CN1409755A - Production of peptides by fedbatch cultivation of microoganism - Google Patents
Production of peptides by fedbatch cultivation of microoganism Download PDFInfo
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- CN1409755A CN1409755A CN00816886A CN00816886A CN1409755A CN 1409755 A CN1409755 A CN 1409755A CN 00816886 A CN00816886 A CN 00816886A CN 00816886 A CN00816886 A CN 00816886A CN 1409755 A CN1409755 A CN 1409755A
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- glucose
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- 238000004519 manufacturing process Methods 0.000 title abstract description 17
- 238000012366 Fed-batch cultivation Methods 0.000 title abstract description 9
- 102000004196 processed proteins & peptides Human genes 0.000 title abstract description 3
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- 239000008103 glucose Substances 0.000 claims abstract description 34
- 238000000034 method Methods 0.000 claims abstract description 25
- 229910052799 carbon Inorganic materials 0.000 claims abstract description 20
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- 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
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- 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
- 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
- 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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- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- General Engineering & Computer Science (AREA)
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Abstract
The invention relates to a method for the production of recombinant peptides by fedbatch 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.
Description
The present invention relates in containing the substratum bio-reactor of (containing organic carbon source, nitrogenous source and inorganic salt), cultivate the method for producing recombinant peptide by microbial fed-batch.This cultivation is added organic carbon source by the fluctuation feed supplement and/or is passed through the fluctuation variation of stirring velocity, for example carries out with square wave (square) mode or sine wave (sinus) mode.The preferred glucose of this organic carbon source, this recombinant peptide preferred growth hormone.
Background
Human recombination protein has become important medicine.The combination of recombinant DNA technology and scale operation has made those can not or can't become possibility from the proteic production that natural origin obtains because of too expensive.For example, by recombinant DNA technology, human growth hormone (hGH), type II interferon, tissue plasminogen activator (tPA) and insulin human have commercially available now.
Yet in extensive biological production, organism will be exposed in the changing environment and will react to this.Find, when increasing the reactor size, the difference of recombinant protein production aspect.For example Riesenberg D etc. (the using microbe biotechnology, 1990, show that 34:77-82) when volume of culture was increased to 250L by 15L, the quantity of human interferon-alpha 1 reduced one times.
When biological production enlarges in proportion, may be in microenvironment, for example concentration and gradient, the aspect can be variant.Because the substrate concentration gradient of (for example the carbon source of adding in restriction feed supplement (fed-batch) is used for titrating ammoniacal liquor of pH and oxygen) can appear in how much and the change of processing parameter and the selection of feed entrance point, expectation.Most of organism can change the metabolism of reacting and can change them in several seconds or less time to quick environment.
In the past after deliberation in fermentor tank, the influence to output and expression of glucose and fluctuation glucose concn is added in pulse.
Glucose is added in pulse, promptly when cultivation reaches stationary phase, adds glucose and can greatly and very rapidly induce gapA expression of gene (Gschaedler, Anne etc., biotechnology biotechnology, 1999,63 (6): 712-720) within several seconds.
The someone has studied the influence of glucose concn to biomass yield and acetate formation of fluctuating in fermentor tank.Conclusion is, when large scale culturing is found that cell has caused lower biomass yield and higher acetic acid formation (Bylund to the reaction of glucose fluctuation in fermentor tank with cultivating on a small scale when comparing, F etc., the biological production engineering, 1999,20 (5): 377-389).
During fed batch cultivation, growth substrate (glucose) is added in (making can have tangible hunger period between two consecutive pulses) pulse in the appropriate timed interval has positive effect (Cheng to stablizing plasmid and increasing protein production, Chinyuan etc., the biotechnology biotechnology, 1997,56 (1): 23-31).In about 6 hours interval, repeat pulse and add 4-5 time.The result shows that the hungry feed supplement strategy of this interim glucose can keep the stable cell proportion that carries plasmid and keep the stable specific production rate of recombinant protein.On the contrary, during fed batch cultivation, do not have glucose hunger, the ratio and the specific production rate that carry plasmid cell will continue to descend, thereby greatly reduce the time length of product production and restriction cultivation valid function.
Wang, (biotechnology biotechnology, 1993,42 (1): show that 95-102) in the latter stage of YPD (eutrophy complex medium) batch culture, the glucose pulse helps the transportation of residual kytoplasm saccharase such as Zhengjun.
United States Patent (USP) 5912133 discloses a kind of fed batch cultivation method, and wherein carbon source concentration remains on following low-level of 5g/L and added carbon source at least in twice feed supplement.After the feed supplement first time and before the feed supplement second time, carbon source should be used up.
