Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/66—Microorganisms or materials therefrom
  • A61K35/74—Bacteria
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/04—Immunostimulants
• • 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
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/02—Preparation of hybrid cells by fusion of two or more cells, e.g. protoplast fusion
  • C12N15/03—Bacteria

Definitions

• the present invention relates to the production of immunostimulating agents based on propionibacteria.
• Preparations for chemotherapy of tumors are known from DE-A 3 011 461 on the basis of the propionibacterial strains DSM 1773, 1772 and ATCC 6919.
• the bacteria are propagated under anaerobic conditions in liquid culture in 1 liter vessels.
• the bacterial cells are then centrifuged, ground, inactivated by boiling, subjected to trypsin treatment, centrifuged again and freeze-dried.
• the preparation so produced can then be resuspended and e.g. administered by intravenous injection.
• the manufacturing process described is complex and is an application of the means e.g. as an immunostimulating agent in the field of animal health in
• the present invention relates to the preparation of an immunostimulating agent based on propionibacteria, which is characterized in that the bacteria are multiplied anaerobically in a liquid culture, that after the fermentation has ended, the bacteria are inactivated in a conventional manner so that the bacteria obtained in this way Cell mass is separated, washed and dried.
• Propionibacterium avidum KP 40 deposited with the German Collection of Microorganisms under the number DSM 1772, is preferably used to produce the immunostimulating agents.
• the whole-line preparations based on Propionibacterium avidum KP 40 (DSM1772) are obtained in a simplified process in liquid culture in fermenters.
• the inoculation material used comes from a master stock. By using this seed principle, the use of a seed with a defined number of passages is guaranteed is identical in its properties to the strain deposited with the German Collection of Microorganisms
• the bacteria are propagated via a pre-culture and a main culture under anaerobic conditions at 30-39 ° C, preferably at 35-37 ° C, in nutrient-rich liquid media, the yeast extracts, which can also be tryptically digested, protein hydrolysates, glucose and contain organic acids such as pyruvate, lacatate or ⁇ -ketoglutarate. Complex media such as brain-heart broth can also be used.
• the preculture takes place in submerged stand cultures of 0.1-150 l volume for 18-30 hours.
• the main culture is inoculated with the preculture.
• the fermentation is carried out in controlled fermenters with a volume of 10-5000 1 with constant movement of the medium.
• the fermentation time is 24-72 hours, preferably 40-56 hours, particularly preferably 48 hours
• the bacteria are inactivated in the usual way by physical processes, for example the action of heat, UV or gamma radiation or chemical processes, for example the action of
• the inactivation by heat takes place in fermenters at 60-120 ° C, preferably at 75-85 ° C for 15-60 minutes, preferably 30-45 minutes.
• Chemical inactivation takes place in a known manner in suitable vessels, for example in fermenters.
• Formaldehyde is preferably used in concentrations of 0.01-0.5%, particularly preferably 0.05-0.2%.
• the inactivation takes place with constant movement of the inactivated material at 30-39 ° C., preferably at 35-37 ° C. over a period of 12-48 hours, preferably 18-30 hours
• the inactivated bacterial cells are separated from the culture broth by filtration or by centrifugation in a batch or flow process
• Filtration is preferably used using the countercurrent flow method using membranes with a pore size of 0.1-1 ⁇ m, preferably 0.22-0.45 ⁇ m.
• This method also allows the bacterial cells to be concentrated and the culture broth and the formaldehyde pressure levels to be washed out aqueous, buffered solutions with physiological salt concentrations, such as 0.9% saline solution or phosphate-buffered saline solution.
• physiological salt concentrations such as 0.9% saline solution or phosphate-buffered saline solution
• the bacterial cells are separated off by centrifugation at a centrifugal acceleration of 5,000-20,000 xg in portions in a batch process or in a flow-through process
• the sediments are resuspended in aqueous, buffered solutions with physiological salt concentrations, such as 0.9% saline solution or phosphate-buffered saline solution. This process is repeated several times to wash out the culture broth and the formaldehyde pressure levels
• the bacterial cells are subjected to a drying process, such as spray drying or freeze-drying. Freeze-drying is preferably used, the resuspended bacterial cells being freeze-dried either in aliquots directly in the removal container or in larger portions and then weighed into removal containers Protective colloids or stabilizers, defoamers and preservatives added
• the dried product is washed with a
• Reconstituted solvents such as aqua dest, aqua purificata or 0.9% saline
• Solution 1 is transferred to the fermenter after complete dissolution of all substances and sterilized together with the fermenter.
• Solutions 2 and 3 are autoclaved separately for 15 minutes immediately after preparation at + 110 ° C.
• Solutions 2 and 3 are added to solution 1 in the fermenter under sterile conditions Setting anaerobic conditions and checking the sterility, the fermenter is operated overnight at + 37 ° C, 100 revolutions per minute (rpm) and 1.5 l nitrogen / hour (N 2 / h). For preculturing, 100 ml culture medium are placed in a 100 ml Schott bottle transfers 1 ml of this medium to
• Fermenter content mixed with 27 ml formaldehyde solution 36.5% (corresponds to 0.1% formaldehyde as the final concentration).
• the inactivation takes place for 24 hours at + 37 ° C and reduced N 2 fumigation.
• the fermenter contents are then drained off and worked up.
• An inactivation control is created from this material (3 passages per week).
• the inactivated material is centrifuged in portions at 10,000 xg for 15 minutes. The supernatant is discarded, the sediments are combined and washed twice in phosphate-buffered saline (PBS).
• PBS phosphate-buffered saline
• the resuspended bacterial cells are then filled into 10 ml aliquots in sterile 25 ml bottles and freeze-dried.
• the finished preparations are stored at + 4 ° C

