Polypeptide production in animal cell culture

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...

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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 osmolality within a desired range has not been proposed by these researchers.

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.

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