Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Using tissue cell culture to make a protein or polypeptide
Patent
1990-02-22
1991-03-26
Schwartz, Richard A.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Using tissue cell culture to make a protein or polypeptide
435212, 435 701, 4352401, C12P 2104
Patent
active
050028774
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
This invention concerns the production of enzymes from cells, particularly from cell lines such as CNCM I-222 which produces tissue plasminogen activator (tPA).
BACKGROUND OF THE INVENTION
In a paper by J. B. Griffiths and A. Electricwala entitled "Amplification of Tissue Phasminogen Activator Expression from Epithelial Lines" given at the 7th General Meeting of ESACTR on Advances in Aninmal Cell Technology: Cell Engineering Evaluation and Exploitation, at Baden, Australia, 1985 and published in Develop. biol. Standard., Volume 66 pages 417-422 (S. Karger, Basel 1987), reference is made to a process for achieving expression of tPA from established epithelial cell lines, one derived from human breast tissue (BEB) and another from guinea pig ear keratocytes (GPK). The GPK cell line has been deposited in the Collection Nationale de Cultures de Microorganismes (CNCM) at the Institute Pasteur in Paris, France under accession number CNCM I-222.
The enzyme was found to be mainly expressed during the cell growth phase rather than from stationary culture cells, and production involved a two-step operation with initial growth to about 70% confluency in the presence of serum followed by a change to serum-free conditions for the final period of growth, after which the enzyme was harvested.
The paper discusses various approaches to increase the enzyme yield. The addition of mitogenic lectins was reported to increase the enzyme yield 15-20 fold.
In a typical enzyme production process based on this approach, cells are inoculated into roller bottles and incubated at 36.5.degree. C. for 72 hours in culture medium containing animal serum, resulting in growth to about 70% confluency. At the end of the 72 hours the growth medium is poured off, the cell washed twice and the medium replaced with half the original volume of culture medium containing no serum but with 50 microgarms/ml of a lectin, the most effective of which was found to be concanavalin A. Experiments showed this to be the optimum concentration of concanavalin A. The cultures are then incubated in the presence of lectin for a further 48 hours at 36.5.degree. C. after which supernatants (containing enzyme) are harvested and the cultures discarded.
SUMMARY OF THE INVENTION
According to the present invention it has been found that by employing less concentrated lectin in the culture medium added to replace the initial growth medium than is used in the prior art process, the culture can be reactivated after the first harvesting of enzyme by the addition of fresh culture medium containing an even lower lestin concentration, and after incubation for a similar period of time a further harvest of supernatants containing the desired enzyme can be harvested. The two harvests of enzyme together can give a greater overall yield than a single harvest after a single induction step employing the previously used higher lectin concentration.
Hence, according to the present invention there is provided a process for producing an enzyme from cells, comprising incubating cells in culture medium at a suitable temperature and for a suitable time to produce growth of the cells; removing the growth medium; adding a further supply of fresh culture medium containing a lectin at a first concentration substantially less than 50 micrograms/ml but containing no serum; incubating the culture at a suitable temperature and for a suitable time for production of enzyme from the cells; harvesting supernatants including the desired enzyme; adding to the culture after harvesting of the supernatants a further supply of fresh culture medium containing a lectin at a second concentration lower than said first concentration but containing no serum; incubating the culture at a suitable temperature and for a suitable time for production of enzyme from the cells; and harvesting supernatants including the desired enzyme.
It has been found that the enzyme yield can be further increased by repeating the last incubation step once or twice, by the addition of fresh serum-free "inducti
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patent: 4889808 (1989-12-01), Rappaport
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Chemical Abstracts, vol. 102, No. 7, Feb. 18, 1985, (Columbus, Ohio, U.S.), Schuyler, M. et al., p. 406, abstract 59984b.
Chemical Abstracts, vol. 92, No. 7, Feb. 18, 1980, (Columbus, Ohio, U.S.), Mochan, E., p. 148, abstract 52788g.
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Brouty-Boye, G. et al., Bio/Technology, Dec., p. 1058, 1984.
Kearns Michael J.
McEntee Ian J.
North John R.
Bond Eugene M.
Porta Marianne
Porton Products Ltd
Public Health Lab. Service BD
Schwartz Richard A.
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