In cell culture apparatus, the external magnetic field that periodic changes and to produce stirring movement also be known method.(DD271850)。Partial concn is too high thereby the material that makes importing can be evenly distributed causes damage.
The none report disclosed organic carbon source with square wave mode or the fluctuation of sinusoidal wave mode multiple, it is characterized in that amplitude and frequency.On the contrary, we can draw such conclusion from prior art, between two continuous feedings, a significantly hunger period (Cheng etc.) should be arranged, or the glucose pulse only should be carried out (Wang etc.) latter stage in batch culture.
The most common technique that reaches high-cell density is the fed batch cultivation of glucose limitation.In the fed-batch production process, all media components (except for example carbon source) resemble in the batch production process all excessive.With substrate solution (often being glucose) feed supplement in bio-reactor, its speed guarantees that this substrate composition is a growth limitation.This substrate restriction can control growing speed and sugared absorption.Absorption by limiting sugar and therefore limited reactions speed can avoid Engineering Bureau sex-limited, for example excessive heat radiation and oxygen restriction.Glucose is taken in and can be divided into three reactions, is used for anabolic glucose, is used for keeping the glucose consumption of daily need of cell and the glucose consumption that is used to grow.The productive rate of latter two reaction is near 1.07g
O2g
-1Sugar.Therefore growth velocity control can be used for controlling and related oxygen depletion and the heat generation of growing.In addition, substrate restriction can be used to carry out metabolism control, can avoid overflow (overflow) metabolism (promptly as the formation of the acetate in intestinal bacteria) and metabolic by-prods inhibition thus.In the yeast production process of bread industry, the whole course of processing all use sugar restriction with the flow metabolism avoiding causing excess ethyl alcohol to produce (George etc., the biological production engineering, 1998,18:135-142).With same method avoided intestinal bacteria inhibition acetate generation (reference example such as Lee, S.Y., biotechnology trend, 1996,14:98-105).
In the production of recombinant protein, oxygen shift and dissolved oxygen tension force to the output of product and quality all be very important (reference example such as Bhattacharya and Dubey, microbial enzyme learns a skill, 1997,20:355-360).Therefore, when the design high-cell density was produced in intestinal bacteria, fed-batch production normally first was selected.Accompanying drawing Fig. 1 is the percentage amounts of pressing the rhGH of criterion calculation for operator scheme Fig. 2.Fig. 3 is the percentage amounts of rhGH mutant.
The present invention
We very surprisingly find, in fed batch cultivation, when the glucose feed supplement changes in the mode of fluctuation feed supplement and/or stirring velocity, can improve quality and the output (promptly reducing undesired product) of rhGH.This speech of fluctuation used herein can be described to pulse or move up and down.
The present invention relates in containing the substratum bio-reactor of (containing organic carbon source, nitrogenous source and inorganic salt), cultivate the method for producing recombinant peptide by microbial fed-batch.This also comprises compound culture medium.
By this statement, microorganism culturing is meant the biology host, for example bacterium, yeast or zooblast, cultivation.
This cultivation changes and carries out by add organic carbon source and/or the fluctuation by stirring velocity with the fluctuation feed supplement.The fluctuation of this fluctuation feed supplement and/or stirring velocity changes and can carry out the production phase in whole culturing process or after recombinant protein is induced.This fluctuation feed supplement can be the mode square wave mode or sinusoidal wave.In the fluctuation phase hungry the generation should do not arranged.The glucose that adds is consumed by cell, but will just be consumed by cell in the time of complete hunger.In this process, this carbon source never should be depleted, and also do not need to measure its concentration in culturing process.In culturing process, cell concn should be very high, more than the preferred 10g/L, more preferably more than the 20g/L, more more preferably more than the 50g/L.In any case, should maintain oxygen condition.
In this experiment, biomass concentration is in this rank of 40g/L.
The preferred glucose of organic carbon source, recombinant peptide preferred growth hormone.
The cycle time that those skilled in the art can determine every kind of albumen at an easy rate, cultivate the curve of cyclical fluctuations of type, substratum etc.The cycle of fluctuation time can be, what for example gave an example in this place is as short as 1 minute like that, but grew to 5,10,30 minutes or longer cycle also within the scope of the invention.
This amplitude can be in approximately variation between ± 5% to ± 100%, for example ± 20, ± 30, ± 40, ± 50 or ± 60%, it also can easily be measured by those skilled in the art.
Can be blocked cycle of fluctuation by the cycle of constant glucose interpolation or constant agitation speed.
