CN114130369B - Virus-expelling composite chromatographic medium and preparation method thereof - Google Patents
Virus-expelling composite chromatographic medium and preparation method thereof Download PDFInfo
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- CN114130369B CN114130369B CN202111479068.9A CN202111479068A CN114130369B CN 114130369 B CN114130369 B CN 114130369B CN 202111479068 A CN202111479068 A CN 202111479068A CN 114130369 B CN114130369 B CN 114130369B
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- 239000012501 chromatography medium Substances 0.000 title claims abstract description 51
- 239000002131 composite material Substances 0.000 title claims abstract description 31
- 238000002360 preparation method Methods 0.000 title abstract description 12
- 239000004005 microsphere Substances 0.000 claims abstract description 122
- 241000700605 Viruses Species 0.000 claims abstract description 69
- 230000007717 exclusion Effects 0.000 claims abstract description 15
- IOQPZZOEVPZRBK-UHFFFAOYSA-N octan-1-amine Chemical compound CCCCCCCCN IOQPZZOEVPZRBK-UHFFFAOYSA-N 0.000 claims abstract description 13
- 238000007259 addition reaction Methods 0.000 claims abstract description 8
- STMDPCBYJCIZOD-UHFFFAOYSA-N 2-(2,4-dinitroanilino)-4-methylpentanoic acid Chemical compound CC(C)CC(C(O)=O)NC1=CC=C([N+]([O-])=O)C=C1[N+]([O-])=O STMDPCBYJCIZOD-UHFFFAOYSA-N 0.000 claims abstract description 5
- 238000000746 purification Methods 0.000 claims description 25
- RBNPOMFGQQGHHO-UHFFFAOYSA-N glyceric acid Chemical compound OCC(O)C(O)=O RBNPOMFGQQGHHO-UHFFFAOYSA-N 0.000 claims description 16
- JGJLWPGRMCADHB-UHFFFAOYSA-N hypobromite Inorganic materials Br[O-] JGJLWPGRMCADHB-UHFFFAOYSA-N 0.000 claims description 14
- -1 allyl glycerol ether Chemical compound 0.000 claims description 11
- 239000003446 ligand Substances 0.000 claims description 8
- 238000000034 method Methods 0.000 claims description 7
- GPLRAVKSCUXZTP-UHFFFAOYSA-N diglycerol Chemical compound OCC(O)COCC(O)CO GPLRAVKSCUXZTP-UHFFFAOYSA-N 0.000 claims description 5
- 125000003277 amino group Chemical group 0.000 claims description 4
- 230000031709 bromination Effects 0.000 claims description 3
- 238000005893 bromination reaction Methods 0.000 claims description 3
- 125000002347 octyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 claims description 3
- 229920000936 Agarose Polymers 0.000 claims description 2
- 229920002307 Dextran Polymers 0.000 claims description 2
- 239000004793 Polystyrene Substances 0.000 claims description 2
- 230000003213 activating effect Effects 0.000 claims description 2
- 230000004913 activation Effects 0.000 claims description 2
- 239000001913 cellulose Substances 0.000 claims description 2
- 229920002678 cellulose Polymers 0.000 claims description 2
- 239000000203 mixture Substances 0.000 claims description 2
- 229920000193 polymethacrylate Polymers 0.000 claims description 2
- 229920002223 polystyrene Polymers 0.000 claims description 2
- 108090000623 proteins and genes Proteins 0.000 abstract description 25
- 102000004169 proteins and genes Human genes 0.000 abstract description 25
- 238000011084 recovery Methods 0.000 abstract description 23
- 125000003903 2-propenyl group Chemical group [H]C([*])([H])C([H])=C([H])[H] 0.000 abstract description 3
- WKBOTKDWSSQWDR-UHFFFAOYSA-N Bromine atom Chemical compound [Br] WKBOTKDWSSQWDR-UHFFFAOYSA-N 0.000 abstract description 3
- GDTBXPJZTBHREO-UHFFFAOYSA-N bromine Substances BrBr GDTBXPJZTBHREO-UHFFFAOYSA-N 0.000 abstract description 3
- 229910052794 bromium Inorganic materials 0.000 abstract description 3
- 150000008282 halocarbons Chemical class 0.000 abstract description 3
- 238000007142 ring opening reaction Methods 0.000 abstract description 3
- 239000000126 substance Substances 0.000 abstract description 3
- 238000013375 chromatographic separation Methods 0.000 abstract description 2
- 125000003700 epoxy group Chemical group 0.000 abstract description 2
- 238000003756 stirring Methods 0.000 description 51
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 44
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 30
- 238000011068 loading method Methods 0.000 description 26
- 238000005303 weighing Methods 0.000 description 25
- 239000012535 impurity Substances 0.000 description 19
