CN114130369A - Virus exclusion composite chromatographic medium and preparation method thereof - Google Patents
Virus exclusion composite chromatographic medium and preparation method thereof Download PDFInfo
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- CN114130369A CN114130369A CN202111479068.9A CN202111479068A CN114130369A CN 114130369 A CN114130369 A CN 114130369A CN 202111479068 A CN202111479068 A CN 202111479068A CN 114130369 A CN114130369 A CN 114130369A
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- 241000700605 Viruses Species 0.000 title claims abstract description 109
- 239000012501 chromatography medium Substances 0.000 title claims abstract description 59
- 230000007717 exclusion Effects 0.000 title claims abstract description 55
- 239000002131 composite material Substances 0.000 title claims abstract description 32
- 238000002360 preparation method Methods 0.000 title abstract description 17
- 239000004005 microsphere Substances 0.000 claims abstract description 127
- 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 10
- 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 21
- 238000000034 method Methods 0.000 claims description 13
- -1 allyl glyceryl ether Chemical compound 0.000 claims description 10
- JGJLWPGRMCADHB-UHFFFAOYSA-N hypobromite Inorganic materials Br[O-] JGJLWPGRMCADHB-UHFFFAOYSA-N 0.000 claims description 10
- 239000003446 ligand Substances 0.000 claims description 8
- 125000003277 amino group Chemical group 0.000 claims description 5
- 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 5
- 230000031709 bromination Effects 0.000 claims description 3
- 238000005893 bromination reaction Methods 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
- GPLRAVKSCUXZTP-UHFFFAOYSA-N diglycerol Chemical compound OCC(O)COCC(O)CO GPLRAVKSCUXZTP-UHFFFAOYSA-N 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 26
- 102000004169 proteins and genes Human genes 0.000 abstract description 26
- 238000011084 recovery Methods 0.000 abstract description 24
- 125000003903 2-propenyl group Chemical group [H]C([*])([H])C([H])=C([H])[H] 0.000 abstract description 3
- 150000008282 halocarbons Chemical class 0.000 abstract description 3
- 125000002887 hydroxy group Chemical group [H]O* 0.000 abstract description 3
- 238000007142 ring opening reaction Methods 0.000 abstract description 3
- 239000000126 substance Substances 0.000 abstract description 3
- WKBOTKDWSSQWDR-UHFFFAOYSA-N Bromine atom Chemical compound [Br] WKBOTKDWSSQWDR-UHFFFAOYSA-N 0.000 abstract description 2
- GDTBXPJZTBHREO-UHFFFAOYSA-N bromine Substances BrBr GDTBXPJZTBHREO-UHFFFAOYSA-N 0.000 abstract description 2
- 229910052794 bromium Inorganic materials 0.000 abstract description 2
- 238000013375 chromatographic separation Methods 0.000 abstract description 2
- 125000003700 epoxy group Chemical group 0.000 abstract description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 49
- 238000003756 stirring Methods 0.000 description 48
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 30
- 238000005303 weighing Methods 0.000 description 28
- 239000012535 impurity Substances 0.000 description 19
- 239000008213 purified water Substances 0.000 description 19
- 238000011068 loading method Methods 0.000 description 14
- 241000711798 Rabies lyssavirus Species 0.000 description 12
- 238000003828 vacuum filtration Methods 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
- 230000000052 comparative effect Effects 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
- 239000007788 liquid Substances 0.000 description 8
- 238000012856 packing Methods 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
- 230000009471 action Effects 0.000 description 6
- 230000002209 hydrophobic effect Effects 0.000 description 6
- 239000011148 porous material Substances 0.000 description 6
- 238000001179 sorption measurement Methods 0.000 description 5
- 238000010407 vacuum cleaning Methods 0.000 description 5
- 239000007853 buffer solution Substances 0.000 description 4
- 239000003480 eluent Substances 0.000 description 4
- 238000001641 gel filtration chromatography Methods 0.000 description 4
- 239000012528 membrane Substances 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000004255 ion exchange chromatography Methods 0.000 description 3
- 150000002500 ions Chemical group 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 238000005086 pumping Methods 0.000 description 3
- 238000002659 cell therapy Methods 0.000 description 2
- 238000011097 chromatography purification Methods 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 238000002523 gelfiltration Methods 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
- 239000002808 molecular sieve Substances 0.000 description 2
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 2
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 2
- 239000011550 stock solution 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
- CPELXLSAUQHCOX-UHFFFAOYSA-N Hydrogen bromide Chemical compound Br CPELXLSAUQHCOX-UHFFFAOYSA-N 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000001516 effect on protein Effects 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 125000003010 ionic group Chemical group 0.000 description 1
- 238000011031 large-scale manufacturing process 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
- 210000002381 plasma Anatomy 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
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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
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- 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
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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
