CN86107102A - The manufacture method of colony stimulating factor - Google Patents
The manufacture method of colony stimulating factor Download PDFInfo
- Publication number
- CN86107102A CN86107102A CN198686107102A CN86107102A CN86107102A CN 86107102 A CN86107102 A CN 86107102A CN 198686107102 A CN198686107102 A CN 198686107102A CN 86107102 A CN86107102 A CN 86107102A CN 86107102 A CN86107102 A CN 86107102A
- Authority
- CN
- China
- Prior art keywords
- csf
- stimulating factor
- colony stimulating
- solution
- molecular weight
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K9/00—Peptides having up to 20 amino acids, containing saccharide radicals and having a fully defined sequence; Derivatives thereof
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Life Sciences & Earth Sciences (AREA)
- Peptides Or Proteins (AREA)
- Separation Using Semi-Permeable Membranes (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
Process for purification from the colony stimulating factor of Urina Hominis.This method is that the flaky material that will be substrate is rolled into helicoidal structure with the cellulose, makes filter cylinder type anion exchanger.Handle the solution that contains from the colony stimulating factor of Urina Hominis with this permutoid, further also will handle above-mentioned solution with the ultrafilter membrane that can remove at least than one of the big material of colony stimulating factor molecular weight and material littler than colony stimulating factor molecular weight.This method can high-purity, high yield reclaims, specific activity is high and remove the former CSF of heating.Compare with existing method, this method is simple to operate, the efficient height, and good in economic efficiency.
Description
The invention relates to high yield, high-purity contains the method that reclaims CSF in the Urina Hominis of colony stimulating factor (hereinafter to be referred as CSF).
CSF is present in biological active substances in the Urina Hominis with denier, is to act on precursor cell in the bone marrow to promote leukocyte to form the glycoprotein of the granule ball at cell place and the division of single ball macrophage, propagation, can expect to have the drug effect of treatment leukopenia.
Process for purification as CSF has the method that adopts adsorbent, the method for the method of anion exchanger and employing ultrafilter membrane etc.
Because CSF content is atomic, it is difficult making CSF separate effectively, concentrate and make with extra care in Urina Hominis.The mineral such as silicate, kieselguhr that make that for example in the past often adopted contact with urine, adsorb CSF, make the method for CSF stripping then, the method for utilizing various anion-exchange chromatographies to reclaim.Their shortcoming is a complicated operation, and because of once the treatable urine of institute is also limited, a large amount of trace or branch CSF of reclaiming, its cost is inevitable very high.
For example, in the chromatography that adopts various anion exchangers, operations such as the utilization filtration of the filling of the adsorbent of cylinder method and batch process or centrifugalize recovery absorption all are very complicated, thereby their are imperfect as the method for effectively obtaining CSF on a large scale.
Moreover, many impurity are arranged in the urine, adopt method in the past, these impurity are easy to be recovered with CSF, and this is very disadvantageous to follow-up refining step.
Purpose of the present invention just provides a kind of from containing the CSF from urine of trace, and contains in the solution of many impurity with high yield, high-purity, and the method for CSF is made in recovery low-cost and simple to operate.
The purpose of this invention is to provide a kind of refining method from the colony stimulating factor in the Urina Hominis, it is characterized in that usually to be that the sheet-shaped material of substrate is rolled into helicoidal structure with fiber, make filter cylinder, as cation exchange column, handle the solution that contains from the colony stimulating factor of Urina Hominis with this anion exchanger.
The used solution that contains CSF among the present invention has more than the aqueous solution that is limited to CSF, for example contains the urine that there is a large amount of impurity at the micro-CSF dawn etc. and also can use.
As anion exchanger, as diethyl amino ethyl group-polydextran gel, QAE (QAE yl)-polydextran gel, diethyl amino ethyl group-cellulose, QAE-cellulose, diethyl amino ethyl group-セ Off ァ ロ-ス, diethyl amino ethyl group-セ Off ァ セ Le, ス Off ェ ロ ッ Le-DEA etc. all can adopt, but preferably adopting the cellulose that contains the anion exchange base is the sheet-shaped material twist up of substrate, makes filter cylinder, comparatively favourable.
