CN102191226A - Separation and purification method for cell culture mixture - Google Patents
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Abstract
The invention discloses a separation and purification method for a cell culture mixture, comprising the step of: filtering the cell culture mixture by a filter, wherein the filter is provided with at least two layers of filtering membranes, the nominal pore diameter of each layer of filtering membrane is gradually reduced in the direction of flow of the filtrate, i.e. the nominal pore diameter of the first layer of filtering membrane is about 2-10mu m, and the nominal pore diameter of the final layer of filtering membrane is about 0.1-0.2mu m. The separation and purification method for a cell culture mixture disclosed by the invention has the advantages of strong pertinence, large treatment capacity, simple separation and purification step, stable product quality, low cost and the like.
Description
Technical field
The present invention relates to a kind of method of separation and purification of biological products, particularly in order to the separation purification method of the cell cultures mixture that obtains the purpose biological products.
Background technology
The separation and purification of the culturing mixt of purpose biological products is one of most essential steps during biological products are produced, it is the hinge step between results purpose culturing mixt and the processing of further downstream, output, quality, repeatability to biological products have direct influence, have also determined the efficient and the cost consumption of biological products integral production technology.
Purpose culturing mixt separation purification method commonly used mostly is the use of uniting as several methods such as centrifugal, gel chromatography, filtrations in the existing biological products production technique, exist many problems more, at first the transfer of culturing mixt between equipment causes the pollution of product easily, secondly, employed tables of equipment batch processed amount is less, is difficult for carrying out industrial scale and enlarges, once more, equipment used is reused and need be cleaned repeatedly, has increased expending of labor force and production cost.In addition, existing technology usually can not fully separation and purification purpose culturing mixt, causes final biological products xenobiotic many.
At present, the conventional and unconventional immunity of the mankind, poultry, domestic animal is the important means that ensures human life, health, so the demand of various virus vacciness is also constantly increasing.And most of virus vacciness all obtain by separation and purification after the cell cultures, so more and more be subjected to people's attention for the demand of high-level efficiency, high-quality ground separation and purification cell cultures mixture.
Summary of the invention
Use existing technique means to come separation and purification cell cultures mixture, owing to there are the problems referred to above, separation and purification process complexity, can not be in a large number, fast and high purity ground separation and purification cell cultures mixture, cause the cost of separation and purification higher, defectives such as production time length, in addition, final biological products xenobiotic is many, and side reaction is bigger during use.As seen, existing separation purification method can't satisfy the needs that reality is produced, lived.
Therefore, the present invention purifies at the separation of cell cultures mixture especially and concentrates on studies, and finds that the cell cultures mixture has generally lower, the initial large usage quantity of content, the different virus granular size that component comprises complexity, purpose product and has characteristics such as certain difference.
For this kind biological product of cell cultures mixture, the inventor has proposed novel method that it is carried out separation and purification in a creative way, has overcome above-mentioned defective.
The invention provides a kind of separation purification method of cell cultures mixture, 1. the separation purification method of a cell cultures mixture, comprise the cell cultures mixture is filtered through filter, this strainer has the different filtering membrane in two-layer at least aperture, the nominal pore size of each tunic of filtering membrane diminishes on the filtrate flows direction step by step, and the nominal pore size of the first layer filtering membrane is about 2～10 μ m, and the nominal pore size of last one deck filtering membrane is about 0.1～0.2 μ m.
2. according to the separation purification method of technical scheme 1 described cell cultures mixture, wherein, the cell cultures mixture is selected from the Hybridoma Cell Culture mixture of animal with virocyte culturing mixt, human virocyte culturing mixt or secretory antibody.
3. according to the separation purification method of technical scheme 1 described cell cultures mixture, wherein, described strainer has three layers, four layers or five layers of filtering membrane.
4. according to the separation purification method of technical scheme 1 described cell cultures mixture, wherein, in filtration procedure, the pressure reduction before and after the filtering membrane is about 0.05-0.1MPa.