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• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Chemical & Material Sciences (AREA)
• General Health & Medical Sciences (AREA)
• Engineering & Computer Science (AREA)
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• Medicinal Chemistry (AREA)
• Molecular Biology (AREA)
• Organic Chemistry (AREA)
• Pharmacology & Pharmacy (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Animal Behavior & Ethology (AREA)
• Immunology (AREA)
• Public Health (AREA)
• Veterinary Medicine (AREA)
• Biotechnology (AREA)
• Zoology (AREA)
• Mycology (AREA)
• Epidemiology (AREA)
• Wood Science & Technology (AREA)
• General Engineering & Computer Science (AREA)
• Biomedical Technology (AREA)
• Biophysics (AREA)
• General Chemical & Material Sciences (AREA)
• Physics & Mathematics (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Plant Pathology (AREA)
• Cell Biology (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Biochemistry (AREA)
• Preparation Of Compounds By Using Micro-Organisms (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)
• Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
• Medicines Containing Material From Animals Or Micro-Organisms (AREA)

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Publications (1)

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Citations (2)

• 1996
  • 1996-07-26 DE DE19630230A patent/DE19630230A1/de not_active Withdrawn
• 1997
  • 1997-07-14 CA CA002261991A patent/CA2261991A1/en not_active Abandoned
  • 1997-07-14 TR TR1998/02777T patent/TR199802777T2/xx unknown
  • 1997-07-14 JP JP10508434A patent/JP2000516922A/ja active Pending
  • 1997-07-14 AU AU36948/97A patent/AU3694897A/en not_active Abandoned
  • 1997-07-14 EP EP97933674A patent/EP0918530A1/de not_active Withdrawn
  • 1997-07-14 PL PL97331146A patent/PL331146A1/xx unknown
  • 1997-07-14 CZ CZ99267A patent/CZ26799A3/cs unknown
  • 1997-07-14 CN CN97196768A patent/CN1226170A/zh active Pending
  • 1997-07-14 BR BR9710562A patent/BR9710562A/pt not_active Application Discontinuation
  • 1997-07-14 SK SK55-99A patent/SK5599A3/sk unknown
  • 1997-07-14 WO PCT/EP1997/003747 patent/WO1998004275A1/de not_active Application Discontinuation
• 1999
  • 1999-01-08 KR KR1019997000078A patent/KR20000023629A/ko not_active Application Discontinuation
  • 1999-01-25 NO NO990325A patent/NO990325D0/no not_active Application Discontinuation

Patent Citations (2)

Non-Patent Citations (4)

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