AU780122B2 - Production of peptides by fedbatch cultivation of a microorganism - Google Patents
Production of peptides by fedbatch cultivation of a microorganism Download PDFInfo
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- AU780122B2 AU780122B2 AU19105/01A AU1910501A AU780122B2 AU 780122 B2 AU780122 B2 AU 780122B2 AU 19105/01 A AU19105/01 A AU 19105/01A AU 1910501 A AU1910501 A AU 1910501A AU 780122 B2 AU780122 B2 AU 780122B2
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- 108090000765 processed proteins & peptides Proteins 0.000 title claims description 19
- 238000004519 manufacturing process Methods 0.000 title claims description 13
- 244000005700 microbiome Species 0.000 title claims description 10
- 238000012366 Fed-batch cultivation Methods 0.000 title claims description 9
- 102000004196 processed proteins & peptides Human genes 0.000 title description 4
- 238000000034 method Methods 0.000 claims description 42
- 230000010355 oscillation Effects 0.000 claims description 39
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 31
- 239000008103 glucose Substances 0.000 claims description 31
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 18
- 229910052799 carbon Inorganic materials 0.000 claims description 18
- 238000003756 stirring Methods 0.000 claims description 15
- 102000002265 Human Growth Hormone Human genes 0.000 claims description 11
- 108010000521 Human Growth Hormone Proteins 0.000 claims description 11
- 239000000854 Human Growth Hormone Substances 0.000 claims description 11
- 241000588724 Escherichia coli Species 0.000 claims description 8
- 102000018997 Growth Hormone Human genes 0.000 claims description 7
- 108010051696 Growth Hormone Proteins 0.000 claims description 7
- 239000000122 growth hormone Substances 0.000 claims description 7
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 6
- 150000003839 salts Chemical class 0.000 claims description 5
- 229910052500 inorganic mineral Inorganic materials 0.000 claims description 4
- 239000011707 mineral Substances 0.000 claims description 4
- 230000005428 wave function Effects 0.000 claims description 4
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 3
- 102000003978 Tissue Plasminogen Activator Human genes 0.000 claims description 3
- 108090000373 Tissue Plasminogen Activator Proteins 0.000 claims description 3
- 229910052757 nitrogen Inorganic materials 0.000 claims description 3
- 229960000187 tissue plasminogen activator Drugs 0.000 claims description 3
- 241000894006 Bacteria Species 0.000 claims description 2
- 101000976075 Homo sapiens Insulin Proteins 0.000 claims description 2
- 102000008070 Interferon-gamma Human genes 0.000 claims description 2
- 108010074328 Interferon-gamma Proteins 0.000 claims description 2
- 210000004102 animal cell Anatomy 0.000 claims description 2
- PBGKTOXHQIOBKM-FHFVDXKLSA-N insulin (human) Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@H]1CSSC[C@H]2C(=O)N[C@H](C(=O)N[C@@H](CO)C(=O)N[C@H](C(=O)N[C@H](C(N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=3C=CC(O)=CC=3)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC=3C=CC(O)=CC=3)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=3C=CC(O)=CC=3)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=3NC=NC=3)NC(=O)[C@H](CO)NC(=O)CNC1=O)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O)=O)CSSC[C@@H](C(N2)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](NC(=O)CN)[C@@H](C)CC)[C@@H](C)CC)[C@@H](C)O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)CC=1C=CC=CC=1)C(C)C)C1=CN=CN1 PBGKTOXHQIOBKM-FHFVDXKLSA-N 0.000 claims description 2
- 210000005253 yeast cell Anatomy 0.000 claims description 2
- 210000004027 cell Anatomy 0.000 description 7
- 230000008569 process Effects 0.000 description 7
- 239000000758 substrate Substances 0.000 description 7
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 6
- 230000035611 feeding Effects 0.000 description 6
- 235000003642 hunger Nutrition 0.000 description 6
- 229910052760 oxygen Inorganic materials 0.000 description 6
- 239000001301 oxygen Substances 0.000 description 6
- 230000037351 starvation Effects 0.000 description 6
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 5
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 5
- 108090000623 proteins and genes Proteins 0.000 description 5
- 102000004169 proteins and genes Human genes 0.000 description 5
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 4
- 238000010923 batch production Methods 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 239000002028 Biomass Substances 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 230000014509 gene expression Effects 0.000 description 3
- 238000004191 hydrophobic interaction chromatography Methods 0.000 description 3
- 230000001965 increasing effect Effects 0.000 description 3
