前苏联专利SU784784A3 Method of protein isolation

专利PDF首页>>前苏联专利

专利附录

专利说明

权利要求

类似技术

同族专利

引用文献

法律状态

优先权

专利摘要:
A novel process of separating from an aqueous medium at least one protein or at least one morphologically organized unit by liquid exclusion chromatography on a solid support previously passivated by treatment with an aqueous solution of a non-proteinic polymer. In this process, the chromatographic support is submitted, prior to the chromatographic operation, to a second passivation consisting in contacting the support with an aqueous solution of a protein which has a molecular weight lower than that of the protein to be separated, and which is adapted to be adsorbed by said support. The support and/or the liquid to be treated are sterilized with antiseptic agent selected from the group constituted by the lower aliphatic halogenated hydrocarbons.
公开号:SU784784A3
申请号:SU782614452
申请日:1978-04-26
公开日:1980-11-30
发明作者:Дюлу Шарль；Пейрузе Андре；Панари Рене；Аннун Клод；Весан Жан
申请人:Сосьете Насьональ ЕЛФ Акитэн (Продюксьон)；Энститю Пастер, Фондасьен Реконню Д" Ютилит Пюблик (Фирма)；
IPC主号:

专利说明:

METHOD FOR PROTEIN ALLOCATION
(54;
The invention relates to a method for isolating proteins with different molecular weights using chromatography, which can be used in microbiology for isolating and purifying viruses. Separative chromatography is widely used for purification and separation of organized fractions of bacterial or viral bodies, macromolecules I, 2. High-purity protein viruses are obtained using chromatography on elastic gels, for example, agar i balls. However, this carrier has significant drawbacks: low mechanical strength, does not inhibit heat sterilization, which limits its use for separating products that must be kept sterile. The closest to the described method is the separation of protein by silica gel chromatography. The silica gel is washed with water, with 1% aqueous solution of carbowax 20 M, during subsequent washing the temperature is reduced to 9 ° C. Then the column is again washed with water and buffer (pH 5.5-7.6), the separation is carried out at a low temperature (9 ° C), the deactivation of silica gel by carbowax 20 M prevents the adsorption of proteins, their separation is carried out due to a gradient of salt concentration in the eluting buffer and pH changes and application of pressurized (several tens of bar). The disadvantage of this method is that proteins can be separated only under the action of strong pressure. In addition, the method is applicable only to analytically pure low molecular weight proteins (25,000-800,000): lysozyme, albumin, catalase, thyroglobulin, cytochrome C. The method is not applicable to high molecular weight proteins (above a million), with which in the isolation and purification of influenza viruses, the average molecular weights of which are several million. The purpose of the present invention is a method for isolating high molecular weight proteins and simplifying the process. The goal is achieved by the described method of isolating protein from an aqueous medium by separation chromatography on a solid carrier, previously passivated with an aqueous solution of the polymer and elution of the protein with a buffer solution of pH 5.5-7.6, consisting in the use of silica gel as a solid carrier alkali metal with particles from 40 to 200 µm in size, pores with a diameter from 5 to 200 nm, the carrier after primary passivation with an aqueous solution of a polymer with a molecular weight of from 5,000 to 30,000, passivating along Torno 0.2-20% aqueous solution of the protein having a molecular weight less than the molecular weight vschel emogo protein.
Preferred variants of the method are the use of polyethylene glycol, polyvinyl pyrrolidone or polypropylene glycol as a polymer for the primary passivation. As a protein for re-passivation, proteins with molecular weight less than 100,000 are albumin, gelatin, peptone or polypeptide decomposition products. Using a buffer solution with the addition of an antiseptic in an amount of from 0.1 to 10 g representing dichloromethane, dibromomethane, diiodomethane, 1,2-dichloroethane, dibromo-1,2-ethane, or trichloroethyl. Used as an isolable peptide of the virus.
Example 1. Preparation of the column. A column with an inner diameter of 10 cm and a height of 120 cm is filled with Sferosil Hove 030 silica powder (rhon-Poulenc) in the form of particles with a size of 100-20-0 µm, with an average pore diameter of nm and a specific surface 50, the pore volume of the filler is 1 ml / g. After steam sterilization, the gel placed in the column is previously passivated, i.e. treated with an aqueous 1% polyethylene glycol solution of a molecular weight of 20,000 for 24 hours to block its g absorbent sites. Then, 500 non-influenza virus of the allanteic fluid is loaded into the column, with the aid of which it is re-passivated.
The circulation of the solution through the columns is carried out at an insignificant BittiieHHH pressure at the inlet of the colo-: well (approximately 1 bar.).
From 450 ml of viral solution: from the result of its chromatographic purification, 1050 ml of eluate containing a virus of high purity is obtained.
In the drawing, the chromatographic curve of the isolation of the influenza A / H53 virus from its allante nutrient medium, the optical densities are plotted along the axis HIcI
the eluate, on the x-axis - the volumes of the eluate. Peak V corresponds to peak virus, peak 1 corresponds to impurities, peak 1 is discontinuous due to a change in the flow rate of the eluate from 9 to 27 l / h. When working with an analytical chromatographic column (diameter 0.8 cm, height 120 cm), filled with the same silica as indicated above f get a solution of pure virus that does not contain impurities (1050 ml)
