Mammallian cell culture process for protein production

Abstract

The present invention describes methods and processes for the production of proteins, particularly glycoproteins, by animal cell or mammalian cell culture, preferably, but not limited to, fed-batch cell cultures. In one aspect, the methods comprise at least two temperature shifts performed during the culturing period, in which the temperature is lower at the end of the culturing period than at the time of initial cell culture. Throughout their duration, the culturing processes of the invention involving two or more downward shifts in temperature sustain a high viability of the cultured cells, and can yield an increased end titer of protein product, and a high quality of protein product, as determined, e.g., by sialic acid content of the produced protein. In another aspect, the methods comprise the delayed addition of polyanionic compound during the culturing period. The delayed addition of polyanionic compound sustains a high viability of the cultured cells, and can extend the growth phase, delay the onset of the death phase, and arrest the death phase.

Description

[0001]This invention claims priority from provisional U.S. application Ser. No. 60 / 436,101 filed Dec. 23, 2002, which is incorporated herein by reference in its entirety.FIELD OF THE INVENTION[0002]The present invention relates to new methods and processes for culturing mammalian cells which produce a protein product, preferably a glycosylated protein product. Performance of the cell culturing methods and processes result in high cell viability and can also result in high product quality and quantity, extension of the growth phase, delay of onset of the death phase, and arrest of the death phase.BACKGROUND OF THE INVENTION[0003]Animal cell culture, notably mammalian cell culture, is preferably used for the expression of recombinantly produced, glycosylated proteins for therapeutic and / or prophylactic applications. Glycosylation patterns of recombinant glycoproteins are important, because the oligosaccharide side chains of glycoproteins affect protein function, as well as the intramo...

AI Technical Summary

Benefits of technology

[0015]The present invention provides new processes for the production of proteins, preferably recombinant protein products, more preferably glycoprotein products, by animal or mammalian cell cultures. These new processes achieve increased cell viability.

[0016]One aspect of this invention concerns the use two or more temperature shifts. In this aspect, cell culture processes of this invention can advantageously achieve an enhanced final titer or concentration of product, e.g., glycoprotein, as well as an enhanced sialic acid content of the glycoprotein produced by the cultured cells. More specifically, in accordance with this invention, two or more temperature shifts during the cell culturing period sustain a high cell viability of the cells in the culture and can provide a high quantity and quality of produced product throughout an entire culture run. Also, according to one aspect of the invention, the two or more temperature shifts comprising the culturing processes can advantageously allow for an extension of the production phase of the culture. During the extended production phase, the titer of the desired product is increased; the product quality, as characterized by sialic acid content, is maintained at a high level; and cell viability is also maintained at a high level. In addition, the extended production phase associated with the culturing processes of the invention allows for the production of product beyond that which is produced during a standard production phase.

[0017]In one aspect of the present invention, multi-step temperature shifts, preferably, timed multi-step temperature shifts comprising two or more downward temperature shifts, are used in the culturing of mammalian cells to produce a desired protein product, particularly, a glycoprotein product. Two or more (i.e., at least two) temperature shifts, which may be performed after the growth phase of the culture, comprise the processes of this invention. With the at least two temperature shifts, preferably with approximately four day increments between the shifts, a high protein yield with a concomitant high sialic acid content of the desired protein product can be achieved. The multiple temperature shifts comprising the culturing methods can achieve both high quality and quantity of protein product, as well as sustain cell viability for the duration of a culturing period.

Problems solved by technology

However, bacterial and yeast cells are limited in their ability to optimally express high molecular weight protein products, to properly fold a protein having a complex steric structure, and / or to provide the necessary post-translational modifications to mature an expressed glycoprotein, thereby affecting the immunogenicity and clearance rate of the product.

Run times of cell culture processes, particularly non-continuous processes, are usually limited by the remaining viability of the cells, which typically declines over the course of the run.

The presence of cell debris and the contents of dead cells in the culture can negatively impact on the ability to isolate and / or purify the protein product at the end of the culturing run.

However, suramin did not protect against apoptosis during the death phase.

As a result, suramin was capable of maintaining high viability during the growth phase, but did not allow for an extension of culture longevity.

Moreover, it has never been reported that dextran sulfate can delay the onset of the death phase, extend the growth phase, or arrest the death phase.

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