- B01J20/28—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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- 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
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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. Hydroxyl of the specific exclusion limit microsphere is subjected to ring-opening reaction with an epoxy group of allyl glycidyl ether under an alkaline condition to obtain microsphere containing allyl, then simple substance bromine is subjected to addition reaction to obtain microsphere with halogenated hydrocarbon at the tail end, and finally the microsphere is subjected to addition reaction with n-octylamine to obtain the virus exclusion composite chromatographic medium containing ionic group amino and hydrophobic group octyl, 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 vaccines, cell therapy and the like, virus separation and purification are mainly carried out in large-scale production by virus exclusion mode chromatography purification through a gel filtration chromatography medium, and the impurity adsorption mode chromatography purification is carried out on the other part of the virus separation and purification through an ion exchange chromatography medium. 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 coming-out volumes is different, so that the purpose of separating the viruses from the impurities is achieved. The gel filtration chromatography medium has the advantages that the activity of the virus is not influenced, the stability of the virus, the protein removal rate and the virus recovery rate can be ensured to the maximum extent, the industrial production efficiency is low, the virus needs to be subjected to concentration treatment of 20-50 times after the culture of the virus, and the sample loading amount is only 10% of the column loading volume of the gel filtration medium. The ion exchange chromatography medium mainly adsorbs impurities in a virus sample through bonded ion functional groups, viruses flow through liquid, and the ion exchange chromatography medium has the advantages that the sample loading amount can reach dozens of times or even dozens of times of the volume of a column filled with the ion exchange medium, the defects are that the protein removal rate and the virus recovery rate are low, the virus cannot enter the internal pore size of the microsphere when the internal pore size of the microsphere is large, and meanwhile, the functional groups on the surface of the microsphere are slightly combined with the viruses, so that the virus loss is caused, the recovery rate is not high, and only part of charged impurities in the virus sample can be adsorbed by the single ion functional groups, the adsorption range of the impurities is narrow, and the protein removal rate is low.
Therefore, how to solve the problems of low protein removal rate and low virus recovery rate in the prior virus purification process has 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 in the prior art, the invention provides a virus exclusion composite chromatographic medium and a preparation method thereof.
The invention adopts the following technical scheme to realize the technical purpose:
the invention provides a virus exclusion composite chromatographic medium, which comprises microspheres and functional ligands on the surfaces of the microspheres, wherein the functional ligands are amino and octyl coupled after being activated by allyl glycidyl ether, and the structural composition of the chromatographic medium is as follows:
the exclusion limit of the microspheres is 10 KD-1000 KD.
The separation principle of the virus exclusion composite chromatographic medium is as follows: when a virus sample passes through the microspheres with a specific exclusion limit, viruses cannot enter the microspheres, and only flow-through liquid between gaps of the microspheres flows out, the virus flow-through liquid is collected, meanwhile, various impurities in the virus sample enter the microspheres, the impurities are adsorbed in the pores inside the microspheres through amino and octyl plasmas and hydrophobic effect inside the microspheres, the sample can be continuously loaded to dozens of times or even dozens of times of the column volume of a chromatographic medium, and finally, the adsorbed impurities are eluted by using 1M NaOH and 30% isopropanol. The virus exclusion composite chromatographic medium has high virus recovery rate due to no adsorption of viruses, and has high protein removal rate due to multiple functions of adsorbing impurities, so that a virus sample with very high purity and yield can be obtained by one-step purification. The specific combination principle is three: firstly, the virus enters a gap between the outer parts of the microspheres and flows out according to the sizes of the virus and impurities under the action of a molecular sieve, and the impurities enter the pore diameter of the inner parts of the microspheres; secondly, under the ionic action of amino groups, the amino groups have positive charges and can be combined with negative charges on the surfaces of impurities in the virus sample, so that the impurities with the negative charges can be adsorbed on ligands in the inner pore diameter of the microspheres; and thirdly, the hydrophobic effect of octyl absorbs impurities in a larger range in the virus on the ligand of the inner pore diameter of the microsphere by combining with the hydrophobic property of the impurities in the virus sample. 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 Wuhan Hui research organisms, with a microsphere exclusion limit of 10-1000 kD.