5. according to the separation purification method of technical scheme 1 to 4 each described cell cultures mixture, wherein, described strainer is that filtering membrane is the strainer of folded form.
6. according to the separation purification method of technical scheme 5 described cell cultures mixtures, wherein, described strainer is provided with
Housing;
Folding filtering membrane is divided into central area and marginarium with enclosure interior.
7. according to the separation purification method of technical scheme 1 to 4 each described cell cultures mixture, wherein, so the cell cultures mixture is selected from: the foot and mouth disease virus culturing mixt, the porcine reproductive and respiratory syndrome virus culturing mixt, the Pestivirus suis culturing mixt, porcine kidney cell (PK cell) and pig testis cell (ST cell); Rabies virus culturing mixt, hepatitis B virus culturing mixt, epidemic encephalitis B virus culturing mixt, hepatitis A virus culturing mixt or poliovirus culturing mixt.
8. according to the separation purification method of technical scheme 1 to 4 each described cell cultures mixture, wherein, the treatment capacity of liquid to be filtered is: the flow range of every square metre of filtering membrane is about 100～200L under every 0.01Mpa pressure reduction.
9. according to the separation purification method of technical scheme 1 described cell cultures mixture, wherein, described cell cultures mixture is the foot and mouth disease virus culturing mixt, described filtering membrane is two-layer filtering membrane, the nominal pore size of the first layer filtering membrane is about 2～10 μ m, and the nominal pore size of second layer filtering membrane is about 0.1～0.2 μ m.
10. according to the separation purification method of technical scheme 1 described cell cultures mixture, wherein, described cell cultures mixture is the porcine reproductive and respiratory syndrome virus culturing mixt, described filtering membrane is the three layer filtration film, the nominal pore size of the first layer filtering membrane is about 2～10 μ m, the nominal pore size of second layer filtering membrane is about 0.2～2 μ m, and the nominal pore size of three layer filtration film is about 0.1～0.2 μ m.
The separation purification method of a kind of cell cultures mixture of the present invention, pointed strong, treatment capacity is big, purification procedures is simple, constant product quality, low cost and other advantages, be particularly suitable for separation and purification foot and mouth disease virus culturing mixt or porcine reproductive and respiratory syndrome virus culturing mixt etc., obtain the higher virus vaccines mixture of purity.
In addition, in the separation purification method of cell cultures mixture of the present invention, can reach bigger single treatment amount, high to several hectolitres, thus be easy to expand the scale of production; And, the separation purification method of cell cultures mixture of the present invention is comparatively thorough to the object separation and purification, cell debris in the product and other big materials all are trapped, thereby reduced the immunization side reaction of biological products, the transfer that has reduced the purpose culture exposes, thereby has reduced the contaminated probability of object.
The separation purification method process control of cell cultures mixture of the present invention is simple and easy, uses filtering element can be disposable product, does not have processes such as cleaning, clean checking, saves labor force and production cost, enhances productivity;
The separation purification method process control of cell cultures mixture of the present invention is simple and easy, can select different big or small filtering elements and combination thereof according to different separation and purification objects, uses flexibly, has wide range of applications.
Embodiment
Term among the present invention " animal " refers to inhuman various animal, particularly preferably is domestic animals or poultry, such as: pig, dog, ox, sheep, chicken, duck and/or goose etc.
Term among the present invention " cell cultures mixture " is meant and cultivates the cell cultures liquid mixture that virus, antibody etc. are obtained under existing condition.Specifically, described cell cultures mixture comprises the Hybridoma Cell Culture mixture of animal with virocyte culturing mixt, human virocyte culturing mixt and secretory antibody.Mainly contain virion (or antibody) in the wherein said cell cultures mixture, and impurity such as culturing cell, culturing cell fragment, protein, nucleic acid, polysaccharide, if used cell culture medium then also contains remaining serum in the gained mixture for containing the serum cell culture medium.Require to have the purpose virus of high level in the gained filtrate, and impurities for example host protein, host's nucleic acid etc. are controlled at lower level, no mushroom pollution as far as possible.