- 230000000670 limiting effect Effects 0.000 description 3
- 230000004060 metabolic process Effects 0.000 description 3
- 235000010755 mineral Nutrition 0.000 description 3
- 238000012546 transfer Methods 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- 108020004511 Recombinant DNA Proteins 0.000 description 2
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 125000002791 glucosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000012092 media component Substances 0.000 description 2
- 230000000737 periodic effect Effects 0.000 description 2
- 239000013612 plasmid Substances 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 102000003839 Human Proteins Human genes 0.000 description 1
- 108090000144 Human Proteins Proteins 0.000 description 1
- 102000006992 Interferon-alpha Human genes 0.000 description 1
- 108010047761 Interferon-alpha Proteins 0.000 description 1
- 102100021941 Sorcin Human genes 0.000 description 1
- 101710089292 Sorcin Proteins 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 238000013019 agitation Methods 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 108010051210 beta-Fructofuranosidase Proteins 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 230000036755 cellular response Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000012786 cultivation procedure Methods 0.000 description 1
- 230000001086 cytosolic effect Effects 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 230000007717 exclusion Effects 0.000 description 1
- 239000011536 extraction buffer Substances 0.000 description 1
- 239000012527 feed solution Substances 0.000 description 1
- 239000006260 foam Substances 0.000 description 1
- 239000000446 fuel Substances 0.000 description 1
- 101150091570 gapA gene Proteins 0.000 description 1
- 230000004190 glucose uptake Effects 0.000 description 1
- 230000020169 heat generation Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 239000001573 invertase Substances 0.000 description 1
- 235000011073 invertase Nutrition 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000036284 oxygen consumption Effects 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 210000001322 periplasm Anatomy 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 230000017854 proteolysis Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 230000000087 stabilizing effect Effects 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 238000004448 titration Methods 0.000 description 1
- 235000013619 trace mineral Nutrition 0.000 description 1
- 239000011573 trace mineral Substances 0.000 description 1
- 230000014616 translation Effects 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- 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
- C12P21/00—Preparation of peptides or proteins
-
- 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
- C12P21/00—Preparation of peptides or proteins
- C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
-
- 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
- C07K14/615—Extraction from natural sources
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- General Engineering & Computer Science (AREA)
- Endocrinology (AREA)
- Microbiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Biophysics (AREA)
- Toxicology (AREA)
- Medicinal Chemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Description
WO 01/42420 PCT/SE00/02373 1 Production of peptides by fedbatch cultivation of a microorganism.
The invention relates to a method for the production of recombinant peptides by fed-batch cultivation of a microorganism in a bioreactor containing a medium comprising organic carbon source, nitrogen source and mineral salts. The cultivation is carried out by the addition of the organic carbon source by oscillation feed and/or by oscillation variation of stirring speed, e.g. in a square or sinus wave pattern. Preferably the organic carbon source is glucose and the recombinant peptide is growth hormone.
BACKGROUND
Recombinant human proteins have become important pharmaceuticals. The combination of recombinant DNA technology and large-scale processes has enabled production of proteins that might otherwise have been impossible or too expensive to obtain from natural sources.
For instance human growth hormone (hGH), immune interferon, tissue plasminogen activator (tPA) and human insulin are now commercial available by recombinant DNA technology.
However, the organism in large-scale bioprocesses is exposed to a changing environment and also respond to this. Differences in the production of recombinant proteins have been observed when increasing the reactor size. E.g. Riesenberg D et al. in Appl Microbiol Biotechnol 34:77-82, 1990, showed that the amount of human interferon alpha 1 was reduced by a factor of two when the culture volume was increased from 15 to 250 L.