Glucose herein adds is set forth with square wave function.Sinusoidal wave function is particularly suitable for stirring velocity.And, also can conversion between sine wave and square wave.
By of the present invention, carbon source and concentration of oxygen change in proportion with the fluctuation of feed supplement and stirring respectively.
Utilized and in aerobic fed batch cultivation method, produced recombinant human growth hormone (rhGH), specifically studied the influence of substrate feed supplement the quality of the recombinant protein that produces by intestinal bacteria as modular system.
Our invention and this are found at Bylund, Castan, Mikkola, Veide and Larsson; Biotechnology and Bioengineering, 2000,69 (2): obtained further confirmation in 119-128 one literary composition.
Materials and methods
Organism and substratum
Intestinal bacteria (W3110) (pBR that contains coding recombinant human growth hormone and antibiotics resistance derive plasmid) are used to this cultivation.This rhGH form is by 191 amino acid 22kDa albumen that form, that two intramolecular disulfide bonds are arranged.This albumen is secreted into pericentral siphon and forms disulfide linkage at that.
In the glucose minimal medium, cultivate.Used substratum Forsberg G etc., journal of biological chemistry, 1997, state (modification is arranged slightly) in 272:12430-12438 one literary composition.
Substratum composition and distilled water together in bio-reactor in 121 ℃ of sterilizations.After the sterilization, add death of monks or nuns respectively and cross the sal epsom of bacterium, trace element and microbiotic.Breox can add when needs carry out foam control.The glucose concn of feed supplement liquid is 590g/l.
Culturing process and bio-reactor
Cell with exponential growth from seeding tank changes the production bio-reactor over to.The inoculation volume is 10% of a final total volume.Begin to carry out the feed supplement of glucose after the transfer step immediately.The feed supplement scheme was made up of three different stages: at first be the index feed supplement phase; When the maximum of the bio-reactor that reaches estimation was led to the oxygen ability, feed supplement transferred constant rate of speed to then; At last when inducing feed rate reduce to its peaked 80%.
Glucose adds and to carry out with square wave function, and itself and standard feed supplement are superimposed, as shown in Figure 1.
With temperature and pH control near the intestinal bacteria growth just when.
All cultivations are all carried out in 151 CF, 3000 Chemap-Fermenter (Switzerland) agitator tank reactors (STR), initial volume 7L.Volume increases to 9L during the feed supplement phase.
Analytical procedure
After the 5ml cell suspension was centrifugal, the thalline of acquisition partly carried out extracting, obtained being used for the sample of product analysis.This method for extracting discharges the albumen of pericentral siphon part.Centrifugally carry out centrifugal 15 minutes at+4 ℃, 5000rpm.Extraction buffer (/L) by Tris-HCl, 0.9837g; Tris-base, 0.4551g; EDTAx2H
20,0.372g forms.The purity of rhGH and amount are measured by hydrophobic interaction chromatography (HIC).HIC carries out with TSK phenyl 5PW post (Tosoh Haas).The salt gradient that four kinds of different rhGH mutant are descended is gradually separated, and detects at the 230hm place with ultraviolet ray: (des-Phel)-and rhGH (LMW), rhGH, (trisulfide Cys182-Cys189)-rhGH and disappearance 142/143-rhGH (deletion mutant).
The result
The result is shown in Fig. 2 and 3.
In Fig. 1-3, rhGH is produced by following method:
(a) standard method,
(b) amplitude is that standard value ± 30% and frequency are the glucose feed supplement of 1 minute square wave mode,
(c) the glucose feed supplement of standard, but its stirring distribution plan (with the speed of 800-1200rpm) is amplitude ± 20% and 1 minute square wave of frequency.
Fig. 2 has shown when preparing rhGH with different methods, the amount of rhGH.
We can be clear that from this experiment, and when the form that changes with fluctuation feed supplement and stirring velocity fluctuation was added glucose, what the amount of rhGH will be high was many.
Fig. 3 has shown the per-cent of rhGH 22kDa total amount, i.e. the rhGH (black) of correct form and the amount of deletion mutant (white) when using (a) and (b) and method (c).We as can be seen, when glucose was added by the form that changes with the fluctuation of fluctuation feed supplement and stirring velocity, the output of the rhGH (being 22kDa) of correct form was higher.
Conclusion
Glucose feed supplement strategy or the optimization (using square wave or sinusoidal wave fluctuation) that stirs distribution mode are not compared when adding with the form of fluctuation feed supplement with organic raw material, have higher protein yield, avoid proteolysis and the easier advantage of carrying out of purifying thus on higher level.
