CN106337077B - Indissoluble fat acid mother liquor of chemically defined high density CHO culture medium and preparation method thereof - Google Patents
Indissoluble fat acid mother liquor of chemically defined high density CHO culture medium and preparation method thereof Download PDFInfo
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- CN106337077B CN106337077B CN201510408597.8A CN201510408597A CN106337077B CN 106337077 B CN106337077 B CN 106337077B CN 201510408597 A CN201510408597 A CN 201510408597A CN 106337077 B CN106337077 B CN 106337077B
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Abstract
The invention belongs to field of biotechnology and chemical field, it is related to a kind of mother liquor base composition for dissolved fat acid, mother liquor and preparation method thereof.A kind of solvent substrate composition for dissolved fat acid, contains cyclodextrin, poly yamanashi esters, poloxamer.The culture medium of invention while the fatty acid dissolution solvent, adipic acid solution and the fatty acids that are supplied to based on the composition.
Description
Technical field
The invention belongs to field of biotechnology and chemical fields, are related to a kind of mother liquor substrate combination for dissolved fat acid
Object, mother liquor and preparation method thereof.
Background technique
Fatty acid is the essential component in many cell culture mediums.For example, in chemically defined high density CHO culture medium,
Fatty acid is a kind of essential component, and the production of growth and antibody to Chinese hamster ovary celI has important role.Chemistry containing fatty acid
Limiting high density CHO culture medium is indispensable for improving Chinese hamster ovary celI antibody producing.
Fatty acid is the organic matter of the aliphatic hydrocarbon chain for the length that a carboxyl is contained in one end, and linear saturated fatty acids are led to
Formula is C (n) H (2n+1) COOH, and rudimentary fatty acid is colourless liquid, irritant smell, and advanced fatty acid is wax-like solid
Body, the smell that can not obviously smell.Fatty acid is practically insoluble in water, is soluble in organic solvent.We attempt fatty acid dissolution
It in dehydrated alcohol, DMSO or chloroform, is then added in culture medium, discovery fatty acid is precipitated again, and culture medium becomes cloudy, subsequent
Filtering also therefore become highly difficult.How to make fatty acid dissolution in the medium, and is allowed to become urgently in clear shape
It solves the problems, such as.
Summary of the invention
It is an object of the present invention to provide a kind of solvent substrate compositions for dissolved fat acid.
The second purpose of the present invention is to provide a kind of solvents for dissolved fat acid.
The three of the object of the invention are to provide a kind of fatty acids solution configured using above-mentioned solvent, such as culture medium
Mother liquor.
The fourth object of the present invention is to provide the preparation method of above-mentioned adipic acid solution.
Above-mentioned purpose that the invention is realized by the following technical scheme.
Invention provide a kind of mother liquor base composition for dissolved fat acid, containing methyl-B-cyclodextrin,F-68, Tween-80.
The weight proportion of each component in composition are as follows: 0.6~6 part of cyclodextrin, poly yamanashi esters surfactant 95~170
Part, 210~300 parts of poloxamer.
The poly yamanashi esters surfactant is Tween 80, can also be polysorbas20, tween 50, polysorbate85 etc..
The cyclodextrin is beta-cyclodextrin；Preferably methyl-B-cyclodextrin.
Alternatively, the difficult lysing adipose tissue acid of the fatty acid；Preferably, the fatty acid is selected from following object
One or more kinds of combinations of matter: (soil)-alpha-lipoic acid, methyl linoleate, linoleic acid, arachidonic acid, DL- α-fertility
Phenol acetate, myristic acid, oleic acid, palmitinic acid, stearic acid, L- α-phosphatidyl choline, gamma-Linolenic acid, palmitoleic acid, gallbladder are solid
Alcohol, sinapic acid, parinaric acid, eicosatrienoic acid, eicosatetraenoic acid, eicosapentaenoic acid, clupanodonic acid,
Docosahexaenoic acid, nisioic acid, elaidic acid.
Invention additionally provides a kind of solvent for dissolved fat acid, contains such as above-mentioned composition.
Invention additionally provides a kind of fatty acids solution, is obtained with above-mentioned solvent dissolved fat acid.Optionally implementing
In scheme, the mass fraction of fatty acid in the solution is 0.003%-0.3%.
Invention additionally provides a kind of preparation method of adipic acid solution, it is characterised in that using molten as claimed in claim 6
Fatty acid is dissolved into the solvent by agent, or fatty acid is dissolved into organic solvent to add institute after basic mother liquor
State solvent dissolution；Preferably, it is obtained again with membrane filtration after dissolution；It is further preferable that the organic solvent is dehydrated alcohol；
It is further preferable that the mass fraction of fatty acid is 3%-30% in the basic mother liquor.