When scaling up bioprocesses, differences in the micro-environment, such as concentration, and gradients, are likely to be expected.
WO 01/42420 PCT/SE00/02373 2 Concentration gradients of substrates, such as the carbon source added at limiting feed (fedbatch), ammonia for pH titration and oxygen, are to be expected as the result of changing geometry and process parameters as well as the choice of feed position. Most organisms respond to rapid environmental changes and can also change their metabolism in a timescale of seconds or less.
Pulse addition of glucose and the oscillating glucose concentration within the fermentor have earlier been studied with regard to yield and expression.
Pulse addition of glucose, i.e. addition of glucose within a few seconds when the culture had reached the steady state, was shown to induce gapA gene expression strongly and very rapidly (Gschaedler, Anne; et al, Biotechnol. Bioeng. (1999), 63(6), 712-720).
The effect of oscillating glucose concentration within the fermentor on biomass yield and acetate formation has been studied. The conclusion was, that the lower biomass yield and the higher acetate formation were caused by the cell response to the glucose oscillation within the fermentor when comparing large and small scale cultivations. (Bylund, F et al, Bioprocess Eng. (1999), 20(5), 377-389).
Pulse addition of the growth substrate (glucose) at appropriate time intervals allowing for significant starvation period between two consecutive pulses during fed-batch cultivation have positive effects on stabilizing plasmid and enhancing protein production. (Cheng, Chinyuan et al.Biotechnol. Bioeng. (1997), 56(1), 23-31). The pulse addition was repeated for 4-5 times at about 6 hours intervals. The result shows that the periodic glucose starvation feeding strategy can maintain a stable plasmid-carrying cell fraction and a stable specific productivity of the recombinant protein. On the contrary, without glucose starvation, the fraction of plasmid-carrying cells and the specific productivity continue to drop during the fed-batch cultivation, which would greatly reduce the product yield and limit the duration that the cultivation can be effectively operated.
WO 01/42420 WO 0142420PCTSEOO/02373 3 Wang, Zhengjun et al. Biotechnol. Bioeng. (1993), 42(1), 95-102 have shown that a glucose pulse at the end of batch culture in YPD (rich complex medium) facilitated the transport of residual cytoplasmic invertase.
US 5912133 discloses a fed-batch cultivation, wherein the carbon source concentration is at a constant low level under 5g/L and wherein the carbon source is added in at least two feedings. The carbon source should be exhausted after the first feeding and before the second feeding.
It is also known to use external magnetic field with periodic variations and product stirring movement in a cell culture vessel. (DD271 850). Thereby introduced material is effectively distributed with no local and potentially damaging concentrations None of the reports disclose a repeated oscillation of organic carbon source in a square or sinus wave pattern, which can be characterized by amplitude and frequency. On the contrary it can be concluded from prior art that there should be a significant starvation period between two consecutive feedings (Cheng et al.) or that that a glucose pulse should only be done at the end of batch culture (Wang et al.).
The most applied technique to achieve high cell densities is the glucose-limnited fed-batch.
In a fed-batch process, all media components are supplied in excess as in a batch process, except for example the carbon source. A feed with a substrate solution, often glucose, is fed to the bioreactor with a rate that ensures that this substrate component is growth limiting.
This substrate limitation allows control of the growth rate and the sugar uptake. By limiting the sugar uptake and thereby the reaction rate, engineering limitations, such as excessive heat evolution and oxygen limitation can be avoided. Glucose uptake can be divided in three reactions, glucose that is used for anabolism, glucose consumption for maintenance, which is the housekeeping requirement of the cell and glucose consumption to fuel growth.