The balance is made by determining the activity of the virus in the solution according to the hemagglutination method (HA). It was found that in 450 ml of the initial alantoic solution, the activity is 1200 HA units / 0.25 ml. The activity of the virus solution after chromatographic purification is 480 HA units / 0.25 ml. The eluate volume is 1050 t-vi. Thus, the activity in the initial solution is 2160000 units of HA, the activity in the purified virus solution is 2016000 units of HA. Therefore, the yield is: (2016000: 2160000) 93.8% This yield is much higher than the yield obtained using a known cleaning method.
This double passivation is followed by washing with sterile aqueous buffer solution of primary potassium phosphate and secondary sodium phosphate with a pH of 7.5, containing NaCI at a concentration of 0.15 M.
Example 2. Isolation of influenza A / x53 strain virus from its allantoic nutrient medium. The solution of the virus from the classical culture in the allantiic cavity of the chicken embryonic egg, after incubation for 10-12 days, contains 1,200 units of HA (hemagglutination method) of 0.25 ml.
450 iviri of this solution are introduced into the column, the solution passing rate of 150 ml / min. During this time, a buffer solution is continuously passed through the column at a rate of 9 l / h until the appearance of one virus at the exit from the column (l / 30 min). The feed rate of the buffer is tripled (up to 27 l / h), which allows the entire process to be completed in 1 hour.
The column, equipped with an automatic device, operates without interruption for the entire time required for the process.
The determination of the components of the eluate at the outlet of the column is carried out by the optical density in ultraviolet; this is at a wavelength of 252 nm.
Example 3. Isolation of the virus by the double adsorption-elution method on erythrocytes, followed by purification by chromatography on silica gel.
SO l VIRAL allanteic fluids, derived from fetal chicken eggs 10–12 days old, are preliminarily clarified by centrifugation to remove insoluble substances. The supernatant liquid has 1200 units of HA in 0.25 ml. To the resulting liquid, 4% by volume of chicken erythrocyte sediment is added. After 8-16 hours at a temperature of + 4 ° C or + 37 ° C, the red blood cells are separated by centrifugation at a speed of 3,000 about min and the virus is eluted with 5 l phosphate buffer. The eluate has a hemagglutinate titer of 12,000 HA units in 0.25 ml. 450 ml of this virus suspension is automatically injected into the column over a period of 3 minutes (with a flow rate of 150 ml / min). During this time, the buffer solution described in the example is continuously passed through the column (the flow rate of this solution is 9 l / h until only one virus appears at the exit from the column. The consumption is triple (27 l / h), when no more virus is detected in the eluate, the eluate This contains contaminated proteins. Each operation lasts 1 hour. Thanks to its automatic device, the column operates continuously, and 5 liters of eluate are consumed. Fractions corresponding to purified viruses are collected and 15 Titer of this solution are obtained in hemagglutinate In each case, 3200 units of HA per 0.25 ml are used, which means a yield of about 100%. Example 4. Starting from a 10 liter solution of the allantoic fluid charged with the influenza virus, like that described in Example 2, the first stage is concentrated - Purification on a calcium polymer complex. To do this, an aqueous 2% (wt.%) solution of polyethylene glycol (20,000 molecular weight) and 12 g of powder are added to the solution. The mixture is homogenized for 30 minutes, the precipitate is separated by decantation and centrifuged , the precipitate is treated with about 500 ml of aqueous 0.2 M solution of disodium salt of ethylenediaminetetraacetic acid, whose pH was adjusted to 7.5 with bn. caustic soda solution. The solution thus obtained is centrifuged to remove the insoluble part and the upper clear liquid (about 500 ml) is collected. The yield of this concentration-purification operation was found close to 65,500 ml of this concentrated solution, was chromatographed in a column with an inner diameter of 10 cm and a height of 120 cm and equipped with the same silica as in example 1, and using the same buffer solution. 500 ml is injected as described in measure 2. The virus is collected by elution in 1500 ml of a solution, the titer of which is 10,800 units of HA / 0.25 ml, which corresponds to a yield of close to 98%. The resulting virus solution is both very clean and concentrated. Example 5. The infected infected liquid, as described above, is concentrated by diafiltration on membranes or hollow fibers. 50 liters is thus brought to a volume of 5 liters or less. After clarification, this concentrate is directly introduced into the column, according to the model described in the previous examples and in the same way. The viral peak contains the totality of hemagglutinate units that have been proposed. This solution, which is very pure in relation to proteins, can be contaminated with yolk sac phospholipids, organized into micelles; this contamination is separated by sucrose gradient ultracentrifugation. When all impurities are removed by separation chromatography, the yield in all operations is increased. Example 6. 500 liters of infected allanteic liquid, as described above, are purified by ultracentrifugation in a sucrose gradient. The volume of the fractions corresponding to the peak of the virus in the graph is 1 l. The output of this operation is 30-80%. This fraction is introduced automatically into a column of silica gel, as described in Example 2, and separation chromatography is carried out according to the method of this Example. Collect 3 liters of eluate. The yield of the chromatography operation is close to 100%. Example 7. The operations implemented in this example are identical to those described in examples 2, 3, 4, 5 and 6. However, the virus suspension was subjected to separation chromatography, previously inactivated by formalin, fi-propiolactone or ultraviolet radiation, and / or treated with an organic solvent . Example 8. A chromatographic column prepared according to Example 1 was used to purify an aqueous, mixed with, catgase solution extracted from a bovine liver. This solution has an activity of 220 international units. The eluate is buffered at pH 7. A solution with an activity of 1800 between national units is obtained, the yield is 86%. Example 9. The operations described in example 2 are repeated using the column described in example 1, but the column filler is glass beads with a size of 80-280 microns with an average pore diameter of 50 nm.