- 230000001276 controlling effect Effects 0.000 description 18
- 239000008213 purified water Substances 0.000 description 18
- 241000711798 Rabies lyssavirus Species 0.000 description 12
- 239000000243 solution Substances 0.000 description 11
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 10
- 239000004280 Sodium formate Substances 0.000 description 10
- 238000004140 cleaning Methods 0.000 description 10
- 230000000052 comparative effect Effects 0.000 description 10
- 238000001914 filtration Methods 0.000 description 10
- HLBBKKJFGFRGMU-UHFFFAOYSA-M sodium formate Chemical compound [Na+].[O-]C=O HLBBKKJFGFRGMU-UHFFFAOYSA-M 0.000 description 10
- 235000019254 sodium formate Nutrition 0.000 description 10
- 238000000926 separation method Methods 0.000 description 9
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 8
- 229960003127 rabies vaccine Drugs 0.000 description 8
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 6
- 230000002209 hydrophobic effect Effects 0.000 description 5
- 230000001105 regulatory effect Effects 0.000 description 5
- 239000007853 buffer solution Substances 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 239000003480 eluent Substances 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- 238000003828 vacuum filtration Methods 0.000 description 4
- 230000009471 action Effects 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000001641 gel filtration chromatography Methods 0.000 description 3
- 238000002523 gelfiltration Methods 0.000 description 3
- 238000004255 ion exchange chromatography Methods 0.000 description 3
- 150000002500 ions Chemical group 0.000 description 3
- 239000011148 porous material Substances 0.000 description 3
- 238000001179 sorption measurement Methods 0.000 description 3
- 238000002659 cell therapy Methods 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 238000005342 ion exchange Methods 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 229960005486 vaccine Drugs 0.000 description 2
- WOUANPHGFPAJCA-UHFFFAOYSA-N 2-[benzyl(methyl)amino]ethanol Chemical compound OCCN(C)CC1=CC=CC=C1 WOUANPHGFPAJCA-UHFFFAOYSA-N 0.000 description 1
- 239000004593 Epoxy Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 230000001516 effect on protein Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical group [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
- B01J20/24—Naturally occurring macromolecular compounds, e.g. humic acids or their derivatives
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
- B01D15/20—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to the conditioning of the sorbent material
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
- B01D15/36—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving ionic interaction
- B01D15/361—Ion-exchange
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
- B01J20/26—Synthetic macromolecular compounds
- B01J20/265—Synthetic macromolecular compounds modified or post-treated polymers
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
- B01J20/28014—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their form
- B01J20/28016—Particle form
- B01J20/28019—Spherical, ellipsoidal or cylindrical
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Organic Chemistry (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention relates to the field of protein chromatographic separation media, in particular to a virus-exclusion composite chromatographic medium and a preparation method thereof. The invention obtains the microsphere containing allyl through ring-opening reaction with epoxy group of allyl glycidyl ether under alkaline condition, obtains the microsphere with halogenated hydrocarbon at the end through addition reaction of simple substance bromine, and finally obtains the virus exclusion composite chromatographic medium containing ionic group amino and hydrophobic group octyl through addition reaction with n-octylamine, wherein the protein removal rate of the virus exclusion composite chromatographic medium can reach more than 46%, and the virus recovery rate can reach more than 87%.
Description
Technical Field
The invention relates to the field of protein chromatographic separation media, in particular to a virus-exclusion composite chromatographic medium and a preparation method thereof.