The yield in the last two reactions is approximately 1.07 9 0 2 Control of the growth rate can therefore be used to control the oxygen consumption and heat generation associated WO 01/42420 PCT/SE00/02373 4 with growth. Furthermore, substrate limitation permits metabolic control by which overflow metabolism acetate formation in case of E. coli) and catabolite repression can be avoided. In the baker's yeast process sugar limitation is used throughout the process to avoid over-flow metabolism that results in excessive ethanol production (George et al.
(1998). Bioprocess Eng 18: 135-142.). Inhibitory acetate production by E. coli is avoided in the same way (See e.g. Lee, S. Y. (1996). Trends Biotechnol 14: 98-105).
In the production of recombinant proteins the oxygen transfer and the dissolved oxygen tension is particularly important for the yield and quality of the product (See e.g.
Bhattacharya and Dubey (1997) Enzyme Microb Technol 20: 355-360).A fed-batch process is therefore often the first choice when designing high-cell-density processes in E.
coli.
FIGURES
Figure 1 shows mode of operation Figure 2 shows amount of rhGH calculated on standard.
Figure 3 shows of rhGH variants.
THE INVENTION We have to our great surprise found that when the glucose feed was varied in oscillation feed and/or the stirring speed was performed in oscillation variation in a fed-batch cultivation, improved rhGH quality and yield i.e. less amount of not wanted products, was obtained. The word oscillation used here can also be described as pulses or by up and down-shifts.
The invention relates to a method for the production of recombinant peptides by fed-batch cultivation of a microorganism in a bioreactor containing a medium comprising organic carbon source, nitrogen source and mineral salts. This includes also a complex cultivation media.
WO 01/42420 PCT/SE00/02373 By the expression cultivation of a microorganism is meant cultivation of a biological host such as bacteria, yeast or animal cell.
The cultivation is carried out by the addition of the organic carbon source in oscillation feed and/or by oscillation variation of stirring speed. The oscillation feed and/or the oscillation variation of stirring speed can be performed during the entire cultivation process or during the production phase after the induction of the recombinant protein. The oscillation feed can have a square wave or sinus wave pattern. No starvation should occur during the oscillation period. The glucose added is consumed by the cells, but not until total starvation. The carbon source should never be exhausted during the process and there is no need for measuring its concentration during cultivation. The density of the cells should be high, preferably above 10 g/L and more preferably above 20 g/L and even more preferably above g/L during cultivation. However, aerobic conditions should be maintained.
The biomass concentration in the experiments was in of the order of 40 g/L.
Preferably the organic carbon source is glucose and the recombinant peptide is preferably growth hormone.
The period time for the oscillation curve can easily be determined for each protein, type of cultivation, medium etc by a person skilled in the art. The period for the oscillation time can be e.g. a short time of one minute as exemplified here, but periods of five, 10 minutes or longer periods are also within the scope of the invention.
The amplitude can be varied from about 5 up to 100 e.g. 20, 30, 40, 50 or 60%, which also easily is to be determined by a person skilled in the art.
Oscillation periods can be interrupted by periods of constant glucose addition or constant stirring speed.
The addition of glucose is here illustrated as a square wave function. The sinus wave function is especially suitable for the stirring speed. Also variation between sinus and square can be used.
According to our invention, the concentration of the carbon source and oxygen changes proportionally with the oscillation of the feed and agitation, respectively.
WO 01/42420 PCT/SE00/02373 6 The influence of substrate feed on the quality of a recombinant protein produced by Escherichia coli has specifically been studied in the production of recombinant human growth hormone (rhGH)in an aerobic fed-batch process as a model system.
Our invention and the findings are further confirmed in Bylund, Castan, Mikkola, Veide and Larsson; Biotechnology and Bioengineering 69 119-128 (2000).
MATERIAL AND METHODS Organism and Medium Escherichia coli (W3110) with a pBR derived plasmid coding for recombinant human growth hormone (rhGH) and antibiotic resistance was used in the performed cultivations.
This rhGH form is a 22 kDa protein consisting of 191 amino acids with two intramolecular disulphide bridges. The protein is secreted to the periplasm where the disulphide bridges are formed.