The virus diameter is usually between 20nm-200nm, and at different virus, adopt different cells to cultivate, the contriver has carefully studied the cell cultures mixture of different virus and antibody, described animal is specific as follows with the virocyte culturing mixt: the foot and mouth disease virus culturing mixt, the about 20-25nm of virus diameter, culturing cell commonly used is young hamster kidney passage cell (a BHK21 cell); The porcine reproductive and respiratory syndrome virus culturing mixt, the about 50-60nm of viral diameter, culturing cell commonly used are the Marc145 cell; The Pestivirus suis culturing mixt, the about 70nm of viral diameter, culturing cell commonly used are bull testis primary cell (BT cell), porcine kidney cell (PK cell) and pig testis cell (ST cell); The rabies virus culturing mixt, virus is about 170-180nm, and wide about 75-80nm, culturing cell commonly used are BHK21.
Described human virocyte culturing mixt is as follows: the rabies virus culturing mixt, and virus is about 170-180nm, and wide about 75-80nm, culturing cell commonly used are African green monkey kidney cell (Vero cell); The hepatitis B virus culturing mixt, the about 22nm of viral diameter, culturing cell commonly used are Chinese hamster ovary cell (Chinese hamster ovary celI); The epidemic encephalitis B virus culturing mixt, the about 40nm of viral diameter, culturing cell commonly used are the Vero cell; The hepatitis A virus culturing mixt, the about 27-32nm of viral diameter, culturing cell commonly used are human diploid cell KMB
17Or 2BS; The poliovirus culturing mixt, the about 27-30nm of viral diameter, culturing cell commonly used are human diploid cell 2BS.
From the above, the diameter of most of viruses is about 20-100nm, and the diameter of indivedual viruses is between 100-200nm.
The pair cell culturing mixt is separated the target mixture that obtains and is required contamination-free, and the content of impurity should control to minimum level as far as possible, wherein said pollutent such as cell, bacterium, mould and/or mycoplasma etc., described impurity such as foreign protein, nucleic acid and/or bovine serum etc., different biological products have different requirements and restriction to this.
The contriver finds that the diameter of all contaminations compares with the diameter of virus, all there is suitable difference, and in the cell cultures mixture, the diameter of zooblast is about 10-100 μ m usually, the bacterial cell diameter is about 0.5-2.0 μ m usually, fungal cell's diameter is about 10-40 μ m usually, and the mycoplasma diameter is about 0.2-0.3 μ m usually.Therefore,, can improve filtration efficiency widely, thereby reduce the step that obtains final product, enhance productivity, reduce cost by selecting appropriate filtration condition.
The invention provides a kind of separation purification method of cell cultures mixture, this method comprises filters the cell cultures mixture through bilayer or bed filtration film, obtain the good filtrate of separation and purification.The nominal pore size of each tunic of this bilayer or bed filtration film diminishes on filtering direction step by step, such as two layers, three layers, four layers, five layers or six layers, be preferably three layers, four layers or five layers of filtering membrane, according to the virus of cell cultures mixture and the characteristics of cell, the nominal pore size of the first layer filtering membrane is about 2～10 μ m, the nominal pore size of last one deck filtering membrane is about 0.1～0.2 μ m, if the nominal pore size of the first layer filtering membrane is less than 2 μ m, so can each duct of very fast obstruction than multi-pollutant or impurity, thereby filtration can't be carried out smoothly, if the nominal pore size of the first layer filtering membrane is greater than 10 μ m, there are pollutents such as part cell can pass through the first layer filtering membrane so, thereby accumulate on the second layer filtering membrane, thereby make the filtration efficiency step-down; If the nominal pore size of one deck filtering membrane can increase filtration resistance less than 0.1 μ m at last, be unfavorable for improving filtration efficiency, if greater than 0.2 μ m, can make the foreign matter content of final filtrate too many, can't reach desired degree of purity.The first layer filtering membrane can fall larger-diameter pollutent or contaminant filter, and last one deck is pollutent and the contaminant filter of minor diameter to be fallen as far as possible, according to the characteristics of cell culture fluid, and filtration successively, thus obtain the higher viral filtrate of purity.