The yield of purified virus is 87%.
Example 10. The E / NC strain virus was purified according to examples 1 and 2, except for the second passivation of the column fill, 500 ml of an aqueous 6% lactalbumin solution of molecular weight about 18,000 were used. Yield 92%.
Example 11. The lactalbumin indicated in Example 10 was replaced with an 8% solution of meat peptone, i.e. products of proteolysis of mac polypeptides. The yield is 93%.
Example 12. After isolation of the virus according to example 2, the obtained product (A) is examined from the point of view of its microbial flora: the number of microbes per ml is indicated below,
This separation is carried out with the difference that 5 g of chloroform per liter is added to the buffer razor: the thief is used to sterilize the medium: the resulting solution B contains very microbes. In operation C, the same antiseptic is added in an amount of 5 g / l to both the buffer solution and the liquid to be treated. The result is similar to B. The virus medium is subjected to zonal ultracentrifugation, after the usual addition of 0.01% methylate and 0.02% formaldehyde.
The following microbial / ml results were reported:
A. Chromatography, without antiseptics 10
B. Chromatography, chloroform in buffer 10
C. Chromatography, chloroform in buffer and in the treated liquid 10 D. Zonal ultracentrifugation of methiol t + formaldehyde 3 000
In addition, the viruses received in operations A, B to C are live, while the virus obtained in operation D is inactive. Moreover, the products obtained as a result of operations in and C, with chloroform, the products retain the same infectious titer and have the same hemagglutinate capacity as the product obtained in operation A, which was carried out without the need for antiseptic.
Example 13 A virus solution similar to that used in experiments A to D in example 1, but corresponding to other strains of the virus, is subjected to separation according to the method described in example 2.
Thus, fluids obtained in 3 influenza viruses were investigated: A / USSR, A / Texas, B / NK
In each case, chromatography was carried out, eluting with a buffer solution, containing 5 g of chloroform in 1 liter or without chloroform. In addition, comparative isolations are carried out by zonal centrifugation.
The output,% relative to the initial fluid, and viral titer (in international units per mg of protein) are given below.
Output, %
A / USSR A / Texas H / NC Chromatography with chloroform 88 77.5 88 Chromatography without
chloroform 81 71.5 81 Zonal ultracentrifugation {metiol t + formaldegnd) 72 50 63
International U / mg
protein chromatography with chloroform 19800 23400 26200 Chromatography without
chloroform 15900 12100 19200 Zonal ultracentrifugation (methiol t + formaldehyde) 12900 13600 15300 The results show that adding chloroform improves both the yield and the virus concentration in the resulting product. From the results obtained during the implementation of the process described in Example 13, it was found that the virus isolated in the presence of chloroform is live and has the same infectious titer and has the same hemagglutinate ability as the virus obtained by chromatography without chloroform. In contrast, the virus isolated by centrifugation with classical sterilization is inactive.
Example 14. In operations similar to B and C of Example 12, bromoform was used at a concentration of about 1 g / l, which corresponds to the maximum solubility of CHBr.j in soda. About a hundred microbes per ml are found in the final fluid.
Example 15. Replacing chloroform in example 12 by 1.1, 2-trichloroethane, 4 g / l each (solubility 4.4 g / at 20 ° C) leads to a significant decrease in the microbial flora, menega 30 microbes / ml.
Example 16. The operations of Example 2 are implemented in the column described in Example 1 but without the second passivation, i.e. without processing filler alantoic. fluid. The virus yield is 70%. Example 17. Operation without second passivation, as in Example 16, with the filler being the same glass beads as in Example 9, yielding a yield of 64% (versus 87%, Example 9). The flow conditions of the eluting agent, the amount of liquid injected into the column, the frequency of this injection described in the examples are not restrictive, they can be varied over a wide range. Example 18. Solutions of allantoic fluid infected with a virus are prepared in a known manner, using dilution strains of the virus. 6 g / l of chloroform is introduced into these solutions. The following shows that chloroform administration does not change the infectious :: titer and hemagglutinate capacity of these solutions. A / Vikto-A / X47 NK 7 ri 75 Title: HA to chloroform 210 160 290 after 24 hours 210 160 280 2 days 180 after 3 days 230 160 after 5 days later Particle diameter, µm 100-200 50-100 10 Average pore diameter, named Surface area, pH of eluent 1 st passivation agent. Polyethylene glycol Molecular weight 20000 2nd passive agent - Egg albumin (nec, alantoic liquid molecular weight Yield,% 93.3 93.0

