a first aspect of the invention concerns a process for isolating a glycosylated polypeptide from eukaryotic cells, wherein the eukaryotic cells are cultured in a suitable medium and the desired polypeptide is isolated from the cells or/and the culture supernatant wherein the process is characterized in that a mixture of at least 2 and preferably at least 3 carbohydrates is added to the culture medium.
The carbohydrates are preferably selected from monosaccharides and disaccharides such as glucose, glucosamine, ribose, fructose, galactose, mannose, sucrose, lactose, mannose-1-phosphate, mannose-1-sulfate and mannose-6-sulfate. Nutrient media are for example suitable which contain glucose or/and mannose or/and galactose. Particularly good results were obtained with nutrient media which contain a mixture of glucose, galactose and mannose for example in a mass ratio of 1:(0.5-3):(1-5) and in particular of 1:(0.7-2.4):(1.8-4.0) where each of the carbohydrates is particularly preferably used in the D(+) form. The total concentration of all sugars during the ferme
Eberhardt Horst
Franze Reinhard
Wallerius Claus
Davis Ruth
Fulbright & Jaworski L.L.P.
Lankford , Jr. Leon B.
Roche Diagnostics GmbH
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