Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/52—Cytokines; Lymphokines; Interferons
  • C07K14/53—Colony-stimulating factor [CSF]
  • C07K14/535—Granulocyte CSF; Granulocyte-macrophage CSF
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/06—Lysis of microorganisms
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
  • Y10S530/82—Proteins from microorganisms
  • Y10S530/825—Bacteria

Definitions

• the present invention relates to extraction of granulocyte/macrophage colony stimulating factor (GM-CSF) from GM-CSF-expressing bacteria.
• GM-CSF granulocyte/macrophage colony stimulating factor
• 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.
• 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.
• 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.
• 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).
• Figure 1 is a construction map of plasmid pAKG-151.
• the present invention provides a method for extracting GM-CSF from GM-CSF-expressing bacterial cells comprising.
• 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.
• 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.
• the enhancing agent can itself be an acid, e.g. trichloroacetic acid, when it may be the sole acid used in the killing step.
• an enhancing agent not only aids in killing the bacterial cells but also often serves to improve the yield of extracted GM-CSF.
• suitable enhancing agents include chaotropic ions (or compounds providing them) , such as trichloroacetate, perchlorate, thiocyanate and guanidinium; non-chaotropic salts, such as sodium chloride, sodium phosphate; and non-ionic chaotropes such as urea. Chaotropic ions are the preferred enhancing agents.
• Trichloroacetate is the most preferred enhancing agent (about 0.1M to 2.0M depending on such variables as cell density, pH and salt composition) .
• the enhancing agent is a chaotropic ion it may be added either as a chaotropic salt, such as sodium thiocyanate or guanidinium chloride, or as a chaotropic acid, such as trichloroacetic acid or perchloric acid. If a chaotropic acid is used, then less or none of the other acid may be necessary.
• a chaotropic salt such as sodium thiocyanate or guanidinium chloride
• a chaotropic acid such as trichloroacetic acid or perchloric acid. If a chaotropic acid is used, then less or none of the other acid may be necessary.
• phosphoric acid is added to the suspension to lower the pH to about 4 to 5, preferably 4.5, and then trichloroacetic acid is added to lower the pH to about 2.0.
• the temperature range for the acid treatment should be from about 10°C to about 40°C, and preferably about 25°C.
• all subsequent steps of the method of this invention are carried out at a temperature of from about 0°C to about 40°C, preferably 0° to 4°C.
• the cells are separated from the treatment liquid by microfiltration, centrifugation or the like, preferably by centrifugation, and resuspended in an aqueous buffer solution or in water.
• buffers that may be used in resuspending the pellet are sodium phosphate, potassium phosphate and tris (hydroxymethyl) aminomethane hydrochloride.
• Preferred buffers are sodium phosphate and especially tris (hydroxymethyl) aminomethane hydrochloride.
• the cell suspension is neutralized to a pH of about 6.0 to 9.0, preferably 7.2 to 7.6.
• suitable bases that may be used in the neutralization step are sodium hydroxide, potassium hydroxide and the like.
• bacteria that can be altered by recombinant DNA techniques to produce GM-CSF and from which GM-CSF may then be extracted using the method of the present invention are E. coli. Bacillus subtilis, Streptomyces coelicolor, and the like. The preferred bacterium is E. coli.
• the method of the present invention may be used with bacteria that express different forms of GM-CSF, for example human GM-CSF [Lee et al. , Proc. Natl. Acad. Sci. USA, Vol. 82, 4060-4064 (1985)], or murine GM-CSF [Burgess et al., J. Biol. Chem., Vol. 252, 1998-2033 (1977)].
• human GM-CSF Lee et al. , Proc. Natl. Acad. Sci. USA, Vol. 82, 4060-4064 (1985)]
• murine GM-CSF Burgess et al., J. Biol. Chem., Vol. 252, 1998-2033 (1977)].
• the following Example describes the invention in detail. It will be apparent to those skilled in the art that modification of materials and methods may be practised without departing from the purpose and intent of this disclosure.
• the human GM-CSF expression plasmid, pAKG-151 used in this example consists of about 3800 base pairs and includes the following sequences (see Figure 1):
• Cultivate a culture of E. coli strain 294 harboring the plasmid pAKG-151 in 200 ml of broth contained in a 2 liter baffled shake-flask at 30°C.
• the broth consists of 30 g/1 of casein hydrolysate, 20 g/1 of yeast extract, 20 g/1 of glycerol, 10 mg/1 of kanamycin, 5 g/1 KH 2 P0 4 1 g/1 MgS0 4 *7H 2 0, 0.1 ml/1 of an antifoa agent, and water.
• the initial pH is adjusted to 7.0 with sodium hydroxide. Agitate until the cellular density of the culture reaches about 4 optical density units (lightpath 1 cm, 660 ym).

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• 1987
  • 1987-05-11 US US07/048,187 patent/US4912200A/en not_active Expired - Lifetime
• 1988
  • 1988-05-11 US US07/435,510 patent/US5136024A/en not_active Expired - Lifetime
  • 1988-05-11 JP JP63503997A patent/JPH0771507B2/ja not_active Expired - Lifetime
  • 1988-05-11 EP EP88304268A patent/EP0291294B1/en not_active Expired - Lifetime
  • 1988-05-11 AT AT88304268T patent/ATE73853T1/de not_active IP Right Cessation
  • 1988-05-11 EP EP88904376A patent/EP0357674A1/en active Pending
  • 1988-05-11 WO PCT/US1988/001489 patent/WO1988008881A1/en not_active Ceased
  • 1988-05-11 ES ES198888304268T patent/ES2033428T3/es not_active Expired - Lifetime
  • 1988-05-11 DE DE8888304268T patent/DE3869195D1/de not_active Expired - Lifetime
• 1992
  • 1992-04-01 GR GR920400597T patent/GR3004227T3/el unknown
• 1994
  • 1994-12-01 HK HK135694A patent/HK135694A/en not_active IP Right Cessation

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