Polypeptide production in animal cell culture

a cell culture and polypeptide technology, applied in the field of improving the production of polypeptides in animal cell culture, can solve the problems of not being able to disclose a method of controlling the osmolality of the cell culture medium, the concept of controlling the glucose in fed-batch cell culture in order to control the osmolality within a desired range has not been proposed by these researchers, and the time required for optimal production of polypeptides is reduced. , to achieve the effect o

Inactive Publication Date: 2005-12-08
GENENTECH INC
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Benefits of technology

[0011] It is a further object of the present invention to provide a method of making a polypeptide which comprises initially culturing animal cells under conditions which enhance cell growth and then, in a production phase distinct from the cell growth phase, culturing animal cells under conditions which increase protein production thereby. This en

Problems solved by technology

However, these publications fail to disclose a method of controlling the osmolality of the cell culture medium by controlling the addition of the primary energy source, glucose, to the cell culture medium.
However, the concept of controlling glucose in fed-batch cell culture in order to control osm

Method used

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  • Polypeptide production in animal cell culture
  • Polypeptide production in animal cell culture
  • Polypeptide production in animal cell culture

Examples

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example 1

Cell Growth and Productivity vs Osmolality

[0091] The impact of osmolality on cell growth and productivity was determined in petri dish culture. CHO cells were transformed with nucleic acid encoding recombinant human deoxyribonuclease (rhDNase) using the techniques disclosed by Shak et al., PNAS, 87: 9188-9192 (1990), expressly incorporated by reference herein. These recombinant CHO cells were grown in petri dish culture in medium consisting essentially of the Super medium referred to in U.S. Pat. No. 5,122,469 (except that NaCl was added to the Super medium at various concentrations in order to achieve an osmolality in the range from about 260-680 mOsm). The glucose concentration was 4.5 g / L. The osmolality was measured using an OSMETTE™ osmometer. The pH of the culture medium was 7.2 and cells were cultured in a 37° C. humidified CO2 incubator. The results are depicted in FIG. 2; the closed circles represent the CHO cell density (expressed as 105 cells / ml) after 5 days and the ope...

example 2

Osmolality Chance vs Glucose Concentration

[0092] The osmolality change during cultivation at various controlled glucose concentrations versus high (batch) glucose concentration was quantified. CHO cells transformed with rhDNase cells (see Shak et al., supra) were cultured in a pH controlled Applikon (Foster City, Calif.) bioreactor at a 2.5 liter working volume. The Super cell culture medium of U.S. Pat. No. 5,122,469 (with adjusted NaCl and glucose concentrations) was used and the CHO cells were stirred at 150 rpm agitation. The initial osmolality was adjusted to about 270-300 mOsm via the amount of NaCl added. The cell seed density was 1×106 cells / ml. The other cell culture medium and culturing conditions were: pH=7.2, dO2=60% and culturing temperature=37° C. Glucose was added either in batch (at a concentration of 12 g / L) at the start of the culturing or was controlled throughout the culturing to be at a concentration of 0.05 g / L, 0.2 g / L or 0.5 g / L, respectively. Glucose contro...

example 3

Cell Growth vs Glucose Concentration

[0093] The effects of glucose concentration and glucose control on growth of CHO cells transformed with TBFβ were investigated. CHO cells were transformed with TGFβ using the techniques disclosed in Brunner et al., Mol. Endocrinol., 6(10): 1691-1700 (1992). The results are depicted in FIG. 4; the symbols represent the experiment wherein on line glucose control (OLGC) at 0.1 g / L using FIA was performed throughout the culturing, the ♦ symbols represent the experiment wherein the glucose was added initially in batch (8 g / L). In the experiments, CHO cells were cultured in an Applikon bioreactor at a 2.5 liter working volume. Super cell culture medium (with adjusted NaCl and glucose concentrations) was used and the CHO cells were stirred at 150 rpm agitation. The initial osmolality was adjusted to 280 mOsm via the addition of NaCl. The osmolality of the medium was measured using an OSMETTE™ osmometer. The cell seed density was 8×105 cells / ml. The oth...

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Abstract

A method of producing a polypeptide in fed batch cell culture is provided which involves an initial cell growth phase and a distinct production phase. In the initial growth stage, animal cells having nucleic acid encoding the polypeptide are cultured at a starting osmolality of about 280-330 mOsm in the presence of a concentration of glucose controlled throughout the culturing to be within a range between about 0.01 and 1 g/L. This is followed by a production phase, where the cultured animal cells of the growth phase are inoculated at a cell seed density of at least 1.0×106 cells/mL and the cells are cultured at a starting osmolarity of about 400-600 mOsm in the presence of a concentration of glucose controlled throughout the culturing to be within a range between about 0.01 and 1 g/L. Preferably, the glutamine concentration in the cell culture medium is simultaneously controlled in order to curtail production of lactic acid and ammonia which result from unnecessarily high glutamine concentrations. During the growth phase, production of potentially detrimental metabolic waste products, such as lactic acid, is controlled thereby curtailing the increase of osmolality due to accumulation and neutralization of waste products. Thus, the cell growth can be improved. In the production phase, the cell culture conditions are modified in order to arrest or reduce cell growth and thereby direct nutrient utilization toward production, as opposed to cell growth. Overall, it is intended that the method results in an improvement in specific productivity, reduction in production run times and/or an increase in final product concentration.

Description

BACKGROUND OF THE INVENTION [0001] I. Field of the Invention [0002] This invention relates to a method of improving polypeptide production in animal cell culture. In particular, it is directed to a method of culturing mammalian cells under conditions wherein the glucose concentration in the cell culture medium and the osmolality of the medium are controlled, so as to either promote cell growth or to promote recombinant polypeptide production. [0003] II. Description of Related Art [0004] With the advent of recombinant DNA technology the number of polypeptides which are able to be produced in recombinant cell culture has greatly increased. While some recombinant DNA techniques rely on bacterial or yeast cells for the production of polypeptides, production of polypeptides in animal cells (especially mammalian cells) is becoming widespread, particularly for the production of mammalian polypeptides. Similarly, cell fusion techniques for preparing hybridomas, which may be cultured to prod...

Claims

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Application Information

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IPC IPC(8): C12N5/00C12N5/02C12N5/071C12P21/02C12P21/06
CPCC12N5/0018C12N2500/32C12N2500/34C12N2500/60C12N2510/02C12P21/02C12P21/06
Inventor CHEN, MARYFORMAN, LAWRENCE W.
Owner GENENTECH INC
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