Invention additionally provides a kind of culture medium containing above-mentioned adipic acid solution.
Technical solution of the present invention is particularly suitable for preparing chemically defined high density CHO culture medium.
In one embodiment of the invention, chemically defined high density CHO training can be dissolved completely in order to solve fatty acid
Support base in this problem, with methyl-B-cyclodextrin,F-68, Tween-80 mixed solution hydrotropy, be configured to containing
The mother liquor of 12 kinds of indissoluble fatty acid can be directly appended in chemically defined high density CHO culture medium, be solved containing there are many rouge
The filtration problem of the chemically defined high density CHO culture medium of fat acid reduces filtering cost, produces suitable for culture medium.
The needs that the preparation of the indissoluble fat acid mother liquor of chemically defined high density CHO culture medium is grown containing Chinese hamster ovary celI
Fatty acid, and mother liquor is completely dissolved, and is easy filtering, used isF-68, methyl-B-cyclodextrin,
The combined aqueous dissolution of Tween-80.
In one embodiment of the invention, describedF-68, methyl-B-cyclodextrin, Tween-80 combination
Concentration used is 100mg/L, 100000mg/L, 22000mg/L respectively, and is clear solution.
In one embodiment of the invention, the indissoluble raw material of fatty acid and its initial concentration are as follows:
Fatty acid used, other than L- α-phosphatidyl choline, others first first must be dissolved into mother liquor with dehydrated alcohol,
It is prepared into the indissoluble fat acid mother liquor of chemically defined high density CHO culture medium again, and L- α-phosphatidyl choline is directly dissolved inF-68, methyl-B-cyclodextrin, Tween-80 combined aqueous solution in.
In one embodiment of the invention, the fatty acid basis female for using dehydrated alcohol to be configured in following fatty acid
Liquid, content of the fatty acid in basic mother liquor are as follows:
In an embodiment of the invention, the system of the indissoluble fat acid mother liquor of chemically defined high density CHO culture medium
Preparation Method is as follows:
A. proportionally by 100mg/LF-68,100000mg/L methyl-B-cyclodextrin, 22000mg/L are spat
Temperature -80 is added in ultrapure water, stirs 15 minutes at room temperature, is configured to as clear as crystal aqueous solution.
B. L- α-phosphatidyl choline powder is added into a solution according to dosage, stirs 15 minutes, until L- α-phosphatidyl gallbladder
Ground caustic end is completely dissolved.
C. the fatty acid of 11 kinds of dehydrated alcohols in claim 5 is successively added while stirring in b solution according to dosage
Mother liquor stirs 15 minutes, sterile to be protected from light -20 DEG C of preservations with 0.22 μm of filter membrane negative pressure filtration.
Three kinds of important components are used in technical solution of the present invention:
Cyclodextrin is the circular hollow cylinder of up-narrow and down-wide both ends open, and cylindrical outer is hydrophilic surface, internal
Non-polar group forms the hydrophobic cavity with certain size.Insoluble drug can be with cyclodextrin inner hydrophobic grouping
It is combined by hydrogen bond, Van der Waals force etc. and forms stable inclusion compound, indissoluble is improved by external hydrophilic group and water molecules
The water solubility of property drug.
Beta-cyclodextrin oral toxicity is very low, and beauty, Deng state pharmacopeia have been recorded to take orally auxiliary material.Methyl-B-cyclodextrin
As the outstanding representative of beta-cyclodextrin derivative, solubility is bigger, has bigger inclusion characteristic.Inventor has found that it is used for
It has better effect in the technical program.
Polysorbate: polyoxyethylene sorbitan fatty acid ester, trade name tween (Tween) can be divided into polysorbas20
~85, it is the ester that polyoxyethylene sorbitan is generated from different fatty acid.Tween 80 often makees the emulsifier of O/W type emulsion, difficult
The solubilizer of soluble drug, the wetting agent etc. of suspension, due to there is more one polyoxyethylene groups of hydrophilic radical in its molecule,
Therefore hydrophily is strong, is nonionic surface active agent.Solubilizer, emulsifier etc. are widely used as in pharmacy.Tween-
80 be oleate, and hydrophily is stronger, and emulsification is more complete, more for drug, the solubilising of food.