With universal support (as cellulose, the glucosan agarose) crosslinked with ethene polymers, import QAE again, diethyl amino ethyl group (DEAE yl), TEAE triethyl aminoethyl (TEAE yl), aminoethyl (AE yl), GE (ion exchange substrate are consulted Fig. 1) such as (GE yls) is rolled into the filter cylinder that helical form promptly can be made into said structure with it.Its best shape as shown in Figure 2.In Fig. 2,1 is ion exchange substrate (bed thickness 0.1-1mm), 2 is water penetration support (bed thickness 0.1-1mm), and this support preferably adopts latticed or the braiding shape, and its material can adopt as polystyrene, nylon, Merlon, polyacetals, polyflon, rustless steel etc.
Fig. 3 is to use the profile of the process for purification embodiment of this structure, from the 3 CSF liquid that enter that enter the mouth, assembles from the periphery to central part 4, discharges from exporting 5.The arrow indication is the flow direction of CSF liquid, and 6 is air vent, and 7 is outlet.Fig. 4 is a cross-sectional view, for the CSF liquid that enters from the periphery to the accumulative state in center.
As service condition of the present invention, it is 5～7 that the liquid that contains CSF is preferably in pH value, and electric conductivity is 20ms/cm(25 ℃) following through cylinder, and CSF is adsorbed.Under such condition, CSF is adsorbed, and most of impurity is not adsorbed and directly passes through.Then, be the following phosphate buffer solution of rare saline solution (for example: electric conductivity is 20ms/cm(25 ℃) of 5～7 with pH) washing.Use this washing operation, can further remove impurity, and still former state absorption and left behind of CSF.
For making the CSF stripping of absorption, feeding pH is 5～9 the above phosphoric acid buffer agent of dense saline solution (for example: electric conductivity is 30ms/cm(25 ℃), salt solution) just achieve the goal at an easy rate.
In the present invention, preferably further utilized filter membrane to handle.The processing of ultrafilter membrane is carried out before aforementioned anion exchanger is handled.
As ultrafilter membrane, preferably adopt ultrafilter membrane (promptly being used for separation of high molecular weight person) a kind of to remove the ultrafilter membrane (promptly being used for separate low molecular amount person) of the lower-molecular substance little and can remove the polymer substance bigger, or two kinds are used all than CSF than CSF.As separate low molecular amount person, adopt 1 * 10 usually
4～4 * 10
4Daltonian ultrafilter membrane.As separation of high molecular weight person, use 10 usually
5～10
6Daltonian ultrafilter membrane.As ultrafilter membrane, preferably adopt polypropylene nitrile ultrafilter membrane and polysulfones ultrafilter membrane and cellulose-based ultrafilter membrane etc.
The solution that contains CSF, the condition during by ultrafilter membrane is generally:
The pH value of treatment fluid is 5～8
Operating pressure is 1～5kg/cm
2
Ultrafiltration membrane treatment method of the present invention can be used for the recovery of CSF, the arbitrary operation in producing, but preferably handles with the supermembrane filter membrane of separate low molecular amount earlier, and then handles with the ultrafilter membrane of separation of high molecular weight.
So, the CSF of recovery preferably further adopts the refining means (as affinity chromatography, gel filtration etc.) of known high accuracy to handle.And heating (as 50～80 ℃ of heating 1～10 hour) sterilization, then, lyophilization after handling like this, can be used as medical CSF preparation.Certainly, also can not carry out lyophilization, and adopt means known to separate, make with extra care.
But adopt the present invention's high-purity, high yield reclaims CSF.And can obtain the specific activity height, remove the former CSF that generates heat.Moreover, compare with existent method, have operability simple and easy, that efficient is high and economy.
In order to illustrate in greater detail the present invention, especially exemplified by going out following embodiment, but the present invention has more than and is limited to these.
Embodiment 1
After removing the low molecular weight impurities in the fresh urine of 50l with polyacrylonitrile ultrafiltration film (can divide molecular weight＜20000 parts), be concentrated into 1l.
Zetaprep QAE-filter cylinder R250 that this concentrated solution is made by AMF Inc., this is the phosphate buffer (pH is 6.5, and electric conductivity is 8.5ms/cm(25 ℃) with previously prepared 0.05M) carry out making after the Balance Treatment.CSF is adsorbed when flowing through this.
Then with behind phosphate buffer (pH the is 6.5) thorough washing of this QAE tube with 0.05M, usefulness contains the 0.05M phosphate buffer (pH is 6.5) (electric conductivity is 8.5ms/cm(25 ℃) of 1.0M NaCl) stripping, just can obtain containing the solution of CSF.Moreover, in order to contrast, in addition fresh urine 50l is diluted and four times after, add again with the diethylaminoethyl cellulose 100g after 0.05M phosphate buffer (pH6.5) balance, stir and made it absorption in one hour, carry out and top identical washing, stripping, can obtain containing the dissolution fluid of CSF.