Background
At present, in the fields of vaccine, cell therapy and the like, virus separation and purification are mainly performed by gel filtration chromatography media in mass production, and the virus separation and purification is performed by ion exchange chromatography media in a virus resistance mode. The gel filtration chromatography medium is mainly separated through gaps among the microspheres and apertures inside the microspheres, viruses can only come out from the external water volume through the gaps among the microspheres, impurities enter the apertures inside the microspheres and come out from the internal water volume, and the order of the volumes is different, so that the purpose of separating the viruses from the impurities is achieved. The gel filtration chromatography medium has the advantages of not affecting the activity of the virus, ensuring the stability of the virus, the protein removal rate and the virus recovery rate to the greatest extent, and has the defects of low industrial production efficiency, the sample is loaded after the virus culture is finished and the loading amount is less than 10 percent of the column loading volume of the gel filtration medium. The ion exchange chromatography medium is mainly used for adsorbing impurities in virus samples through bonding ion functional groups, the virus can flow through liquid, the ion exchange chromatography medium has the advantages that the sample loading amount can reach more than ten times or even tens times of the volume of the ion exchange medium column, the defects are low protein removal rate and virus recovery rate, when the inner aperture of the microsphere is larger, the virus can not enter the inner aperture of the microsphere, and meanwhile, the surface of the microsphere is provided with a small amount of functional groups for combining the virus, so that the virus is lost, the recovery rate is not high, and the single ion functional groups can only adsorb part of charged impurities in the virus samples, the adsorption range of the impurities is narrower, and the protein removal rate is lower.
Therefore, how to solve the problems of lower protein removal rate and low virus recovery rate in the current virus purification process is of great significance in providing a high-efficiency large-scale virus separation chromatography medium for vaccine production and cell therapy enterprises.
Disclosure of Invention
Aiming at the technical problems existing in the prior art, the invention provides a virus-exclusion composite chromatography medium and a preparation method thereof.
The invention adopts the following technical scheme to realize the technical purposes:
The invention provides a virus-exclusion composite chromatography medium, which comprises microspheres and functional ligands on the surfaces of the microspheres, wherein the functional ligands are amino groups and octyl groups which are coupled after being activated by allyl glycidyl ether, and the structural composition of the chromatography medium is as follows:
the exclusion limit of the microsphere is 10 KD-1000 KD.
The separation principle of the virus-exclusion composite chromatography medium is as follows: when a virus sample passes through the microsphere with a specific exclusion limit, the virus cannot enter the microsphere, and can only flow out from the flowing-through liquid between the gaps of the microsphere, the virus flowing-through liquid is collected, meanwhile, various impurities in the virus sample enter the microsphere, the impurities are adsorbed in the inner pore diameter of the microsphere through the amino, octyl and the like in the microsphere and the hydrophobic effect, the impurities can be continuously loaded to tens times or even tens times of the column volume of the chromatographic medium, and finally the adsorbed impurities are eluted by using 1M NaOH and 30% isopropanol. The virus-exclusion composite chromatographic medium has high recovery rate of virus because the virus is not adsorbed, and simultaneously, the impurity is adsorbed by multiple functions, the protein removal rate is higher, and a virus sample with very high purity and yield can be obtained by one-step purification. The specific combination principle is three: firstly, molecular sieve function, according to the size of virus and impurity, virus enters the gap between the outside of microsphere and flows out, and impurity enters the aperture of microsphere interior; secondly, the ionic action of the amino group, because the amino group has positive charges, the impurities with negative charges can be adsorbed on ligands of the inner pore diameter of the microsphere by combining with the negative charges on the surfaces of the impurities in the virus sample; thirdly, the hydrophobic effect of octyl, through combining with the hydrophobic property of the impurities in the virus sample, the impurities in the virus in a larger range are adsorbed on the ligand of the inner pore diameter of the microsphere. The protein removal rate of the virus-exclusion composite chromatographic medium provided by the invention can reach more than 46%, and the virus recovery rate can reach more than 87%.
As an embodiment of the present invention, the microsphere is selected from one or more of agarose microsphere, dextran microsphere, cellulose microsphere, polystyrene microsphere, and polymethacrylate microsphere. Such as Focurose microspheres from the wuhan sink research organism, with a microsphere exclusion limit of 10-1000KD.
The invention also provides a preparation method of the virus-exclusion composite chromatographic medium, which comprises the following steps:
s1, activating the microspheres with allyl glycerol ether under alkaline conditions to obtain glycerol ether activated microspheres;
S2, brominating the glyceryl ether activated microspheres with bromine water to obtain brominated microspheres;
s3, adding the brominated microspheres and n-octylamine to react, thus obtaining the product.