Cultivations were performed in a glucose mineral salt medium. The media used, with some minor modifications, is described by Forsberg G et al. in J. Biol. Chem. 272 (1997) 12430- 12438 Media components together with distilled water were sterilised in the bioreactor at 121 oC.
After sterilisation, sterilised magnesium sulphate, trace elements and antibiotics were added separately. Breox was added when needed for foam control. The feed solution had a glucose concentration of 590 g/1.
Cultivation Procedure and Bioreactors Exponentially growing cells from the seed fermentor were transferred to the production bioreactor. The inoculum volume was about 10 of final total volume. After the transfer procedure the glucose feed was immediately started. The feed profile consisted of three different phases: firstly, there was an exponential feed phase; secondly, the feed was turned to constant rate when the estimated maximum oxygen transfer capacity of the bioreactor WO 01/42420 PCT/SE00/02373 7 was approached and finally the feed rate was decreased to 80 of its maximum value at the time for induction.
The addition of glucose was made as a square wave function that was superimposed to the standard feed as illustrated in Figure 1.
Temperature and pH were controlled near the optimal value for E.coli growth.
All cultivations were performed in a 15 1 CF 3000 Chemap-Fermenter (Switzerland) stirred tank reactor (STR) with a start volume of 7 1. The volume increased during the feed phase up to 9 1.
Analytical Procedures.
Samples for product analyses were achieved from the extraction of the pellet fraction of ml centrifuged cell suspension. This extraction method releases the periplasmic fraction of proteins. Centrifugation was performed at 5000 rpm for 15 min at +4 0 C. Extraction buffer consisted of (per litre): Tris-HC1, 0.9837 g; Tris-base, 0.4551 g; EDTAx2H20, 0.372 g.
Purity and quantity of rhGH were determined by hydrophobic interaction chromatography (HIC). HIC was performed on a TSK phenyl 5PW column, Tosoh Haas. Four different variants of rhGH are separated by a decreasing salt gradient and detected with UV at 230 nm: (des-Phel)-rhGH (LMW), rhGH, (trisulfide Cysl82-Cysl89)-rhGH and clipped 142/143-rhGH (clipped variant).
RESULTS
The result is shown in Figures 2 and 3.
In the Figures 1-3, rhGH is produced by a standard method by a glucose feed that follow a square wave with an amplitude of 30% of the standard value and a frequency of 1 minute and P %OPERUEH\Rcs ClmsV00\40D 2533632 cIms do.O-3/1ZIL2 -8by a standard glucose feed but an stirring profile (at a rate of 800 1200 rpm) that follow a square wave with an amplitude of± 20% and a frequency of 1 minute.
Figure 2 shows the amount of rhGH when rhGH is prepared by the different methods. It is clear from the experiment that the amount of rhGH was much higher when glucose was added in an oscillation feed and by oscillation variation of stirring speed.
Figure 3 shows the amount in of total of rhGH 22kDa, i.e. the proper form of rhGH (bloack) and the amount of clipped variants (white) when the methods and are used. It can be seen that the yield of the proper form of rhGH, i.e. 22 kDa is higher when glucose was added in an oscillation feed and by oscillation variation of stirring speed.
CONCLUSIONS
Optimisation of the glucose feeding strategy or stirring profile by using oscillation in 15 square or sinus wave pattern gives the advantages that the yield of the protein is higher, that proteolysis could be avoided at a higher level and that the purification thereby is easier S•than when the organic source is fed without oscillation feed.
The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that that prior art forms part of the common S-general knowledge in Australia.
Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will 25 be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.
Claims (28)
1. Method for the production of recombinant peptide by fed-batch cultivation of a microorganism in a bioreactor containing a medium comprising organic carbon source, nitrogen source and mineral salts, wherein the cultivation is carried out by the addition of the organic carbon source in oscillation feed and/or by oscillation variation of stirring speed, without exhaustion of the organic carbon source during the oscillation period, wherein the oscillation has a wave period of from about 1 to about 30 minutes, wherein the microorganism is a biological host selected from the group consisting of bacteria, yeast and animal cell, and wherein the cultivation conditions remain aerobic.