Poloxamer is that a kind of PEO-PPO-PEO being made of polyoxyethylene (PEO), polyoxypropylene (PPO) is non-ionic
Triblock copolymer, Families of poloxamers include more than the 30 kinds of polymer containing different PPO, PEO block ratios, and it is living to all have surface
Property.F-68 (PF68) is one of novel form and the main drug administration carrier of new transmission system research in recent years.F-68 itself is water-soluble good, and unique hydrophobic inner core-hydrophilic outer shell is allowed to have amphipathic, and has good
Surface-active.
Although methyl-B-cyclodextrin is widely used in the dissolution of drug and fatty acid, but the technique of its inclusion is excessively multiple
Miscellaneous, cost and time do not comply in the mass production for being applied to culture medium, and Tween-80 andF-68 is to Mr. Yu
A little indissoluble fatty acid do not achieve the effect that all to dissolve, finally above-mentioned, the present invention selected methyl-B-cyclodextrin,The optimal combination that F-68, Tween-80 are used in mixed way quickly can be completely dissolved indissoluble fatty acid again, be configured to
The indissoluble fat acid mother liquor of chemically defined high density CHO culture medium accords with chemically defined high density CHO culture medium mass production
Requirement.
Three kinds of above-mentioned components are that inventor is finally determined by solubility experiment and cell toxicological experiment.It is used to dissolve
Fatty acid, dissolubility is good and SOLUTION PROPERTIES is stablized, and does not have cytotoxicity.
Specific embodiment
Configuration of 1 present invention of embodiment for the solvent of dissolved fat acid
Three kinds of cosolvent methyl-B-cyclodextrins,The optium concentration combination of F-68, Tween-80, specific concentration
It is as follows:
F-68 100mg/L
Methyl-B-cyclodextrin 100000mg/L
Tween-80 22000mg/L
The configuration (can be used as configuring the fatty acid mother liquor of culture medium) of 2 adipic acid solution of embodiment
Fatty acid of the present invention has 12 kinds, cannot be completely dissolved in aqueous solution, in three kinds of solvent methyl-β-ring paste
Essence,F-68, Tween-80 combination solution in can be completely dissolved, and dissolved mother liquor can be with aperture
0.22 μm of membrane filtration is added in chemically defined high density CHO culture medium and nor affects on its physicochemical properties.
The component and dosage of 1. fatty acid of table
The above fatty acid is both soluble in organic solvents such as ethyl alcohol, DMSO, chloroforms, but it is organic molten to be dissolved in ethyl alcohol etc.
It will appear the phenomenon that fatty acid is precipitated when the fatty acid of agent is added in aqueous solution, cause fatty acid that cannot be dissolved completely in water
In solution.
Fatty acid, which is dissolved in dehydrated alcohol, in existing 11 other than L- α-phosphatidyl choline of the present invention is configured to basis female
Liquid.
Fatty acid and the content being dissolved in dehydrated alcohol:
A. proportionally by 100mg/LF-68,100000mg/L methyl-B-cyclodextrin, 22000mg/L are spat
Temperature -80 is added in ultrapure water, stirs 15 minutes at room temperature, is configured to as clear as crystal aqueous solution.B. according to dosage into a solution
L- α-phosphatidyl choline powder is added, stirs 15 minutes, until L- α-phosphatidyl choline powder is completely dissolved.C. exist according to dosage
The fatty acid basis mother liquor of above-mentioned 11 kinds of dehydrated alcohols is successively added in b solution while stirring, stirs 15 minutes, with 0.22 μm
Fat acid mother liquor is made in filter membrane negative pressure filtration, sterile to be protected from light -20 DEG C of preservations.
The turbidity for the indissoluble fat acid mother liquor prepared according to the present invention is 10NTU, the membrane filtration for being 0.22 μm with aperture
The indissoluble fat acid mother liquor turbidity for showing as being easy filtering, and being directly dissolved in water is 50NTU, the filter membrane mistake for being 0.22 μm with aperture
Filter shows as sad filter, easily blocks fenestra.
The preparation of 3 culture medium of embodiment
The chemically defined height of not fatty acids is added according to proper ratio for the indissoluble fat acid mother liquor prepared according to the present invention
In density CHO culture medium (by the independent development of Guangzhou Ke Ruite company), with the water-soluble indissoluble fat for being directly added into same ratio
The change of fatty acid is also not added in the chemically defined high density CHO culture medium (by the independent development of Guangzhou Ke Ruite company) of acid mother liquor
Learn limit high density CHO culture medium (by the independent development of Guangzhou Ke Ruite company) turbidity, filtering, to Chinese hamster ovary celI (cell Lai
From hundred biotechnology Guangzhou Co., Ltds difficult to understand safe) growth and antibody production comparing result it is as shown in table 2.