The invention obtains the microsphere containing allyl through ring opening reaction with epoxy group of allyl glycidyl ether under alkaline condition to the hydroxyl of the specific exclusion limit microsphere, then obtains the microsphere with halogenated hydrocarbon at the end through addition reaction of simple substance bromine, finally obtains the virus exclusion composite chromatography medium containing ionic group amino and hydrophobic group octyl through addition reaction with n-octylamine, the preparation principle is as follows:
s1, a chemical reaction process of the glycerol ether activated microsphere:
S2, a chemical reaction process of bromination of glycerol ether microspheres:
s3, the chemical reaction process of the brominated microsphere and n-octylamine:
In one embodiment of the present invention, the mass ratio of the microsphere to the allyl glycerol ether in step S1 is: 1:0.01 to 1:0.5, preferably 1:0.05 to 1:0.2.
In one embodiment of the present invention, the conditions for the activation of step S1 are: the temperature is 20-60 ℃.
In one embodiment of the present invention, the mass ratio of the glycerol ether activated microsphere to the bromine water in step S2 is: 1:0.01 to 1:0.2, preferably 1:0.01 to 1:0.05.
In one embodiment of the present invention, the bromination conditions of step S2 are: the temperature is 20-60 ℃.
In one embodiment of the present invention, the mass ratio of the brominated microsphere to n-octylamine in step S3 is: 1:0.001 to 1:0.2, preferably 1:0.001 to 1:0.05.
In one embodiment of the present invention, the conditions of the addition reaction in step S3 are: the pH value is 9-11, and the temperature is 20-60 ℃.
The virus-exclusion composite chromatographic medium provided by the invention has the following advantages:
(1) The method has strong applicability, can be suitable for the separation and purification of whole viruses, virus-like particles and the like, has no adsorption and influence on the viruses because the viruses cannot enter the microspheres, has almost no influence on the titer and activity of the viruses, and has broad-spectrum adsorption effect on proteins and other impurities in virus samples;
(2) The product has higher separation and purification efficiency and better purification effect, integrates gel filtration, ion exchange and hydrophobic chromatography, viruses pass through the specific exclusion limit of the microspheres and flow through gaps among the microspheres, impurities are adsorbed on a chromatographic medium through the combined action of ions and hydrophobic ligands, the sample loading volume can be tens of times as well, the purification efficiency is improved by hundreds of times compared with the purification efficiency of the traditional gel filtration chromatographic medium, and the multiple actions of the ligands on the impurities are improved, so that the protein removal rate and the virus recovery rate in virus flow through liquid are improved;
(3) The industrial application cost of the product is lower, the industrial separation and purification production time can be shortened to be within 8 hours due to the improvement of the purification efficiency, the investment of equipment and chromatographic media in production is reduced by a plurality of times, and the investment of millions to tens of millions of cost can be saved.
Drawings
FIG. 1 is a diagram of a purified rabies vaccine with virus-exclusion complex-type chromatography medium exclusion prepared in example 1 of the invention;
FIG. 2 is a diagram of a purified rabies vaccine with virus-exclusion complex-type chromatography medium exclusion prepared in example 2 of the invention;
FIG. 3 is a diagram of a purified rabies vaccine with virus-exclusion complex-type chromatography medium exclusion prepared in example 3 of the invention;
FIG. 4 is a photograph of a purified rabies vaccine with virus-exclusion complex type chromatography medium exclusion prepared in comparative example 1 of the present invention.
Detailed Description
The present invention will be described in further detail with reference to specific examples so as to more clearly understand the present invention by those skilled in the art.
The following examples are given by way of illustration of the invention and are not intended to limit the scope of the invention. All other embodiments obtained by those skilled in the art without creative efforts are within the protection scope of the present invention based on the specific embodiments of the present invention.
In the examples of the present invention, all raw material components are commercially available products well known to those skilled in the art unless specified otherwise; in the embodiments of the present invention, unless specifically indicated, all technical means used are conventional means well known to those skilled in the art.