2. Method according to claim 1, wherein the organic carbon is glucose.
3. Method according to claim 1, wherein the microorganism is E. Coli. oo*
4. Method according to claim 1, wherein the oscillation feed has a square wave pattern.
5. Method according to claim 1, wherein the oscillation feed has a sinus pattern.
6. Method according to claim 1, wherein the recombinant peptide is growth hormone.
7. Method according to claim 1, wherein the recombinant peptide is human growth hormone.
8. Method according to claim 1, wherein the oscillation feed has a square wave function of of standard and a wave period of 1 minute.
9. Method according to claim 1, wherein the oscillation feed has a wave amplitude of from about to of standard.
P OPERVEH\Rn CImsUD 2S133632 lms doO 3I 2AJ4 oooo eeeee e Method according to claim 1, wherein the oscillation variation in stirring speed is of standard with a square wave period of 1 minute.
11. Method according to claim 1, wherein the microorganism is E. coli and wherein the recombinant peptide is human growth hormone.
12. Method according to claim 1, wherein the recombinant peptide comprises recombinant human growth hormone, immune interferon, tissue plasminogen activator, or human insulin.
13. Method according to claim 1, wherein the oscillation feed and/or oscillation variation in stirring speed is from about to of standard.
14. Method according to claim 2, wherein the recombinant peptide is human growth hormone.
15. Method according to claim 2, wherein the recombinant peptide is growth hormone.
16. Method according to claim 2 wherein the microorganism is E. Coli.
17. Method according to claim 2, wherein the oscillation feed has a square wave pattern.
18. Method according to claim 2, wherein the oscillation feed has a sinus wave pattern.
19. Method according to claim 3, wherein the oscillation feed has a square wave pattern.
Method according to claim 3, wherein the oscillation feed has a sinus wave pattern.
21. Method according to claim 3, wherein the recombinant peptide is growth hormone. P QOPERUEH\Ru Clns\2OO4' 233632 c.1 dmOC-31I2/4 -11
22. Method according to claim 3, wherein the recombinant peptide is human growth hormone.
23. Method according to claim 4, wherein the recombinant peptide is growth hormone.
24. Method according to claim 4, wherein the recombinant peptide is human growth hormone.
Method according to claim 5, wherein the recombinant peptide is growth hormone.
26. Method according to claim 5, wherein the recombinant peptide is human growth hormone.
27. Method according to claim 8, wherein the oscillation variation in stirring speed is of standard with a square wave period of 1 minute.
28. A method according to any one of claims 1 to 27 substantially as hereinbefore described with reference to the Figures. DATED this 3rd day of December, 2004 PHARMACIA AB by DAVIES COLLISON CAVE Patent Attorneys for the Applicant(s) 55 S 5555 S S 555
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| SE9904502A SE9904502D0 (en) | 1999-12-09 | 1999-12-09 | Production of peptides |
| SE9904502 | 1999-12-09 | ||
| PCT/SE2000/002373 WO2001042420A1 (en) | 1999-12-09 | 2000-11-29 | Production of peptides by fedbatch cultivation of a microorganism |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| AU1910501A AU1910501A (en) | 2001-06-18 |
| AU780122B2 true AU780122B2 (en) | 2005-03-03 |
Family
ID=20418057
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| AU19105/01A Ceased AU780122B2 (en) | 1999-12-09 | 2000-11-29 | Production of peptides by fedbatch cultivation of a microorganism |
Country Status (11)
| Country | Link |
|---|---|