权利要求:
Claims (5)
[1]
Claims 1. Method for separating protein from an aqueous medium by separation chromatography on a solid support, previously passivated with an aqueous solution of the polymer and elution of the protein with a buffer solution of pH 5.5-7.6, different 60 140 50 230 7.5 7.0
so that, in order to simplify the process-. ca, silica gel or alkali metal silicate with a part of 40 to 200 µm in size, pores with a diameter of 9 5 D ° 200 nm, carrier after primary passivation with an aqueous solution of a polymer with a molecular mass of exept r: without chloroform with chloroform Example 19. Experimental tests that were conducted at least 13 with various antiseptics in a buffer solution give the following results. Concentration - Microbes, Antiseptic qi, g / l found per ml (average rounded values) No 6,200 Dichloromethane 6,100 Dibromomethane. 640 Diiodomethane Dichloro-1,2-ethane Dibrom-1,2 ethane Trichloroethyl 1300 flax 20-23. According to the method, examples of maintaining examples 1 and 2 were carried out by chromatographic analyzes, and this was done by changing some factors that are indicated in the person, where the results of example 2 are given as a comparison. 0-200 40-80 50-150 30 10 70 420 6.5 6.7 Polyprolivinyl pyropropylenelidene glycol 6000 29000 Lactalar female (Pimpness) tone, bumin 44000 18000 92.6 92.9 90.7
from 5,000 to 30,000, passivate repeatedly with 0.2–20% m aqueous solution of gprotein with a molecular weight less than the molecular weight of the released; protein
[2]
2. The method according to claim 1, characterized in that polyethylene glycol, polyvinylpyrrolidone or polypropylene glycol is used as the polymer for primary passivation.
[3]
3. Method pop. 1, which is different from the fact that the protein used in re-passivation has a molecular weight less than 100 00 and is extin, gelatin, peptone or decomposition products of polypeptides.
[4]
4. The method according to claim 1, characterized in that an antiseptic is added to the buffer solution in an amount of from 0.1 to 10 g, which represents
Dichloromethane, dibromomethane, diiodomethane, 1,2-dichloroethane, dibromo-1,2-ethane or trichlorethylene.
[5]
5. The method according to claim 1, wherein the virus is used as the secreted protein.
Priority points:
04/26/07 to para. 1, 2, 3, 5.
04/12/78 According to paragraph 4.
Sources of information taken into account in the examination
1.S. Bengtsson, L. Philipson, C h roma tog of animal viruses on pearl-condensed.agar, Blochim. Biophss. Acta E6, 79, 399.
2, Jshaiuhu Shechter, Separation of Proteins and High-Speed Pressure liguid Ch roma tog ra ph-j,. Biochemistry, 197, 58, 30.