The invention provides a preparation method of a virus-exclusion composite chromatographic medium, which comprises the following steps:
(1) Adding the microspheres into a three-neck flask, adding purified water with the mass of about 1/5 of the microspheres, stirring, adding allyl glycerol ether, ensuring that the mass ratio of the microspheres to the allyl glycerol ether is 1:0.01-1:0.5, simultaneously adding sodium hydroxide, ensuring that the solution is alkaline, controlling the temperature between 20 and 60 ℃, controlling the stirring rotation speed between 200 and 500rpm/min, and reacting for 16 to 24 hours to obtain glycerol ether activated microspheres;
(2) Vacuum filtering and cleaning the obtained glyceryl ether activated microsphere with purified water with the volume of 10 times, adding the purified water with the mass of about 1/5 of that of the glyceryl ether activated microsphere into a new three-neck flask, stirring, adding bromine water to ensure that the mass ratio of the glyceryl ether activated microsphere to the bromine water is 1:0.01-1:0.2, controlling the temperature to be 20-60 ℃, controlling the stirring rotation speed to be 200-500 rpm/min, and reacting for 10-60 min;
(3) Then adding sodium formate to ensure that the mass ratio of the sodium formate to the glycerol ether activated microsphere is 1:0.01-0.2, controlling the temperature to be 20-60 ℃, and reacting for 10-60 min at the stirring speed of 200-500 rpm/min to obtain brominated microspheres;
(4) The brominated microspheres are filtered and washed by vacuum with 10 times of purified water, then are added into another new three-neck flask, purified water with the mass of about 1/5 of the mass of the brominated microspheres is added for stirring, then n-octylamine is added to ensure that the mass ratio of the brominated microspheres to the n-octylamine is 1:0.001-1:0.2, meanwhile, the pH is regulated to 9-11, the temperature is controlled to be 20-60 ℃, the stirring rotation speed is controlled to be 200-500 rpm/min, the reaction is carried out for 16-24 h, and then the purified water with the volume of 10 times is used for vacuum filtration and washing, thus obtaining the virus-elimination composite chromatographic medium.
Example 1
The embodiment provides a preparation method of a virus-exclusion composite chromatographic medium, which comprises the following steps:
(1) Weighing 500g Focurose microspheres, adding the microspheres into a 1000mL three-neck flask, and then adding 100g of pure water and stirring;
(2) 25g of allyl glycerol ether is weighed and added into the three-neck flask, 100g of sodium hydroxide is added at the same time, the pH value is more than 7, the temperature is controlled at 30 ℃, the stirring speed is 300rpm/min, and the stirring is carried out for 24 hours;
(3) Vacuum filtering and cleaning the microspheres with purified water with the volume of 10 times to obtain glycerol ether activated microspheres;
(4) Weighing 500g of glycerol ether activated microspheres, adding the microspheres into a 100mL three-necked flask, and then adding 100g of pure water and stirring;
(5) Weighing 20g of bromine water, adding the bromine water into the three-neck flask in the step (4), controlling the temperature in a water bath kettle at 30 ℃, stirring at 300rpm/min, and stirring for 30min;
(6) Weighing 5g of sodium formate, adding the sodium formate into the three-neck flask in the step (5), controlling the temperature in a water bath kettle at 30 ℃, stirring at 300rpm/min, and stirring for 30min;
(7) Vacuum filtering and cleaning the microspheres with purified water with the volume of 10 times to obtain brominated microspheres;
(8) Weighing 500g of brominated microspheres, adding the brominated microspheres into a 1000mL three-necked flask, and then adding 100g of pure water and stirring;
(9) Weighing 2g of n-octylamine, adding into the three-neck flask in the step (8), regulating the pH to 10.0, controlling the temperature in a water bath kettle to be 30 ℃, stirring at 300rpm/min, and stirring for 24 hours;
(10) Vacuum filtering and cleaning with 10 times of purified water, sampling and detecting chloride ion load and BSA protein load, and the results are shown in Table 1;
(11) And (5) loading a column, purifying a rabies virus sample, and verifying the protein removal rate and the virus recovery rate.
The embodiment also provides a method for separating and purifying rabies virus samples by utilizing the virus-exclusion composite chromatography medium, which comprises the following specific steps:
a. preassembling 1mL preassembled column;
b. buffer solution:
Balancing solution: 20mM PB,0.15M NaCl,pH7.35;
Eluent: 30% isopropanol, 1M NaOH;
the solution was prepared and filtered through a 0.45um aqueous filter.
C. Sample pretreatment
The pH of the original sample was measured to be 7.31 (25 ℃ C.) and the conductivity was measured to be 12.45ms/cm, and the sample was filtered through a 0.45 μm filter before loading.