The nominal pore size of intermediary filtering membrane is between the nominal pore size of the first layer filtering membrane and last one deck filtering membrane, the nominal pore size of intermediate filtered film can be adjusted according to the characteristics of cell cultures mixture, such as when using the three layer filtration film, the nominal pore size of the first layer filtering membrane is about 2～10 μ m, the nominal pore size of second layer filtering membrane is about 0.2～2 μ m, the nominal pore size of three layer filtration film is about 0.1～0.2 μ m, use the structure of bed filtration film, can reach the purpose of Depth Filtration.The first layer filtering membrane that the nominal pore size is about 2～10 μ m can filter the thicker impurity of major part, such as: cell, cell debris; Through filtering step by step layer by layer, finally reach degerming, remove other useful component filtration filtering membrane of mycoplasma level, form quality filtered liquid product up to standard substantially, this product can be used for further aftertreatment, such as: add adjuvant, lyophilize, branch packing etc.
Above-mentioned filtering membrane can be selected the suitable filtering membrane in all apertures on the present market for use.The material of filtering membrane is preferably fiber and inorganic filter aids such as pearl carbon and diatomite and adhesive bonds and forms, described fiber can be selected from the blend fiber of polypropylene fibre, glass fibre, cellulose acetate, nitrocellulose and above fiber etc., the material of each layer filtering membrane can be identical or different, and preferably the fiber that uses is polypropylene fibre.The nominal pore size that marks on the filtering membrane product that " nominal pore size " refers in this article.
The bed filtration film can be the bed filtration film that is combined with each other, and also can be that the single layer filter film in suitable aperture stacks together and the bed filtration film that forms, also can be the filtering membrane with a plurality of different apertures filtering layer that is made into integration.As long as meeting relevant aperture requires.
Treatment capacity can determine that for the above cell cultures mixture that exemplifies, the preferably treatment amount is about 100～200L/ (m according to the selection of filtering membrane and the characteristics of cell cultures mixture
2.h.100mbar), i.e. the flow range of every square metre of filtering membrane under 100mbar (0.01Mpa) pressure reduction, preferred about 120～180L/ (m
2.h.100mbar), further preferred about 140～170L/ (m
2.h.100mbar).At this 100～200L/ (m
2.h.100mbar) in the scope, can obtain stay-in-grade product usually.If too high, the undesirable impurity of part may enter filtered liquid, and filter effect is reduced; If too low, filtration efficiency is unfavorable for improving.
For above-mentioned cell cultures mixture, be preferably animal and use the virus culture mixture, for example: foot and mouth disease virus culturing mixt, porcine reproductive and respiratory syndrome virus culturing mixt, Pestivirus suis culturing mixt.
According to the difference of animal with the virus of being cultivated in the virus culture mixture, can suitably select different filtering membranes, particularly, for being the foot and mouth disease virus culturing mixt, because the about 20-25nm of its viral diameter, diameter is less, so consider from the angle that improves filtration efficiency, the preferred double medium filtration film that uses, the nominal pore size of the first layer filtering membrane is about 2～10 μ m, and the nominal pore size of the 2nd layer of filtering membrane is about 0.1～0.2 μ m.
For the porcine reproductive and respiratory syndrome virus culturing mixt, the about 50-60nm of virus diameter, 3 layers of filtering membrane of preferred use, the nominal pore size of the first layer filtering membrane is about 2～10 μ m, the nominal pore size of second layer filtering membrane is about 0.2～2 μ m, and the nominal pore size of last one deck filtering membrane is about 0.1～0.2 μ m.