类似技术:

公开号 | 公开日 | 专利标题

SU784784A3|1980-11-30|Method of protein isolation

US5177194A|1993-01-05|Process for purifying immune serum globulins

RU2484135C2|2013-06-10|Method of cleaning intended to produce cleaned virus of vesicular stomatitis from cellular culture

EP0751988B1|2000-02-09|Highly efficient production and isolation of viral particles

US4732683A|1988-03-22|Purification method for alpha interferon

AU621148B2|1992-03-05|Removal of process chemicals from labile biological mixtures by hydrophobic interaction chromatography

CA1277272C|1990-12-04|Purification of pichia produced lipophilic proteins

US6149917A|2000-11-21|Industrial production process for a vaccine against Japanese encephalitis virus and vaccine produced

Lockart Jr1963|Production of an interferon by L cells infected with Western equine encephalomyelitis virus

KR19980081114A|1998-11-25|Influenza vaccine

Wertz et al.1970|Interferon production and inhibition of host synthesis in cells infected with vesicular stomatitis virus

JP2002034556A|2002-02-05|Method for inactivating virus

Karger et al.1998|Simple and rapid purification of alphaherpesviruses by chromatography on a cation exchange membrane

Jacobson et al.1980|Viral pathogenesis and resistance to defective interfering particles

US4206014A|1980-06-03|Process for removing detergents from virus-antigen suspensions

Fujinami et al.1981|Failure to cleave measles virus fusion protein in lymphoid cells. A possible mechanism for viral persistence in lymphocytes

US4485038A|1984-11-27|Method for the production and purification of human leukocyte interferon

US5731187A|1998-03-24|Process for preparing hepatitis A | antigens and vaccines

EP0138167A1|1985-04-24|Method for purification of HBs antigen

Barton1977|An examination of permeation chromatography on columns of controlled pore glass for routine purification of plant viruses