D. the purification process comprises the following steps: the loading flow rate during purification was 0.33mL/min.
E. The protein removal rate and the virus recovery rate are detected, the result of the virus protein removal rate is shown in table 3, the result of the virus recovery rate is shown in table 4, and the rabies virus sample purification map is shown in fig. 1.
Example 2
The embodiment provides a preparation method of a virus-exclusion composite chromatographic medium, which comprises the following steps:
(1) Weighing 500g Focurose microspheres, adding the microspheres into a 1000mL three-neck flask, and then adding 100g of pure water and stirring;
(2) Weighing 100g of allyl glycerol ether, adding 100g of sodium hydroxide into the three-neck flask, measuring pH to be more than 7, controlling the temperature to be 30 ℃, stirring at 300rpm, and stirring for 24 hours;
(3) Vacuum filtering and cleaning the microspheres with purified water with the volume of 10 times to obtain glycerol ether activated microspheres;
(4) Weighing 500g of glycerol ether activated microspheres, adding the microspheres into a 100mL three-necked flask, and then adding 100g of pure water and stirring;
(5) Weighing 20g of bromine water, adding the bromine water into the three-neck flask in the step (4), controlling the temperature in a water bath kettle at 30 ℃, stirring at 300rpm/min, and stirring for 30min;
(6) Weighing 5g of sodium formate, adding the sodium formate into the three-neck flask in the step (5), controlling the temperature in a water bath kettle at 30 ℃, stirring at 300rpm/min, and stirring for 30min;
(7) Vacuum filtering and cleaning the microspheres with purified water with the volume of 10 times to obtain brominated microspheres;
(8) Weighing 500g of brominated microspheres, adding the brominated microspheres into a 1000mL three-necked flask, and then adding 100g of pure water and stirring;
(9) Weighing 10g of n-octylamine, adding into the three-neck flask in the step (8), regulating the pH to 9-11, controlling the temperature in a water bath kettle at 30 ℃, stirring at 300rpm/min, and stirring for 24 hours;
(10) The microspheres were washed with 10 volumes of purified water by vacuum filtration and sampled for chloride ion loading and BSA protein loading, the results are shown in table 1;
(11) And (5) loading a column, purifying a rabies virus sample, and verifying the protein removal rate and the virus recovery rate.
The embodiment also provides a method for separating and purifying rabies virus samples by utilizing the virus-exclusion composite chromatography medium, which comprises the following specific steps:
a. preassembling 1mL preassembled column;
b. buffer solution:
Balancing solution: 20mM PB,0.15M NaCl,pH7.35;
Eluent: 30% isopropanol, 1M NaOH;
the solution was prepared and filtered through a 0.45um aqueous filter.
C. Sample pretreatment
The pH of the original sample was measured to be 7.31 (25 ℃ C.) and the conductivity was measured to be 12.45ms/cm, and the sample was filtered through a 0.45 μm filter before loading.
D. the purification process comprises the following steps: the loading flow rate during purification was 0.33mL/min.
E. The protein removal rate and the virus recovery rate are detected, the result of the virus protein removal rate is shown in table 3, the result of the virus recovery rate is shown in table 4, and the rabies virus sample purification map is shown in fig. 2.