2. different culture medium of table and comparing result
In terms of upper table result, chemically defined high density CHO is not influenced with the indissoluble fat acid mother liquor that the present invention prepares and is cultivated
The turbidity of base and filtering, moreover it is possible to greatly promote the yield of cell growth and antibody.
Claims (10)
1. a kind of solution for dissolved fat acid, which is characterized in that the solution is grouped as by the group of following concentration: methyl-
Beta-cyclodextrin 100000mg/L,F-68 100mg/L, Tween-80 22000mg/L.
2. solution as described in claim 1, it is characterised in that the difficult lysing adipose tissue acid of the fatty acid.
3. solution as described in claim 1, it is characterised in that the fatty acid be selected from following substance one kind or it is a kind of with
On combination: (soil)-alpha-lipoic acid, methyl linoleate, linoleic acid, arachidonic acid, DL- alpha-tocopherol acetate, nutmeg
Acid, oleic acid, palmitinic acid, stearic acid, L- α-phosphatidyl choline, gamma-Linolenic acid, palmitoleic acid, cholesterol, sinapic acid, 18 carbon
Tetraenoic acid, eicosatrienoic acid, eicosatetraenoic acid, eicosapentaenoic acid, clupanodonic acid, docosahexaenoic acid,
Nisioic acid, elaidic acid.
4. a kind of fatty acids solution, it is characterised in that obtained with the acid of solution dissolved fat described in claim 1.
5. fatty acids solution as claimed in claim 4, it is characterised in that the mass fraction of the fatty acid in the solution is
0.003%-0.3%.
6. a kind of preparation method of adipic acid solution, it is characterised in that solution as described in claim 1 is used, by fatty acid dissolution
It is dissolved into the solution, or by fatty acid with organic solvent after basic mother liquor, to add the solution dissolution.
7. preparation method as claimed in claim 6, which is characterized in that obtained again with membrane filtration after dissolution.
8. preparation method as claimed in claim 6, which is characterized in that the organic solvent is dehydrated alcohol.
9. preparation method as claimed in claim 6, which is characterized in that the mass fraction of fatty acid is in the basic mother liquor
3%-30%.
10. a kind of culture medium containing the adipic acid solution as described in claim 6-9 is any.
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| CN201510408597.8A CN106337077B (en) | 2015-07-10 | 2015-07-10 | Indissoluble fat acid mother liquor of chemically defined high density CHO culture medium and preparation method thereof |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1793330A (en) * | 2005-11-07 | 2006-06-28 | 浙江工商大学 | Process for preparing high actived alctic acid bacteria product |
| CN101724600A (en) * | 2009-11-30 | 2010-06-09 | 中国人民解放军军事医学科学院生物工程研究所 | Blood serum-free culture medium for supporting suspension culture of CHO cells with large scale and high density |
| CN102311989A (en) * | 2011-07-05 | 2012-01-11 | 贾晓斌 | Surfactant-containing biotransformation method |
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2015
- 2015-07-10 CN CN201510408597.8A patent/CN106337077B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1793330A (en) * | 2005-11-07 | 2006-06-28 | 浙江工商大学 | Process for preparing high actived alctic acid bacteria product |
| CN101724600A (en) * | 2009-11-30 | 2010-06-09 | 中国人民解放军军事医学科学院生物工程研究所 | Blood serum-free culture medium for supporting suspension culture of CHO cells with large scale and high density |
| CN102311989A (en) * | 2011-07-05 | 2012-01-11 | 贾晓斌 | Surfactant-containing biotransformation method |
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Effective date of registration: 20180629 Address after: 510530 Guangdong Guangzhou high tech Industrial Development Zone, Science City, open source road 11, A6 fifth. Applicant after: Bio-Thera Solutions, Ltd. Address before: 510530 Guangdong City, Guangzhou, Guangzhou hi tech Industrial Development Zone, Science City, 11 yuan A6 road six floor. Applicant before: Ke Ruite bio tech ltd, Guangzhou |
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Address after: 510530 Guangzhou High-tech Industrial Development Zone, Guangzhou, Guangdong Province, Fifth Floor, Building A6, 11 Kaiyuan Avenue, Science City Applicant after: BAOTAI BIOLOGICAL PHARMACEUTICAL CO., LTD. Address before: 510530 Guangdong Guangzhou high tech Industrial Development Zone, Science City, open source road 11, A6 fifth. Applicant before: Bio-Thera Solutions, Ltd. |
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