| EP (1) | EP1244769A1 (en) |
| JP (1) | JP2003516140A (en) |
| KR (1) | KR20020060258A (en) |
| CN (1) | CN1267562C (en) |
| AU (1) | AU780122B2 (en) |
| CA (1) | CA2392268A1 (en) |
| HK (1) | HK1053326A1 (en) |
| IL (1) | IL149624A0 (en) |
| NZ (1) | NZ518919A (en) |
| SE (1) | SE9904502D0 (en) |
| WO (1) | WO2001042420A1 (en) |
Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004026251A2 (en) * | 2002-09-20 | 2004-04-01 | Pharmacia Corporation | Process for decreasing aggregate levels of pegylated protein |
| TWI281864B (en) * | 2002-11-20 | 2007-06-01 | Pharmacia Corp | N-terminally monopegylated human growth hormone conjugates and process for their preparation |
| US20100047870A1 (en) * | 2004-12-21 | 2010-02-25 | Usv Limited | Low cell density fermentation process for the production of heterologous recombinant proteins in microorganisms |
| WO2008010005A1 (en) * | 2006-07-14 | 2008-01-24 | Abb Research Ltd | A method for on-line optimization of a fed-batch fermentation unit to maximize the product yield. |
| EP2619222B1 (en) * | 2010-09-21 | 2016-01-06 | Ferring B.V. | Improved process for production of recombinant human growth hormone |
| CN106222309A (en) * | 2016-07-28 | 2016-12-14 | 山东金朗生物科技有限公司 | A kind of fermentable produces the control of additive raw material method improving L alanine yield |
| CN108624516B (en) * | 2017-03-20 | 2022-08-26 | 华东理工大学 | Method for improving metabolite amount in fermentation cells and preparing IDMS standard substance |
| GB202010934D0 (en) * | 2020-07-15 | 2020-08-26 | Ipsen Biopharm Ltd | Controlling operation of a bioreactor vessel |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5912113A (en) * | 1990-11-30 | 1999-06-15 | Ajinomoto Co., Inc. | Method and apparatus for controlling carbon source concentration in aerobic cultivation of a microorganism |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DD271850A1 (en) * | 1988-05-12 | 1989-09-20 | Adw Ddr | MIXING DEVICE FOR FLUID DISTRIBUTION AND / OR FOR GAS ADMISSION IN BIOLOGICAL CULTURAL SUSPENSIONS WITH PENDULUM ROTORS |
-
1999
- 1999-12-09 SE SE9904502A patent/SE9904502D0/en unknown
-
2000
- 2000-11-29 NZ NZ518919A patent/NZ518919A/en unknown
- 2000-11-29 JP JP2001544297A patent/JP2003516140A/en not_active Withdrawn
- 2000-11-29 WO PCT/SE2000/002373 patent/WO2001042420A1/en active IP Right Grant
- 2000-11-29 IL IL14962400A patent/IL149624A0/en unknown
- 2000-11-29 AU AU19105/01A patent/AU780122B2/en not_active Ceased
- 2000-11-29 EP EP00982029A patent/EP1244769A1/en not_active Withdrawn
- 2000-11-29 CA CA002392268A patent/CA2392268A1/en not_active Abandoned
- 2000-11-29 KR KR1020027007309A patent/KR20020060258A/en not_active Application Discontinuation
- 2000-11-29 CN CNB008168865A patent/CN1267562C/en not_active Expired - Fee Related
-
2003
- 2003-08-01 HK HK03105539A patent/HK1053326A1/en not_active IP Right Cessation
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5912113A (en) * | 1990-11-30 | 1999-06-15 | Ajinomoto Co., Inc. | Method and apparatus for controlling carbon source concentration in aerobic cultivation of a microorganism |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2001042420A1 (en) | 2001-06-14 |
| EP1244769A1 (en) | 2002-10-02 |
| CN1267562C (en) | 2006-08-02 |
| CN1409755A (en) | 2003-04-09 |
| CA2392268A1 (en) | 2001-06-14 |
| AU1910501A (en) | 2001-06-18 |
| HK1053326A1 (en) | 2003-10-17 |
| KR20020060258A (en) | 2002-07-16 |
| NZ518919A (en) | 2003-10-31 |
| IL149624A0 (en) | 2002-11-10 |
| JP2003516140A (en) | 2003-05-13 |
| SE9904502D0 (en) | 1999-12-09 |
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