About these dissolution fluids, measure their CSF activity respectively, obtain their specific activity and the response rate, it the results are shown in table 1-1 and 1-2.
Moreover the activity of CSF is to adopt the colony forming method that utilizes the monolayer agar plate method to measure, and what the monolayer agar plate method was used is C57BL/6N Os Mus myelocyte.Promptly with containing the 2%(volume ratio) distilled water of Ox blood serum suitably dilutes test portion, after the aseptic filtration (0.45 μ film filter), above-mentioned solution is splashed into respectively in three surface plates with 0.1ml, add therein again that to contain 0.3%(heavy) agar, 20%(volume) the cattle fetal blood clear and contain the Mc Coy ' s 5A culture medium solution 1ml of 10 of C57BL/6N Os Mus myelocytes, after fully mixing, contain the 7.5%(volume being in) in the couveuse under the atmosphere of carbon dioxide, cultivated seven days down at 37 ℃.Cultivate the back and measure the number of the colony of forming by the cell more than 50, represent the activity of CSF, that is, represent active unit of CSF with colony of every formation with the colony number of formation at microscopically.
As show shown in 1-1 and the table 1-2, the method that the present invention and past adopt is compared, owing to adopted ultrafilter membrane to make with extra care, removes impurity in advance, though thereby the response rate identical, specific activity (purity) improves more than more than ten times.
Moreover the method that is adopted when absorption, because the way of utilization dilution is adjusted different electric degree, thereby increased amount of liquid in the past; In contrast, method of the present invention is because it is refining as pretreatment to have introduced ultrafilter membrane, thereby amount of liquid is changed on a small quantity, make the electric conductivity adjustment both simple simultaneously, the operating characteristics when operation utilizes anion exchanger to handle after can improving again also can be improved working performance.Moreover, because the ultrafiltration apparatus of introducing is the filter cylinder type, can further improve the transaction capabilities when adopting the anion exchange chromatography.
Table 1-1 data of the present invention
CSF
The test portion title
Specific activity, the U/E280nm response rate, %
Urinate 3.0 100
Concentrated solution 72 88
QAE-7,500 84
The liquid of tube stripping
The data of table 1-2 previous method
CSF
The test portion title
Specific activity, the U/E280nm response rate, %
Urinate 3.0 100
Diethyl amino ethyl group-350 84.8
The cellulose dissolution fluid
With the tabular ultrafilter membrane (can divide molecular weight＞1000000 parts) of polysulfones, the solution 4000ml that will contain CSF separates, and circulating filtration 4000ml solution needs 30 minutes.
Then, identical with embodiment 1, adopt QAE-tube to carry out the separation of ion exchange chromatography.
To the liquid before filtering, liquid after the filtration and QAE-tube stripping liquid adopts the method for embodiment 1, measures the CSF activity, obtains gross activity then, the specific activity and the response rate.According to the Japanese Pharmacopoeia of the 9th revision, utilize rabbit to measure the former removal efficient of heating again, it the results are shown in table 2.
As shown in table 2, can reclaim CSF by high-recovery, also improved specific activity (purity) simultaneously.Moreover, about CSF, owing to remove clogged material with ultrafilter membrane, so also improved activity.
Moreover, remove the heating former aspect, obvious effects is also arranged.
Table 2
The former experiment of generating heat
The test portion title (goes up and heats up
The specific activity U/E280nm response rate, the % degree, ℃)
Liquid 6,500 100 0.6,0.8,0.7 before filtering
Add up to 2.1
Filter back liquid 21,000 135.2 0.2,0.3,0.1
Add up to 0.6
QAE-85,000 118 0,0,0.1
The tube dissolution fluid adds up to 0.1
The simple declaration of accompanying drawing
The 1st figure is the structure diagram after the sheet-shaped material that will contain the anion exchange base is rolled into the spirillum shape;
The 2nd figure is the cross-sectional view of said structure;
The 3rd figure is the vertical cross-section diagram that sheet-shaped material is rolled into an embodiment of the anion exchanger after the helical form;
The 4th figure is the cross-sectional view of the foregoing description.
Wherein:
The C-carrier;
The P-ethene polymers;
Q-QAE or diethyl amino ethyl group;
The 1-ion exchange substrate;
2-water penetration support;
The 3-inlet;
The 4-central part;
The 5-outlet;
The 6-air vent;
The 7-outlet.