Claims (7)
1. in the bio-reactor of the substratum that contains organic carbon source, nitrogenous source and inorganic salt, the method of cultivate producing recombinant peptide by microbial fed-batch, wherein this cultivations changes by fluctuate feed supplement interpolation organic carbon source and/or the fluctuation by stirring velocity and carries out.
2. the described method of claim 1, wherein this organic carbon source is a glucose.
3. claim 1 or 2 described methods, wherein this microorganism is intestinal bacteria.
4. any one described method in the claim 1 to 3, wherein this fluctuation feed supplement is the square wave mode.
5. any one described method in the claim 1 to 3, wherein this fluctuation feed supplement is sinusoidal wave mode.
6. any one described method in the claim 1 to 5, wherein this recombinant peptide is a tethelin.
7. any one described method in the claim 1 to 6, wherein this recombinant peptide is a human growth hormone.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| SE99045023 | 1999-12-09 | ||
| SE9904502A SE9904502D0 (en) | 1999-12-09 | 1999-12-09 | Production of peptides |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1409755A true CN1409755A (en) | 2003-04-09 |
| CN1267562C CN1267562C (en) | 2006-08-02 |
Family
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB008168865A Expired - Fee Related CN1267562C (en) | 1999-12-09 | 2000-11-29 | Production of peptides by fedbatch cultivation of microoganism |
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) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN108624516A (en) * | 2017-03-20 | 2018-10-09 | 华东理工大学 | A kind of metabolite amount in raising fermented cells and the method for preparing IDMS standard items |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2498886C (en) * | 2002-09-20 | 2013-06-25 | Pharmacia Corporation | Process for decreasing aggregate levels of pegylated protein |
| PA8588901A1 (en) * | 2002-11-20 | 2005-02-04 | Pharmacia Corp | CONJUGATES OF N-TERMINAL HUMAN GROWTH HORMONE HORMONE AND PROCESS FOR PREPARATION |
| EP1828375B1 (en) * | 2004-12-21 | 2008-05-07 | USV Limited | Low cell density fermentation process for the production of heterologous recombinant proteins in microorganisms |
| CN101484572A (en) * | 2006-07-14 | 2009-07-15 | Abb研究有限公司 | A method for on-line optimization of a fed-batch fermentation unit to maximize the product yield |
| CN103119056B (en) * | 2010-09-21 | 2015-11-25 | 辉凌公司 | For the preparation of the modification method of recombinant human somatropin |
| GB202010934D0 (en) * | 2020-07-15 | 2020-08-26 | Ipsen Biopharm Ltd | Controlling operation of a bioreactor vessel |
Family Cites Families (2)
| 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 |
| IT1256566B (en) * | 1990-11-30 | 1995-12-11 | Ajinomoto Kk | PROCEDURE AND EQUIPMENT TO CONTROL THE CONCENTRATION OF THE CARBON SOURCE IN AN AEROBIC CULTURE OF A MICROORGANISM |
-
1999
- 1999-12-09 SE SE9904502A patent/SE9904502D0/en unknown
-
2000
- 2000-11-29 CN CNB008168865A patent/CN1267562C/en not_active Expired - Fee Related
- 2000-11-29 EP EP00982029A patent/EP1244769A1/en not_active Withdrawn
- 2000-11-29 KR KR1020027007309A patent/KR20020060258A/en not_active Application Discontinuation
- 2000-11-29 AU AU19105/01A patent/AU780122B2/en not_active Ceased
- 2000-11-29 CA CA002392268A patent/CA2392268A1/en not_active Abandoned
- 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 NZ NZ518919A patent/NZ518919A/en unknown
-
2003
- 2003-08-01 HK HK03105539A patent/HK1053326A1/en not_active IP Right Cessation
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN108624516A (en) * | 2017-03-20 | 2018-10-09 | 华东理工大学 | A kind of metabolite amount in raising fermented cells and the method for preparing IDMS standard items |
| 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 |
Also Published As
| Publication number | Publication date |
|---|---|
| NZ518919A (en) | 2003-10-31 |
| AU1910501A (en) | 2001-06-18 |
| KR20020060258A (en) | 2002-07-16 |
| IL149624A0 (en) | 2002-11-10 |
| JP2003516140A (en) | 2003-05-13 |
| HK1053326A1 (en) | 2003-10-17 |
| AU780122B2 (en) | 2005-03-03 |
| CA2392268A1 (en) | 2001-06-14 |
| EP1244769A1 (en) | 2002-10-02 |
| CN1267562C (en) | 2006-08-02 |
| SE9904502D0 (en) | 1999-12-09 |
| WO2001042420A1 (en) | 2001-06-14 |
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