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WO2002064752A1 - Procede de production d'acides nucleiques - Google Patents

Procede de production d'acides nucleiques Download PDF

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Publication number
WO2002064752A1
WO2002064752A1 PCT/EP2002/000290 EP0200290W WO02064752A1 WO 2002064752 A1 WO2002064752 A1 WO 2002064752A1 EP 0200290 W EP0200290 W EP 0200290W WO 02064752 A1 WO02064752 A1 WO 02064752A1
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WO
WIPO (PCT)
Prior art keywords
cultivation
medium
concentration
nucleic acids
bacterial cells
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/EP2002/000290
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German (de)
English (en)
Inventor
Carsten Voss
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
PlasmidFactory GmbH and Co KG
Original Assignee
PlasmidFactory GmbH and Co KG
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by PlasmidFactory GmbH and Co KG filed Critical PlasmidFactory GmbH and Co KG
Publication of WO2002064752A1 publication Critical patent/WO2002064752A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • 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, e.g. protozoa; 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
    • C12N1/20—Bacteria; Culture media therefor
    • 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
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/67—General methods for enhancing the expression
    • C12N15/69—Increasing the copy number of the vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides

Definitions

  • the additional, defined nitrogen-containing component that enhances the product metabolism is a preliminary step in the biosynthesis of the nucleotide bases of the nucleic acids.
  • Complex components of nutrient media are undefined natural products or extracts obtained from them for the cultivation of microorganisms, such as yeast autolysate, yeast extract, peptones, malt or meat extracts.
  • the transformed bacterial cells are cultivated in a medium which contains the defined, synthetic constituents, organic carbon source, inorganic nitrogen source, mineral salt mixture and an additional, defined nitrogen-containing component which enhances the product metabolism. During cultivation, the transformed bacterial cells produce nucleic acids.
  • feed processes in which only small amounts of nutrients are available to the bacterial cells at the beginning of the cultivation and when these nutrients are used fresh medium flows in.
  • feed processes are known and correspond to the prior art (see, for example, Chen et al, J. Ind. Microbiol. Biotechnol. 18 (1997), 43-48, Lahijani et al, Hum. Gen. Ther. 7 (1996), 1971-1980, Schmidt et al WO 99/61633 (1999)).
  • the method according to the invention for producing plasmid-containing biomass in a synthetic medium is therefore superior to the prior art.
  • the defined components, which are produced synthetically can be used in the highest purity without contamination with undesirable substances and also do not vary in their composition.
  • the balanced composition of the defined, synthetic medium from organic carbon source, inorganic nitrogen source, mineral salt mixture and an additional, defined nitrogen-containing component, which enhances the product metabolism, is a decisive advantage of the method according to the invention and allows the transformed bacterial cells to metabolize the substrates and Produce nucleic acids in large quantities without the growth-inhibiting by-products are formed.
  • the defined organic carbon source is preferably from the group of the monosaccharides, disaccharides, polysaccharides or poryols, in particular CHucose, maltose, raffinose, galactose, trehalose, sucrose, fructose, mannose, sorbose, fucose, rhamnose, arabinose, xylose, ribose, manitol , Sorbitol or glycerin used as an organic carbon source.
  • the carbon source in the medium is used in a concentration of from 1 to 500 g / L, preferably 10 to 300 g / L and in particular 50 to 100 g / L.
  • the carbon sources can be chemically synthesized in the highest purity levels and are therefore not contaminated with undesirable substances or animal components.
  • the mineral salt mixture is additionally a trace element solution consisting of iron (ITI) chloride, zinc sulfate, manganese sulfate, cobalt sulfate, copper chloride, boric acid, sodium molybdate and citric acid, in particular iron (m) chloride in a concentration of 20 mM , Zinc sulfate in a concentration of 5 mM, manganese sulfate in a concentration of 11 mM, cobalt sulfate in a concentration of 2 mM, copper chloride in a concentration of 1 mM, boric acid in a concentration of 16 mM sodium molybdate in a concentration of 11 mM and citric acid in a concentration of 24 mM.
  • ITI iron