The mode that above-mentioned filtration is preferably pressurizeed before filtration and/or reduced pressure after filtration improves filtration velocity.Such as using force (forcing) pump (as peristaltic pump) or compressed gas that culturing mixt is pressurizeed, make it pass through filtering membrane, can use also that a side is provided with pressure regulating equipments such as vacuum pump after filtration, impel liquid to pass through filtering membrane, in order to improve filtration velocity, do not influence the quality of filtrate again, the pressure reduction of whole filtering membrane both sides (before and after promptly filtering) preferably is about 0.05-0.1MPa, more preferably from about 0.06-0.08MPa.Certainly,, can suitably adjust,, improve filtration efficiency most possibly and be as the criterion with under the prerequisite of ensuring the quality of products according to the aperture of filtering membrane and the quantity of filtering membrane for this pressure reduction.
For the specific form of filtering membrane, can be provided with as required, in order to increase the area of the filtering membrane that makes unit volume, can use the strainer of folded form, strainer comprises housing, folding filtering membrane is divided into central area and marginarium with enclosure interior.Fluid to be filtered flows into the marginarium of housing, flows into the intermediate zone through folding filtering membrane, thereby obtains target product.The filtration area of the strainer of such folded form can be about 0.6m
2～1.8m
2/ 10 ", such filtration area can reach bigger single treatment amount, high supreme hectolitre, thus be easy to expand the scale of production.
For above embodiment of the present invention further clearly is described, following examples are provided.This embodiment only in order to further specify method of the present invention, does not play the qualification effect to protection scope of the present invention.
Embodiment 1
With Depth Filtration method separation and purification BHK21 cell, foot and mouth disease virus culturing mixt
Recovery BHK21 cell from cell work seed liquid nitrogen storehouse, the first amplification of in square vase, going down to posterity, then the amplification cultivation successively of the order by rolling bottle, 5L bio-reactor, 120L bio-reactor is made cell suspension with tryptic digestion afterwards, is inoculated in CLAVORUS
TMThe 650L reactor carries out extensive amplification cultivation in (sky, Beijing and auspicious biotechnology company), the working volume of bio-reactor is 400L, cultivate after 5 days, on the BHK21 of gained cell, inoculate foot and mouth disease virus, cell culture fluid in the bio-reactor is replaced by virus-culturing fluid, cultivate postvaccinal BHK21 cell, the disposable results in back virus liquid about bioreactor culture 12-24hr, with 4 ℃ of low-temperature storage of 400L culturing mixt of being gathered in the crops in container for storing liquid.Virus concentration is about 4.5 μ g/mL in the gained culturing mixt, wherein contain BHK21 cell, BHK21 cell debris, albumen, nucleic acid, polysaccharide and bovine serum etc., above-mentioned culturing mixt is divided into two parts of equivalent, and portion is used for following experiment, and another part is used for Comparative Examples 1.
Three layers of different apertures filtration module are filled in the strainer, and the nominal pore size of each layer of filtering membrane is respectively: 3 μ m, 0.2 μ m, the filtering membrane total area are 1m
2Utilize aseptic compressed air with the 200L culture supernatant in the container for storing liquid through connecting tube is squeezed into space between filter body and the filtering element by fluid inlet in, supernatant liquor filters step by step by filter membrane, handled all above-mentioned 200L culturing mixt through 20 minutes, obtain filtrate, during the pressure reduction of filtering membrane both sides be increased to about 0.06MPa from about 0.05MPa.Collect the liquid in the collection container, can be further used for downstream reaction.Virus concentration is about 3.7 μ g/mL in the gained filtrate, compares with nutrient solution to have 80% the rate of recovery, and is wherein acellular through identifying, the pollution of no bacterium, mould, mycoplasma, the foreign protein clearance reaches 99%, residual DNA content＜100pg/mL, endotoxin content＜10EU/ml.
Carry out bacterium, mycoplasma check according to steriling test method, mycoplasma method of inspection in the 3rd appendix of in 2005 version of People's Republic of China's veterinary drug allusion quotation, utilize euzymelinked immunosorbent assay (ELISA) to measure foreign protein content, the spectrographic determination nucleic acid content, gel method is measured endotoxin content.
