Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Lymphokines – e.g. – interferons – interlukins – etc.
Patent
1989-10-18
1992-08-04
Draper, Garnette D.
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Lymphokines, e.g., interferons, interlukins, etc.
530395, 530412, 530418, 530419, 530422, 530427, 530825, 435 695, C07K 312
Patent
active
051360241
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
The present invention relates to extraction of granulocyte/macrophage colony stimulating factor (GM-CSF) from GM-CSF-expressing bacteria.
Granulocyte/macrophage colony stimulating factor is believed to be a potential therapeutic agent against infection and cancer. Clinical testing and widespread use of GM-CSF have been delayed owing to the unavailability of sufficient quantities of the material and the great expense of obtaining GM-CSF from natural sources. Recombinant DNA techniques have been used to create bacteria capable of expressing GM-CSF; see, for example, DeLamarter et al., EMBO J., Vol. 4, 2575-2581 (1985). Fermentation of such bacteria is expected to yield sufficient quantities of GM-CSF at substantially lower cost than would be possible utilizing natural sources of GM-CSF. However, clinical use of GM-CSF also requires high purity material that is not contaminated by cell constituents or cell debris of the GM-CSF-expressing bacteria. Contamination by such impurities could result in adverse reactions or in test results that are not reproducible. Accordingly, extraction of GM-CSF from the cells of GM-CSF-expressing bacteria in sufficiently high purity and yield for clinical use has been a major problem.
SUMMARY OF THE INVENTION
The present invention is based on the discovery that GM-CSF can be extracted from GM-CSF-expressing bacteria in high yield and purity by treating a suspension of GM-CSF-containing bacterial cells with an acid and an enhancing agent, or with an acid that is itself an enhancing agent, removing and discarding substantially all of the suspension liquid from the cells, preparing a second suspension of the treated cells, neutralizing said second suspension and separating the GM-CSF-containing liquid from the suspended cells. In accordance with the method of the present invention, GM-CSF is obtained from the cells without the need for mechanical or enzymatic disruption of the cell surface. The method of this invention allows recovery of GM-CSF in a manner which significantly reduces contamination by cell constituents, and subsequent purification is easier and less expensive.
The acid in the killing step is supplemented with an "enhancing agent" that increases the kill at a given pH and that also preferably helps the escape of GM-CSF from the cells.
The word "neutralizing" in the foregoing paragraph means that the second suspension is rendered approximately neutral (e.g. pH 6.0 to 8.0) or weakly alkaline (e.g. up to about pH 9.0).
BRIEF DESCRIPTION OF THE DRAWING
FIG. 1 is a construction map of plasmid pAKG-151.
DETAILED DESCRIPTION
The present invention provides a method for extracting GM-CSF from GM-CSF-expressing bacterial cells comprising: and an enhancing agent, or with an acid that is itself an enhancing agent; cells; cells.
In carrying out the method of the present invention, acid is added to a suspension of GM-CSF-expressing cells to adjust the pH to a lethal value for the cells, i.e. to about 1.5 to 3.0, preferably to about 2.0 to 2.2. Examples of suitable acids that can be utilized in this invention are hydrochloric acid, nitric acid, phosphoric acid and sulfuric acid. Phosphoric acid is the preferred acid.
It should be noted that acid alone at pH 3.0 will kill the bacteria, but the low pH required for complete kill (down to pH 1.5) can cause damage to the GM-CSF by denaturation or decomposition (e.g. deamination). However, the use of an "enhancing agent" provides complete kill of the bacteria at a higher pH, e.g. in particular 2.0 to 2.2, where the likelihood of damage to the GM-CSF is much lower. Killing all the bacteria at this stage is highly desirable to ensure containment of a genetically-engineered microorganism.
The enhancing agent can itself be an acid, e.g. trichloroacetic acid, when it may be the sole acid used in the killing step.
The use of an enhancing agent not only aids in killing the bacterial cells but also often serves to improve the yield of extracted GM-CSF. Examples of suitable enhancing agents include chaotropic
REFERENCES:
patent: 4364863 (1982-12-01), Leibowitz et al.
patent: 4675387 (1987-06-01), Korant
patent: 4801686 (1989-01-01), Kronheim
patent: 5047504 (1991-09-01), Boone
Miyajima et al., EMBD 1986, vol. 5, pp. 1193-1197.
Marston, Biochem. J. 1986, 240, pp. 1-12.
Davis et al., Nature, vol. 238, 1980, pp. 443-438.
Rosen, TIBTECH., 1986, pp. 251-252.
Light, BioTechniques vol. 3(4) 1985, pp. 298-306.
Hochhauser, High Technology Feb. 1983, pp. 55-60.
Pharmacia Bulletin #5001339, 1986, pp. 1-7 (Separation News).
Menge et al., Develop Biol. Standard, vol. 66, 1987, pp. 391-401.
Cantrell et al., Proc., Natl. Acad. Sci. U.S.A. 82:6250 (1985).
Clark et al., Science 236:1229 (1987).
DeLamarter et al., EMBO J. 4:2575 (1985).
Nunokawa, Chem. Abstracts 95:5416M (1981).
Alroy Yair
Leibowitz Paul
Draper Garnette D.
Dulak Norman C.
Nelson James R.
Schering Corporation
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