  • host strain-specific supplements e.g. Vitamins or other components that the host strain used cannot synthesize itself are added.
  • Numerous bacterial strains have, for example, thiamine auxotrophy, such as E. coli DH1 or E. coli DH5 ⁇ , which is why the vitamin is added to the medium when these host strains are used.
  • a particular advantage of the present invention is the fact that no antibiotics have to be added to the medium to increase the plasmid stability in order to cultivate the transformed bacterial cells. This is particularly advantageous for use of the purified nucleic acids as an active ingredient in cell and gene therapy or genetic vaccination.
  • the cultivation takes place in a pH range of 6.0-8.0, preferably in the range of 6.5 to 7.5.
  • the pH is regulated during the cultivation by adding an acid and / or base.
  • anti-foaming agents can optionally be added during the cultivation in order to avoid over-foaming.
  • Figure 7 Quality analysis of plasmid pUT649 from cultivation in a 30 L bioreactor using capillary gel electrophoresis.
  • Figure 8 plasmid concentration, plasmid mass fraction and dry biomass concentration in the cultivation of pUK21CMVß in E. coli DH5 ⁇ in the synthetic medium in the 7 L bioreactor.
  • Figure 9 Glutamate, glycerin, ammonium ion and acetate concentration during the cultivation of pUK21CMVß in E. coli DH5 ⁇ in the synthetic glycerin medium in the 7 L bioreactor.
  • Figure 12 Plasmid, dry biomass concentration and plasmid mass fraction during the cultivation of pUK21CMVß in E. coli DH5 ⁇ in semi-synthetic full medium with glutamate in the 7 L bioreactor.
  • the cultivation was carried out in a 7 L bioeactor (MBR, Wetzikon, Switzerland) in an aqueous, antibiotic-free, defined synthetic medium from 60 g L "1 GHycerin (87%), 2 g L “ 1 ammonium chloride, 30 g L "1 sodium L-GHutamate, 1.5 g L 1 KH 2 P04, 2.3 g L " 1 K 2 HPO 4 , 2.5 g L 1 NaCl, 5 g L 1 citric acid x H 2 0, 1.0 g L 1 MgSO 4 x 7 H 2 O, 10 mg L "1 thiamine hydrochloride and 2 mL L '1 trace element concentrate
  • Trace element concentrate consisted of 5.4 g L "1 FeCl 3 x 6 H 2 O, 1.38 g L " 1 ZnSO 4 x 7 H 2 O, 1.85 g L "1 MnSO 4 x H 2 O, 0.56 g L 1 CoSO 4 x 7 H 2 O, 0.17 g L "1
  • the dry biomass concentration and the plasmid concentration were determined by capillary electrophoresis (see Schmidt et al, J Biotechnol 49 (1996), 219-229) after isolation with a commercially available kit according to the manufacturer's instructions, and the concentration of medium constituents was monitored, and the typical course of the most important cultivation parameters, stirrer frequency, dissolved oxygen concentration (pO 2 ) and The course of the dry biomass, the plasmid concentration and the plasmid mass fraction during this cultivation is shown in Figure 2.
  • Example 2 Preparation of the plasmid pUT649 by bacterial cells in a defined synthetic medium using the batch method in a 30 L bioreactor
  • Example 4 Preparation of the plasmid pUK21CMVß by bacterial cells in synthetic M9 minimal medium using the batch method in a 7 L bioreactor
  • This example shows set cultivation in a synthetic medium which corresponds to the prior art.
  • the plasmid pUK21CMVß in E. coli DH5a was obtained by culturing in the described in the literature, synthetic M9 minimal medium (Sambrook et al, Molecular Cloning, CSH Press, 2 nd edition, 1989, Cold Spring Harbor New York), consisting of 11 g L "1 glucose x H 2 O, 1.0 g L " 1 Na2HPO 4 x 7 H 2 O, 3.0 g L '1 KH 2 PO 4 , 0.5 g L "1 NaCl, 2 , 5 g L "1 (NH4) 2 S04, 1.4 mg L 1 ZnSO 4 x 7 H 2 O, 5.4 mg L '1 FeCl 2 x 6 H 2 O, 1.6 mg L 1 MnSO 4 , 0.167 mg L "1 CuCl 2 , 0.56 g L " 1 CoSO 4 x 7 H 2 O, 0.22 g
  • the plasmid pUK21CMVß in E. coli DH5 ⁇ was prepared in a 7 L bioreactor in a rich semi-synthetic medium containing glycerol, soybean peptone and yeast extract, with and without glutamate.
  • 30 g L "1 glutamate was added to the semisynthetic medium in another

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  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Virology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne un procédé de production d'acides nucléiques, notamment d'acides nucléiques superspiralisés. La mise en culture des cellules bactériennes transformées, pour obtenir des densités cellulaires élevées, s'effectue de préférence par lots, dans un milieu aqueux synthétique défini qui ne contient pas de constituants complexes ou de constituants provenant d'animaux. Les acides nucléiques isolés à partir des cellules bactériennes et purifiés s'utilisent en génothérapie virale, en thérapie cellulaire ou en inoculation génétique.
PCT/EP2002/000290 2001-02-13 2002-01-14 Procede de production d'acides nucleiques Ceased WO2002064752A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE10106493.4 2001-02-13
DE2001106493 DE10106493B4 (de) 2001-02-13 2001-02-13 Verfahren zur Herstellung von Nukleinsäuren