Embodiment 2
With Depth Filtration method separation and purification Marc145 cell and porcine reproductive and respiratory syndrome disease Poison (reproductive and respiratory syndrome virus) culturing mixt
Recovery Marc145 cell from cell work seed liquid nitrogen storehouse, the first amplification of in square vase, going down to posterity, then the amplification cultivation successively of the order by rolling bottle, 5L bio-reactor, 120L bio-reactor is made cell suspension with tryptic digestion afterwards, is inoculated in CLAVORUS
TMThe 650L reactor carries out extensive amplification cultivation in (sky, Beijing and auspicious biotechnology company), the working volume of bio-reactor is 300L, cultivate after 4 days, on the Marc145 of gained cell, inoculate porcine reproductive and respiratory syndrome virus, cell culture fluid in the bio-reactor is replaced by virus-culturing fluid, cultivate postvaccinal Marc145 cell, begin perfusion culture behind the virus culture 6hr, and gathered in the crops viral liquid since first day, with 4 ℃ of low-temperature storage of 800L culturing mixt of being gathered in the crops in container for storing liquid.Contain Marc145 cell, Marc145 cell debris, albumen, nucleic acid, polysaccharide and bovine serum etc. in the gained culturing mixt except that virus, above-mentioned culturing mixt is divided into two parts of equivalent, portion is used for following experiment, and another part is used for Comparative Examples 2.
The nominal pore size of each layer of filtering membrane is respectively: 8 μ m, 0.45 μ m, 0.1 μ m.Operating pressure difference is 0.05-0.08MPa.Promptly handled all nutrient solutions in 30 minutes.Can reach about 83% the rate of recovery, the pollution of acellular, no bacterium, fungi, mycoplasma, the foreign protein clearance reaches 99%, residual DNA content＜100pg/mL, endotoxin content＜10EU/ml.Carry out bacterium, mycoplasma check according to steriling test method, mycoplasma method of inspection in the 3rd appendix of in 2005 version of People's Republic of China's veterinary drug allusion quotation, utilize euzymelinked immunosorbent assay (ELISA) to measure foreign protein content, the spectrographic determination nucleic acid content, gel method is measured endotoxin content.
Comparative example 1
With the other 200L culturing mixt that obtains among the embodiment 1 is the single layer filter film direct filtration of 0.45 μ m by the nominal pore size, the pressure reduction of filtering membrane both sides is increased to 0.5MPa from 0.05MPa during the warp, only handle the culturing mixt of 100L, can't finish whole purifies and separates.The character of collecting the liquid in the collection container is: virion, residual DNA, protein, intracellular toxin, residual DNA content 500pg/mL wherein, foreign protein clearance only 50%, endotoxin content is 30EU/ml.
Comparative example 2
With other 400L Marc145 cell and the porcine reproductive and respiratory syndrome virus culturing mixt that obtains among the embodiment 2, be the single layer filter film direct filtration of 0.1 μ m by the nominal pore size, only the 20min filtering membrane promptly stops up, and can't finish whole purifies and separates.
Claims (10)
1. the separation purification method of a cell cultures mixture, comprise the cell cultures mixture is filtered through filter, this strainer has the different filtering membrane in two-layer at least aperture, the nominal pore size of each tunic of filtering membrane diminishes on the filtrate flows direction step by step, and the nominal pore size of the first layer filtering membrane is 2～10 μ m, and the nominal pore size of last one deck filtering membrane is 0.1～0.2 μ m.
2. the separation purification method of cell cultures mixture according to claim 1, wherein, the cell cultures mixture is selected from the Hybridoma Cell Culture mixture of animal with virocyte culturing mixt, human virocyte culturing mixt or secretory antibody.
3. the separation purification method of cell cultures mixture according to claim 1, wherein, described strainer has three layers, four layers or five layers of filtering membrane.
4. the separation purification method of cell cultures mixture according to claim 1, wherein, in filtration procedure, the pressure reduction before and after the filtering membrane is 0.05-0.1MPa.
5. according to the separation purification method of each described cell cultures mixture of claim 1 to 4, wherein, described strainer is that filtering membrane is the strainer of folded form.
6. the separation purification method of cell cultures mixture according to claim 5, wherein, described strainer is provided with
Housing;
Folding filtering membrane is divided into central area and marginarium with enclosure interior.