Claims (1)
- Process for purification from the colony stimulating factor of Urina Hominis, it is characterized in that this method is that the sheet-shaped material that will be substrate is rolled into helicoidal structure with the cellulose, make filter cylinder type anion exchanger, handle the solution that contains from the colony stimulating factor of Urina Hominis with this permutoid, further also will handle above-mentioned solution with the ultrafilter membrane that can remove one of following two kinds of materials at least; Promptly than little lower-molecular substance of colony stimulating factor molecular weight and the polymer substance bigger than colony stimulating factor molecular weight.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP60234335A JPS6293238A (en) | 1985-10-18 | 1985-10-18 | Production of colony stimulating factor |
| JP234335/85 | 1985-10-18 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN86107102A true CN86107102A (en) | 1987-08-12 |
Family
ID=16969380
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN198686107102A Pending CN86107102A (en) | 1985-10-18 | 1986-10-17 | The manufacture method of colony stimulating factor |
Country Status (3)
| Country | Link |
|---|---|
| JP (1) | JPS6293238A (en) |
| KR (1) | KR930008097B1 (en) |
| CN (1) | CN86107102A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1083488C (en) * | 1996-06-05 | 2002-04-24 | 杭州九源基因工程有限公司 | Method for production of recombining human grunulocyte colony stimulation factor |
-
1985
- 1985-10-18 JP JP60234335A patent/JPS6293238A/en active Granted
-
1986
- 1986-10-16 KR KR1019860008679A patent/KR930008097B1/en not_active IP Right Cessation
- 1986-10-17 CN CN198686107102A patent/CN86107102A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1083488C (en) * | 1996-06-05 | 2002-04-24 | 杭州九源基因工程有限公司 | Method for production of recombining human grunulocyte colony stimulation factor |
Also Published As
| Publication number | Publication date |
|---|---|
| JPS6293238A (en) | 1987-04-28 |
| KR930008097B1 (en) | 1993-08-25 |
| KR870004055A (en) | 1987-05-07 |
| JPH0535160B2 (en) | 1993-05-25 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN1065464A (en) | The method of residual monomer and oligopolymer in the removal amine-containing polymer | |
| CN1387458A (en) | Method for purification of amino acid contg. solutions by electrodialysis | |
| CN1266158C (en) | Method for separating and extracting D-ribose from fermented liquid by film separating technology | |
| CN100339351C (en) | Method for separating remaining sugar and extracting organic acid from organic acid fermentation liquor and corresponding organic acid mother liquor | |
| CN1006550B (en) | Lipoprotein sorbent used in external circulating therapy and process for producing the same | |
| CN102477094A (en) | Separation and purification process for synthetic thymosin alpha 1 | |
| CN86107102A (en) | The manufacture method of colony stimulating factor | |
| CN1141289C (en) | Clean productive process for extracting citric acid from citric acid fermentation liquid | |
| CN105254746A (en) | Method for desalinating thymopeptide alpha 1 | |
| CN1250567C (en) | Method of purifying calcium ion-binding protein | |
| CN1974418A (en) | Sea water desalting agent based on silver carrying acid zeolite and its prepn process | |
| CN1179781C (en) | Electro ultrafiltration liquation crystallization separation purification method of biomacromolecule | |
| CN113214160A (en) | Method for efficiently purifying histidine bulk drug without ammonia nitrogen discharge | |
| CN1083488C (en) | Method for production of recombining human grunulocyte colony stimulation factor | |
| CN1226418C (en) | Natural active protein and peptide separating and purifying process | |
| CN1272342C (en) | Novel process for preparing recombinaant CHO cell hepatitis B vaccine | |
| CN1228099C (en) | Adsorbent device for body fluid treatment | |
| CN1965863A (en) | Method for preparing compound amino acid oral liquid by using animals placenta | |
| CN1049356C (en) | Protein-containing aqueous solutions | |
| JPS6160050B2 (en) | ||
| CN1101823C (en) | Shark liver irritant for preventing and curing hepatism and its preparing process | |
| JP4841749B2 (en) | Method for producing purified antigen | |
| RU93018605A (en) | METHOD FOR PRODUCING SODIUM SALT OF DEXOXYRIBONUCLEIC ACID FROM ANIMAL RAW MATERIAL | |
| CN1482250A (en) | Method for producing acrylamide using film technique microbiological transformation | |
| CN1059237C (en) | Method for producing recombined human liver cytokinin and its use for curing serious hepatic disease |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C12 | Rejection of a patent application after its publication | ||
| RJ01 | Rejection of invention patent application after publication |