Publications (1)

Publication Number Publication Date
WO2002064752A1 true WO2002064752A1 (fr) 2002-08-22

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PCT/EP2002/000290 Ceased WO2002064752A1 (fr) 2001-02-13 2002-01-14 Procede de production d'acides nucleiques

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DE (1) DE10106493B4 (fr)
WO (1) WO2002064752A1 (fr)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2007532102A (ja) * 2004-04-08 2007-11-15 ベーリンガー インゲルハイム オーストリア ゲゼルシャフト ミット ベシュレンクテル ハフツング 大腸菌ｋ−１２株ｊｍ１０８の発酵により製造規模でプラスミドｄｎａを生産する方法
WO2011007005A2 (fr) 2009-07-16 2011-01-20 Boehringer Ingelheim Rcv Gmbh & Co Kg Procédé pour réguler le nombre de copies plasmidiques dans e. coli
US8633211B2 (en) 2007-03-06 2014-01-21 Novartis Ag Bicyclic organic compounds suitable for the treatment of inflammatory or allergic conditions
US9969969B2 (en) 2004-04-08 2018-05-15 Boehringer Ingelheim Rcv Gmbh & Co Kg Fed-batch fermentation process and culture medium for the production of plasmid DNA in E. coli on a manufacturing scale
WO2019018270A1 (fr) 2017-07-21 2019-01-24 Conagen, Inc. Système de dépendance à un plasmide pour diriger l'expression d'un gène désiré
CN117431149A (zh) * 2023-12-22 2024-01-23 北京艺妙神州医药科技有限公司 一种菌体的洗涤方法

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DD239222A1 (de) * 1985-07-10 1986-09-17 Dresden Arzneimittel Verfahren zur herstellung von plasmid-dna vom cole1-typ
WO1998037179A2 (fr) * 1997-02-20 1998-08-27 Dsm N.V. Production fermentative de composes interessants, a l'echelon industriel et a l'aide de milieu definis chimiquement
WO1999061633A2 (fr) * 1998-05-25 1999-12-02 Qiagen Gmbh Methode d'isolement de l'adn plasmidique ccc

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DD239222A1 (de) * 1985-07-10 1986-09-17 Dresden Arzneimittel Verfahren zur herstellung von plasmid-dna vom cole1-typ
WO1998037179A2 (fr) * 1997-02-20 1998-08-27 Dsm N.V. Production fermentative de composes interessants, a l'echelon industriel et a l'aide de milieu definis chimiquement
WO1999061633A2 (fr) * 1998-05-25 1999-12-02 Qiagen Gmbh Methode d'isolement de l'adn plasmidique ccc

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
O'KENNEDY R D ET AL: "Effects of growth medium selection on plasmid DNA production and initial processing steps.", JOURNAL OF BIOTECHNOLOGY, vol. 76, no. 2-3, 21 January 2000 (2000-01-21), pages 175 - 183, XP002198241, ISSN: 0168-1656 *
ROTHEN S A ET AL: "Growth characteristics of Escherichia coli HB101(pGEc47) on defined medium.", BIOTECHNOLOGY AND BIOENGINEERING, vol. 58, no. 1, 5 April 1998 (1998-04-05), pages 92 - 100, XP002198242, ISSN: 0006-3592 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2007532102A (ja) * 2004-04-08 2007-11-15 ベーリンガー インゲルハイム オーストリア ゲゼルシャフト ミット ベシュレンクテル ハフツング 大腸菌ｋ−１２株ｊｍ１０８の発酵により製造規模でプラスミドｄｎａを生産する方法
US9969969B2 (en) 2004-04-08 2018-05-15 Boehringer Ingelheim Rcv Gmbh & Co Kg Fed-batch fermentation process and culture medium for the production of plasmid DNA in E. coli on a manufacturing scale
US8633211B2 (en) 2007-03-06 2014-01-21 Novartis Ag Bicyclic organic compounds suitable for the treatment of inflammatory or allergic conditions
WO2011007005A2 (fr) 2009-07-16 2011-01-20 Boehringer Ingelheim Rcv Gmbh & Co Kg Procédé pour réguler le nombre de copies plasmidiques dans e. coli
WO2019018270A1 (fr) 2017-07-21 2019-01-24 Conagen, Inc. Système de dépendance à un plasmide pour diriger l'expression d'un gène désiré
CN117431149A (zh) * 2023-12-22 2024-01-23 北京艺妙神州医药科技有限公司 一种菌体的洗涤方法
CN117431149B (zh) * 2023-12-22 2024-03-08 北京艺妙神州医药科技有限公司 一种菌体的洗涤方法

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DE10106493A1 (de) 2002-08-14
DE10106493B4 (de) 2010-11-04

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