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
  • C07K14/575—Hormones
  • C07K14/61—Growth hormone [GH], i.e. somatotropin
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
  • C07K1/14—Extraction; Separation; Purification
• • 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
  • C07K14/575—Hormones
  • C07K14/62—Insulins
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
  • C12M29/04—Filters; Permeable or porous membranes or plates, e.g. dialysis
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
  • C12M29/18—External loop; Means for reintroduction of fermented biomass or liquid percolate
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
  • C12M47/10—Separation or concentration of fermentation products
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
  • C12M47/12—Purification
• • 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
  • C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
• • 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
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
  • Y02P20/00—Technologies relating to chemical industry
  • Y02P20/50—Improvements relating to the production of bulk chemicals
  • Y02P20/582—Recycling of unreacted starting or intermediate materials

Definitions

• a process and an apparatus for the recovery of a poly ⁇ peptide from a fermentation broth A process and an apparatus for the recovery of a poly ⁇ peptide from a fermentation broth.
• the present invention concerns a process for recovering a polypeptide from a fermentation broth.
• Polypeptides which were previously recovered from ani ⁇ mal tissue or organs, e.g. by extraction, are now increas ⁇ ingly produced by bio-technjcal processes using micro ⁇ organisms, such as bacteria or yeast cells, transformed by engineering in a manner such as to form the desired polypeptide as a fermentation product.
• the desired polypeptide In fermentation of a substrate by means of the transformed microorganisms the desired polypeptide is in certain cases secreted to the medium.
• the polypeptide will occur in a low concentration, e.g. 10-200 mg/1, and will be mixed with proteolytic enzymes which have simultaneous ⁇ ly been secreted from the microorganism.
• the fermenta- tion is usually carried out at a relatively high tempera ⁇ ture, such as from 25-40°C, with a consequent risk of pro ⁇ teolytic degradation as well as de ⁇ aturation of the poly ⁇ peptide.
• isolation of the desired polypeptide from the fer ⁇ mentation broth is desirable - preferably by a continuous and gentle process comprising a minimum of unit operations, and so that the isolated product is present in a form which lends itself to further purification.
• the present invention is based on the finding that the po ⁇ lypeptide can be extracted directly from the fermentation broth under specific conditions, if desired even during the very fermentation.
• the invention concerns a process for recovering a polypeptide from a fermentation broth, said polypeptide having been formed by fermentation with a microorganism, said fermentation broth being treated with a chromato ⁇ graphic material containing hydrophobic groups and adsorb- ing the polypeptide contained in the fermentation broth, and the process is characterized by passing the fermenta ⁇ tion broth with its content of polypeptide and optional ⁇ ly also the microorganism directly through a bed of the said chromatographic material containing hydrophobic groups, and then eluting the polypeptide adsorbed on the chromatographic material with an aqueous medium which, if desired, may contain water miscible organic solvent.
• the process is carried out as stated, it is possible, in a single step, to recover the desired polypeptide di ⁇ rectly, with a high yield and a high concentration fac ⁇ tor. Owing to its process-technical simplicity the pro ⁇ cess is very advantageous.
• the desired polypeptide is obtained in the eluate in a high yield and great puri- ty, so that the subsequent purification can be performed without complications.
• the fermentation broth is treated continuously with the micro ⁇ organism in a suspended state and during the very fermen ⁇ tation, the broth being circulated through the bed of the chromatographic material, expediently in a fluidized state, and recycled for further fermentation.
• the continuous re ⁇ moval of the fermentation product i.e. the desired poly ⁇ peptide, reduces the risk of degradation, thus giving an optimum yield.
• This effect can be intensified if the broth is kept cooled to 1-12°C, preferably 4-6°C, during the cir ⁇ culation.
• the process of the invention has been found to be useful in the recovery to proinsulin or other insulin-like material, called ILM, from a fermentation broth formed by cultivation of a correspondingly transformed yeast cell.
• the fermentation broth is admixed with ions having a high protein salting-out effect in a concentra ⁇ tion of 0.01 - 1 molar, preferably 0.05-0.4 molar.
• Suitable ions are P0. 3-, SO. 2-, CCrH-U,COO NH
• Human growth hormone, hGH has been recovered in a cor ⁇ responding manner from a fermentation broth formed by cul ⁇ tivation of a substrate with a microorganism.
• the chromatographic material must have a suitable speci ⁇ fic affinity to the desired polypeptide.
• chro- matographic materials suitable in connection with ILM or hGH are sepharose containing aryl or alkyl groups, e.g. octyl sepharose and phenyl sepharose.
• ILM binds so strong ⁇ ly to these gels that it is not necessary to add ions hav ⁇ ing protein salting-out effect to the fermentation broth.
• Phenyl sepharose is the preferred chromatographic material for the recovery of ILM since this protein binds suit ⁇ ably strongly, without rendering the subsequent elution di fficult .
• An eluate is formed in the process of the invention, con* taining polypeptides in a concentrated form, and the de ⁇ sired polypeptide can be isolated from the eluate in a pure state in a manner known per se.