Example 3
The embodiment provides a preparation method of a virus-exclusion composite chromatographic medium, which comprises the following steps:
(1) Weighing 500g Focurose microspheres, adding the microspheres into a 1000mL three-neck flask, and then adding 100g of pure water and stirring;
(2) 25g of allyl glycerol ether is weighed and added into the three-neck flask, 100g of sodium hydroxide is added at the same time, the pH value is more than 7, the temperature is controlled at 30 ℃, the stirring rotation speed is 300rpm, and the stirring is carried out for 24 hours;
(3) Vacuum filtering and cleaning the microspheres with purified water with the volume of 10 times to obtain glycerol ether activated microspheres;
(4) Weighing 500g of glycerol ether activated microspheres, adding the microspheres into a 100mL three-necked flask, and then adding 100g of pure water and stirring;
(5) Weighing 20g of bromine water, adding the bromine water into the three-neck flask in the step (4), controlling the temperature in a water bath kettle at 30 ℃, stirring at 300rpm/min, and stirring for 30min;
(6) Weighing 5g of sodium formate, adding the sodium formate into the three-neck flask in the step (5), controlling the temperature in a water bath kettle at 30 ℃, stirring at 300rpm/min, and stirring for 30min;
(7) Vacuum filtering and cleaning the microspheres with purified water with the volume of 10 times to obtain brominated microspheres;
(8) Weighing 500g of brominated microspheres, adding the brominated microspheres into a 1000mL three-necked flask, and then adding 100g of pure water and stirring;
(9) Weighing 10g of n-octylamine, adding into the three-neck flask in the step (8), regulating the pH to 9-11, controlling the temperature in a water bath kettle at 30 ℃, stirring at 300rpm/min, and stirring for 24 hours;
(10) The microspheres were washed with 10 volumes of purified water by vacuum filtration and sampled for chloride ion loading and BSA protein loading, the results are shown in table 1;
(11) And (5) loading a column, purifying a rabies virus sample, and verifying the protein removal rate and the virus recovery rate.
The embodiment also provides a method for separating and purifying rabies virus samples by utilizing the virus-exclusion composite chromatography medium, which comprises the following specific steps:
a. preassembling 1mL preassembled column;
b. buffer solution:
Balancing solution: 20mM PB,0.15M NaCl,pH7.35;
Eluent: 30% isopropanol, 1M NaOH;
the solution was prepared and filtered through a 0.45um aqueous filter.
C. Sample pretreatment
The pH of the original sample was measured to be 7.31 (25 ℃ C.) and the conductivity was measured to be 12.45ms/cm, and the sample was filtered through a 0.45 μm filter before loading.
D. the purification process comprises the following steps: the loading flow rate during purification was 0.33mL/min.
E. the protein removal rate and the virus recovery rate are detected, the result of the virus protein removal rate is shown in table 3, the result of the virus recovery rate is shown in table 4, and the rabies virus sample purification map is shown in fig. 3.
Comparative example 1
The comparative example provides a preparation method of a virus-exclusion composite chromatographic medium, which comprises the following steps:
(1) Weighing 500g Focurose microspheres, adding the microspheres into a 1000mL three-neck flask, and then adding 100g of pure water and stirring;
(2) 25g of allyl glycerol ether is weighed and added into the three-neck flask, 100g of sodium hydroxide is added at the same time, the pH value is more than 7, the temperature is controlled at 30 ℃, the stirring rotation speed is 300rpm, and the stirring is carried out for 24 hours;
(3) Vacuum filtering and cleaning the microspheres with purified water with the volume of 10 times to obtain glycerol ether activated microspheres;
(4) Weighing 500g of glycerol ether activated microspheres, adding the microspheres into a 100mL three-necked flask, and then adding 100g of pure water and stirring;
(5) Weighing 20g of bromine water, adding the bromine water into the three-neck flask in the step (4), controlling the temperature in a water bath kettle at 30 ℃, stirring at 300rpm/min, and stirring for 30min;
(6) Weighing 5g of sodium formate, adding the sodium formate into the three-neck flask in the step (5), controlling the temperature in a water bath kettle at 30 ℃, stirring at 300rpm/min, and stirring for 30min;
(7) Vacuum filtering and cleaning the microspheres with purified water with the volume of 10 times to obtain brominated microspheres;
(8) Weighing 500g of brominated microspheres, adding the brominated microspheres into a 1000mL three-necked flask, and then adding 100g of pure water and stirring;
(9) Weighing 10g N-benzyl-N-methylethanolamine, adding into the three-neck flask in the step (8), regulating the pH to 9-11, controlling the temperature in a water bath kettle at 3 ℃, stirring at 300rpm/min, and stirring for 24 hours;
(10) The microspheres were washed with 10 volumes of purified water by vacuum filtration and sampled for chloride ion loading and BSA protein loading, the results are shown in table 1;
(11) And (5) loading a column, purifying a rabies virus sample, and verifying the protein removal rate and the virus recovery rate.
The comparative example also provides a method for separating and purifying rabies virus samples by utilizing the virus-exclusion composite chromatography medium, which comprises the following specific steps:
a. preassembling 1mL preassembled column;
b. buffer solution:
Balancing solution: 20mM PB,0.15M NaCl,pH7.35;
Eluent: 30% isopropanol, 1M NaOH;
the solution was prepared and filtered through a 0.45um aqueous filter.