7. according to the separation purification method of each described cell cultures mixture of claim 1 to 4, wherein, described cell cultures mixture is selected from: the foot and mouth disease virus culturing mixt, the porcine reproductive and respiratory syndrome virus culturing mixt, the Pestivirus suis culturing mixt, porcine kidney cell (PK cell) and pig testis cell (ST cell); Rabies virus culturing mixt, hepatitis B virus culturing mixt, epidemic encephalitis B virus culturing mixt, hepatitis A virus culturing mixt or poliovirus culturing mixt.
8. according to the separation purification method of each described cell cultures mixture of claim 1 to 4, wherein, the treatment capacity of liquid to be filtered is: the flow range of every square metre of filtering membrane is 100～200L under every 0.01Mpa pressure reduction.
9. the separation purification method of cell cultures mixture according to claim 1, wherein, described cell cultures mixture is the foot and mouth disease virus culturing mixt, described filtering membrane is two-layer filtering membrane, the nominal pore size of the first layer filtering membrane is 2～10 μ m, and the nominal pore size of second layer filtering membrane is 0.1～0.2 μ m.
10. the separation purification method of cell cultures mixture according to claim 1, wherein, described cell cultures mixture is the porcine reproductive and respiratory syndrome virus culturing mixt, described filtering membrane is the three layer filtration film, the nominal pore size of the first layer filtering membrane is 2～10 μ m, the nominal pore size of second layer filtering membrane is 0.2～2 μ m, and the nominal pore size of three layer filtration film is 0.1～0.2 μ m.
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| PCT/CN2011/071472 WO2011110073A1 (en) | 2010-03-08 | 2011-03-03 | Isolation and purification method of cell culture mixture |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103614339A (en) * | 2013-11-22 | 2014-03-05 | 武汉友芝友医疗科技有限公司 | Cell separation method |
| CN113423832A (en) * | 2019-02-15 | 2021-09-21 | 雷沃卢金有限公司 | Purification method |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1370831A (en) * | 2001-02-23 | 2002-09-25 | 国家***第一海洋研究所 | Purificaltion process of sea fish lymphocysis virus |
| CN101060859A (en) * | 2004-10-06 | 2007-10-24 | 米迪缪尼疫苗股份有限公司 | Refrigerator-temperature stable influenza vaccine compositions |
| CN101098959A (en) * | 2004-10-22 | 2008-01-02 | 昂科利蒂克斯生物科技公司 | Improved viral purification methods |
| CN101426905A (en) * | 2006-04-20 | 2009-05-06 | 惠氏公司 | Purification processes for isolating purified vesicular stomatitis virus from cell culture |
Family Cites Families (3)
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| US20040167320A1 (en) * | 2003-02-24 | 2004-08-26 | Couto Daniel E. | Methods of tangential flow filtration and an apparatus therefore |
| CN1297313C (en) * | 2004-09-16 | 2007-01-31 | 中国医学科学院医学生物学研究所 | After treatment method for attenuated strain polio inactivated vaccine |
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| CN1370831A (en) * | 2001-02-23 | 2002-09-25 | 国家***第一海洋研究所 | Purificaltion process of sea fish lymphocysis virus |
| CN101060859A (en) * | 2004-10-06 | 2007-10-24 | 米迪缪尼疫苗股份有限公司 | Refrigerator-temperature stable influenza vaccine compositions |
| CN101098959A (en) * | 2004-10-22 | 2008-01-02 | 昂科利蒂克斯生物科技公司 | Improved viral purification methods |
| CN101426905A (en) * | 2006-04-20 | 2009-05-06 | 惠氏公司 | Purification processes for isolating purified vesicular stomatitis virus from cell culture |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103614339A (en) * | 2013-11-22 | 2014-03-05 | 武汉友芝友医疗科技有限公司 | Cell separation method |
| CN113423832A (en) * | 2019-02-15 | 2021-09-21 | 雷沃卢金有限公司 | Purification method |
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