• the invention also concerns an apparatus for carrying out the process of the invention, said apparatus being characterized by the features defined in the character-, izing portion of claim 10.
• the apparatus comprises a fermentation container 1 which can be supplied with substrate or nutrient fluid from a supply tank 15 through a supply pipe 16. The amount of supplied nutrient fluid can be adjusted by means of a valve 16a.
• a pipe 2 leads from the container 1 through a pump 3 to an ultrafiltration apparatus 5.
• a conduit 6 for retentate ⁇ _aleads back to the tank 1,' and a conduit 7 for filtrate leads to two columns 8, 9 containing chromato ⁇ graphic material.
• a (not shown) heat exchanger may be in ⁇ serted in the conduits 2 and 6 to recover heat, if the temperature in the ultrafiltration apparatus 5 has been adjusted considerably lower than the temperature of the fermentation broth in the container 1.
• conduits 17, 19, 22 connect the contai ⁇ ner with a pump 18 and a centrifuge 20, which is provided with a drain pipe 21 for yeast concentrate.
• the columns 8, 9 are connected with a tank 11 for equili ⁇ bration liquid or elution liquid, which can be passed to one or the other of the columns 8, 9 by adjustment of a change-over valve 12 and valves 12a and 12b.
• a valve 13a or 13b By opening of a valve 13a or 13b, the eluate can be passed through a pipe 13 to a storage tank 14 for eluate.
• the fermentation broth was passed through an ultrafiltra ⁇ tion system 5 of the Millipore Pellican Casette Svstem type with an 0.5 ⁇ diaphragm.
• the retentate consisting of a concentrated suspension of the yeast cells, was re ⁇ cycled through the pipe 6 to the fermentation container 1.
• 25 1 of a human proinsulin-producing yeast culture (Sacc- haromyces cerevisiae, AB 103-1 containing the plasmid py- BCA 5) were cultivated to an optical density of 4.3 at 37°C, measured at 610 n in the fermentation container 1.
• the fermentation broth contained human semi-synthetic insulin in a concentration of 100 mg/1.
• the fermentation broth including yeast cells and insulin, was passed through an ultrafiltration apparatus 5 of the Pellicon Casette System type with an 0.5 ⁇ diaphragm and recycled to the fermentation container 1.
• the eluate from the column 8 was recycled via the conduit 10 to the fermentation container 1.
• the above-mentioned change between the two columns 8 and 9 was repeated for a total of 4 times.
• the overall volume in the storage tank 14 was measured to 1.2 1 with an insulin concentration of 1.8 mg/ml, corresponding to a recovery percentage of 88.
• the yeast suspension - cultivated to an optical density of 16.6 - was applied from below and up through a 1.6 x 5 cm column, containing suspended phenyl sepharose pa rt icles to form a fluidizing bed. Amount of flow 400 ml/hour. Temperature 20°C The column had been equilibrated before ⁇ hand with 0.3 M ammonium phosphate, pH 8.0. The applica ⁇ tion was performed as a cylic process during 16 hours, and then flushing was effecting with 65 ml of an equ--.li- bration buffer. The top piston was then pressed down to the surface of the gel, and elution was performed with 100 ml of H Von0 in a direction from top toward bottom.
• the amount of hGH present in the application liquid, flush ⁇ ing liquid and eluate were determined by RP-HPLC.
• the application liquid contained 5 mg of BhGH, the flus ⁇ hing liquid contained 0.2 mg of BhGH, and the eluate con ⁇ tained 4.8 mg of hGH.
• 96? ⁇ of the present hGH was thu-s: eluted in an aqueous solution without yeast cells with a con ⁇ centration factor of 5.
• the broth was applied at 4°C to a column from Pharmacia, diameter 5.0 cm, height 15 cm, packed with phenyl sepha ⁇ rose CL 4E , equilibrated with a buffer 0.1 M (NH ⁇ ) S0 4 , adjusted to a pH of 7.2. Elution of the insulin-like ma- terial during application could not be detected. After completed application, flushing was performed with 3 co ⁇ lumn volumes of an equilibration buffer, followed by elu ⁇ tion of the insulin-like material with H consult0.
• the yeast suspension - cultivated to an optical density of 17 - was applied from below and up through a 1.6 x 5 cm column containing suspended phenyl sepharose particles to form a fluidized bed.
• flushing was performed with 65 ml of an equilibration buffer, and then the flow was stopped to sediment the gel.
• the top piston was then pressed down to the surface of the gel, and a column volume of H ? 0 was applied in the direction from top toward bottom. The flow was then stopped for 30 minutes, followed by elution with 25 ml of H ? 0. The eluate was a clear, slightly yellowish insulin solu ⁇ tion without yeast cells.
• the amount of insulin present in the application liquid and eluates was determined on RP-HPLC. In the applica ⁇ tion, 25.5 mg were bound to the column, and then the co ⁇ lumn was apparently saturated. During the flushing pro ⁇ cess an amount corresponding to 2.9 mg was detected in the eluate. Then 25.0 mg of insulin were elu ted with H-0.

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• Biotechnology (AREA)
• Biochemistry (AREA)
• General Health & Medical Sciences (AREA)
• Microbiology (AREA)
• General Engineering & Computer Science (AREA)
• Sustainable Development (AREA)
• Biomedical Technology (AREA)
• Molecular Biology (AREA)
• Endocrinology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
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• 1984
  • 1984-06-25 DK DK309184A patent/DK309184D0/da unknown
• 1985
  • 1985-06-25 EP EP85903232A patent/EP0185078A1/en active Pending
  • 1985-06-25 JP JP60503018A patent/JPS61502585A/ja active Pending
  • 1985-06-25 AU AU46079/85A patent/AU4607985A/en not_active Abandoned
  • 1985-06-25 WO PCT/DK1985/000062 patent/WO1986000339A1/en not_active Application Discontinuation
• 1986
  • 1986-02-24 DK DK84386A patent/DK84386A/da not_active Application Discontinuation

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