Polypeptide production in animal cell culture

Details Polypeptide production in animal cell culture Polypeptide production in animal cell culture

1998-05-04

2001-01-30

Stole, Einar (Department: 1652)

Chemistry: molecular biology and microbiology

Animal cell, per se ; composition thereof; process of...

Rodent cell, per se

C435S325000

Reexamination Certificate

active

06180401

ABSTRACT:

BACKGROUND OF THE INVENTION
I. Field of the Invention
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.
II. Description of Related Art
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 produce monoclonal antibodies (MAbs), are widely used.
Accordingly, techniques have been developed for enhancing cell growth and/or polypeptide production by such genetically modified animal cells. Several groups have looked at the effects of osmolality on cell growth and polypeptide production. See, for example, Stubblefield et al.,
Cancer Research,
20:1646-1655 (December 1960); Garcia-Perez et al.,
Journal of Biological Chemistry,
264(28):16815-16821 (1989); Miner et al.,
Invasion Metastasis,
1:158-174 (1981); GB 2,251,249; EP 481,791; U.S. Pat. No. 5,151,359; U.S. Pat. No. 4,724,206; U.S. Pat. No. 5,122,469; and WO 89/04867. Various osmolality ranges for cell growth or polypeptide production are recommended and, generally, the osmolality of the cell culture medium is increased via the addition of NaCl or amino acids. 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.
Others have discussed the effect of glucose concentration on cell growth and/or polypeptide production in recombinant cell culture. See, for example, Park et al.,
Biotechnology and Bioengineering,
40:686-696 (1992); Huang et al.,
Journal of Biotechnology,
18:161-162 (1991); EP 387,840; Reuveny et al.,
Journal of Immunological Methods,
86:53-59 (1986); Fine et al.,
In Vitro,
12(10):693-701 (1976); Dircks et al.,
Exp. Eve Res.,
44:951-958 (1987); Mizutani et al.,
Biochemical and Biophysical Research Communications,
187(2):664-669 (September 1992); Sugiura
Biotechnology and Bioengineering,
39:953-959 (1992); WO 88/01643 Graf et al.,
DECHEMA Biotechnol. Conf.,
3:615-618 (1989); Japanese Patent Appln No. JP 1-101882; U.S. Pat. No. 3,926,723; WO 87/00195; and Fleischaker, Jr., Ph.D. Thesis,
Massachusetts Institute of Technology,
pp. 196-229 (June 1982).
Glacken et al.,
Biotechnol. Bioeng.,
28: 1376-1389 (1986) have also studied the effect of glutamine on cell cultures.
However, the concept of controlling glucose in fed-batch cell culture in order to control osmolality within a desired range has not been proposed by these researchers.
Accordingly, it is an object of the present invention to provide a method of controlling fed batch cell culture conditions for growth of animal cells so as to maintain high cell viability or extend the period of rapid cell growth. It is a further object to control production of potentially detrimental metabolic waste products, such as lactic acid, during culturing of mammalian cells. Also, it is an object to curtail the increase of osmolality, due to accumulation and neutralization of waste products and subsequent replacement of consumed glucose. Thus, cell viability can be improved by controlling the osmolality and production of waste products.
It is another object of the invention to provide a method of manipulating fed batch cell culture conditions to increase production of a polypeptide by animal cells which have nucleic acid encoding the polypeptide. In the production phase, the cell culture conditions are modified in order to arrest or curtail cell growth and thereby direct nutrient utilization toward production, as opposed to cell replication. 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.
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 enables the growth phase of the production culture to be reduced or eliminated, thereby resulting in a concomitant decrease in the time required for optimal production of the polypeptide of interest by the cell culture. This is particularly desirable for products such as DNase which tend to undergo deamidation in cell culture over protracted periods.
It is a further object of the invention to control the osmolality of an animal cell culture to be substantially maintained within a desired range, via control of the glucose and, optionally, glutamine concentration in the culture medium. This is particularly desirable insofar as the optimal osmolalities for animal cell growth and polypeptide production by animal cells have been identified herein.
Other objects and advantages of the present invention will become apparent to one of ordinary skill in the art.
SUMMARY OF THE INVENTION
In accordance with the objects of the invention, a method of growing animal cells in fed batch cell culture is provided which comprises the steps of culturing the cells at a starting osmolality of about 280-330 mOsm and controlling the glucose concentration in the cell culture to be between about 0.01 and 1 g/L throughout the culturing.
In another aspect, the invention relates to a method of producing a polypeptide by animal cells which contain nucleic acid encoding the polypeptide in fed batch cell culture, wherein the starting osmolality of the cell culture is about 400-600 mOsm and the glucose concentration in the cell culture is controlled to be between about 0.01 and 1 g/L throughout the culturing. The starting cell density of the cell culture is at least about 1.0×10
6
cells/mL, preferably at least about 3.0×10
6
cells/mL.
In yet a further aspect, 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 phase, animal cells having nucleic acid encoding the polypeptide are cultured at a starting osmolarity 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 about 1.0×10
6
cells/mL and the cells are cultured at a starting osmolality 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 control production of lactic acid and ammonia, which results from unnecessarily high glutamine concentrations.
In the preferred embodiment, the cells are mammalian cells, such as chinese hamster ovary (CHO) cells and the polypeptide is DNase or TGF-&bgr;.


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