US3892627A|1975-07-01|Feline panleukopenia vaccine

US3197374A|1965-07-27|Process for the disaggregation and purification of viruses

US4136168A|1979-01-23|Process for the preparation of neuraminidase from viral sources and methods of utilizing same

US3478145A|1969-11-11|Chromatographic purification of virus with brushite modified by autoclaving

Schneider et al.1971|Purification of rabies virus from tissue culture

同族专利:

公开号 | 公开日

JPS53140097A|1978-12-06|

FR2422720B2|1983-02-04|

GB1595378A|1981-08-12|

FR2422720A2|1979-11-09|

CA1111415A|1981-10-27|

ES469156A1|1979-11-01|

US4199450A|1980-04-22|

DE2817871A1|1978-12-07|

引用文献:

公开号 | 申请日 | 公开日 | 申请人 | 专利标题

RU2606609C2|2010-10-27|2017-01-10|Филип Моррис Продактс С.А.|Methodfor capturing particles of interest from mixture|US3549524A|1965-11-10|1970-12-22|Wolfgang Haller|Material and method for performing steric separations|

SE334862B|1966-04-14|1971-05-10|Pharmacia Ab|

SE333824B|1966-06-22|1971-03-29|Pharmacia Ab|

GB1342409A|1970-07-23|1974-01-03|Ciba Geigy Ag|Flowable pancreatin preparation of low microorgan ism content and a process for its manufacture|

US3701609A|1971-05-13|1972-10-31|David G Bailey|Apparatus for automatically adding preselected patterns of eluent solutions to a chromatographic column and monitoring and collecting eluted fractions|

CA987230A|1972-12-05|1976-04-13|Jean C. Gilker|Process for the filtration of a suspension containing a protein such as an influenza virus vaccine|

US3926800A|1974-01-25|1975-12-16|Beckman Instruments Inc|Temperature stabilized water jacket chromatographic column|

US4123931A|1976-08-03|1978-11-07|The Dow Chemical Company|Method for uniformly coating liquid phases on glass beads and applications thereof|FR2475572B1|1980-02-11|1982-02-19|Pasteur Institut|

DE3005495C2|1980-02-14|1983-03-31|Institut Pasteur, 75724 Paris|Production of fragments of viruses with lipid envelopes and pharmaceutical preparations containing them|

FR2483779B1|1980-06-05|1983-07-22|Synthelabo|

US4468331A|1982-09-13|1984-08-28|E. I. Du Pont De Nemours And Company|Method and system for liquid choromatography separations|

HU192642B|1984-12-19|1987-06-29|Forte Fotokemiai Ipar|Process for the extraction of nucleinic acids and purine bases from gelatine|

JP3112682B2|1992-06-19|2000-11-27|セプラコアインコーポレーテッド|Surface-treated and stabilized porous support, its production and use|

US5451660A|1993-12-13|1995-09-19|Genentech, Inc.|Method for purifying polypeptides|

DE19926041A1|1999-06-08|2000-12-21|Octapharma Ag Lachen|Removing or separating viruses from potentially virus-containing samples using gel permeation chromatography on hydrophilic supports|

DE19964015A1|1999-12-30|2001-08-09|Octapharma Ag Lachen|Removing or separating viruses from potentially virus-containing samples using gel permeation chromatography on hydrophilic supports|

WO2014032285A1|2012-08-31|2014-03-06|西安奥岚科技开发有限责任公司|Multidimensional liquid chromatography separation system and separation method for protein separation|

WO2016196906A1|2015-06-05|2016-12-08|W. R. Grace & Co.-Conn.|Adsorbent bioprocessing clarification agents and methods of making and using the same|

法律状态:

优先权:

申请号 | 申请日 | 专利标题

FR7712518A|FR2388585B1|1977-04-26|1977-04-26|

FR7810769A|FR2422720B2|1977-04-26|1978-04-12|

[返回顶部]