C. Sample pretreatment
The pH of the original sample was measured to be 7.31 (25 ℃ C.) and the conductivity was measured to be 12.45ms/cm, and the sample was filtered through a 0.45 μm filter before loading.
D. the purification process comprises the following steps: the loading flow rate during purification was 0.33mL/min.
E. The protein removal rate and the virus recovery rate are detected, the result of the virus protein removal rate is shown in table 3, the result of the virus recovery rate is shown in table 4, and the rabies virus sample purification map is shown in fig. 4.
Analysis of results:
(1) The results of the detection of chloride ion loading and BSA protein loading of the virus-exclusion composite chromatography media prepared in examples 1 to 3 and comparative example 1 are shown in table 1 below:
table 1 chloride ion loading and BSA protein loading of the chromatographic media prepared in examples and comparative examples
(2) The results of the standard curve of BSA protein are shown in Table 2 below, and the results of the protein removal rates of the virus-exclusion composite chromatographic medium separation and purification of rabies vaccine viruses prepared in examples 1 to 3 and comparative example 1 are shown in Table 3 below:
TABLE 2 BSA protein standard curve detection results
The standard curve for the BSA protein was: y=0.0004x+0.5348, r 2 = 0.9934.
TABLE 3 protein removal Rate of purified rabies vaccine virus by separation of chromatography media prepared in examples and comparative examples
Remarks: protein removal = 1- [ M (flow through)/M (stock solution) ]%100%
(3) The results of virus recovery for separating and purifying rabies vaccine virus using the virus-exclusion composite chromatography medium prepared in examples 1 to 3 and comparative example 1 are shown in table 4 below:
TABLE 4 recovery of virus from purified rabies vaccine virus by chromatography medium prepared in examples and comparative examples
Remarks: without standard, virus recovery = OD 450 (flow through)/OD 450( stock) ×100% of the total
From the results, the invention obtains the microsphere containing allyl through ring opening reaction of hydroxyl of the specific exclusion limit microsphere and epoxy of allyl glycidyl ether, obtains the microsphere with halogenated hydrocarbon at the end through addition reaction of simple substance bromine, and finally obtains the virus exclusion composite chromatographic medium containing ionic group amino and hydrophobic group octyl through addition reaction of n-octylamine.
It should be noted that the above examples are only for further illustrating and describing the technical solution of the present invention, and are not intended to limit the technical solution of the present invention, and the method of the present invention is only a preferred embodiment and is not intended to limit the scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (7)
1. The application of the virus-exclusion composite chromatography medium in virus purification is characterized in that the virus-exclusion composite chromatography medium comprises microspheres and functional ligands on the surfaces of the microspheres, wherein the functional ligands are amino groups and octyl groups which are coupled after being activated by allyl glycidyl ether, and the structural composition of the chromatography medium is as follows:
the exclusion limit of the microsphere is 10 KD-1000 KD;
The microsphere is one or more selected from agarose microsphere, dextran microsphere, cellulose microsphere, polystyrene microsphere and polymethacrylate microsphere.
2. The use according to claim 1, wherein the method for preparing the virus-exclusion composite chromatographic medium comprises the steps of:
s1, activating the microspheres with allyl glycerol ether under alkaline conditions to obtain glycerol ether activated microspheres;
S2, brominating the glyceryl ether activated microspheres with bromine water to obtain brominated microspheres;
s3, adding the brominated microspheres and n-octylamine to react, thus obtaining the product.
3. The use according to claim 2, wherein the mass ratio of microspheres to allyl glycerol ether in step S1 is: 1:0.01-1:0.5.
4. The use according to claim 2, wherein the conditions for the activation of step S1 are: the temperature is 20-60 ℃.
5. The use according to claim 2, wherein the mass ratio of the glycerol ether activated microspheres to the bromine water in step S2 is: 1:0.01-1:0.2, the bromination treatment conditions are as follows: the temperature is 20-60 ℃.
6. The use according to claim 2, wherein the mass ratio of brominated microspheres to n-octylamine in step S3 is: 1:0.001-1:0.2.
7. The use according to claim 2, wherein the conditions of the addition reaction in step S3 are: the pH value is 9-11, and the temperature is 20-60 ℃.
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