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 glyceryl ether under an alkaline condition to obtain glyceryl ether activated microspheres;
s2, brominating the glycerol ether activated microspheres with bromine water to obtain brominated microspheres;
s3, carrying out addition reaction on the brominated microspheres and n-octylamine to obtain the product.
The preparation method comprises the following steps of carrying out ring-opening reaction on hydroxyl of a specific exclusion limit microsphere and an epoxy group of allyl glycidyl ether under an alkaline condition to obtain a microsphere containing allyl, carrying out addition reaction on elemental bromine to obtain a microsphere with a halogenated hydrocarbon at the tail end, and finally carrying out addition reaction on the microsphere and n-octylamine to obtain a virus exclusion composite chromatographic medium containing an ionic group amino group and a hydrophobic group octyl, wherein the preparation principle is as follows:
s1, chemical reaction process of the glycerol ether activated microspheres:
s2, chemical reaction process of bromination of the glycerol ether microspheres:
s3, chemical reaction process of the brominated microspheres and n-octylamine:
in one embodiment of the present invention, the mass ratio of the microspheres to the allyl glyceryl ether in step S1 is: 1: 0.01-1: 0.5, preferably 1: 0.05-1: 0.2.
In one embodiment of the present invention, the activation conditions of step S1 are: the temperature is 20-60 ℃.
In one embodiment of the present invention, the mass ratio of the glycerol ether activated microspheres to the bromine water in step S2 is: 1: 0.01-1: 0.2, preferably 1: 0.01-1: 0.05.
In one embodiment of the present invention, the bromination process in step S2 is performed under the following conditions: the temperature is 20-60 ℃.
In one embodiment of the present invention, the mass ratio of the brominated microspheres to the n-octylamine in step S3 is: 1: 0.001-1: 0.2, preferably 1: 0.001-1: 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, is suitable for 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, almost has no influence on the titer and the activity of the viruses, and also has broad-spectrum adsorption effect on proteins and other impurities in a virus sample;
(2) the product has higher separation and purification efficiency and better purification effect, integrates gel filtration, ion exchange and hydrophobic chromatography, viruses flow through gaps among microspheres through the specific exclusion limit of the microspheres, impurities are adsorbed on a chromatography medium through the composite action of ions and hydrophobic ligands, the volume of the sample loading volume can reach dozens of times, the purification efficiency is improved by hundreds of times compared with that of the traditional gel filtration chromatography medium, and the protein removal rate and the virus recovery rate in virus flow-through liquid are improved by adding multiple actions of the ligands on the impurities;
(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 chromatography media in production is reduced by several times, and the cost investment of millions to tens of millions can be saved.
Drawings
FIG. 1 is a graph of virus exclusion complex chromatographic medium exclusion purified rabies vaccine prepared in example 1 of the present invention;
FIG. 2 is a graph of virus exclusion complex chromatographic medium exclusion purified rabies vaccine prepared in example 2 of the present invention;
FIG. 3 is a graph of virus exclusion complex chromatographic medium exclusion purified rabies vaccine prepared in example 3 of the present invention;
FIG. 4 is a graph of virus exclusion complex chromatographic medium exclusion purified rabies vaccine prepared in comparative example 1 of the present invention.
Detailed Description
The present invention is further described in detail below with reference to specific examples so that those skilled in the art can more clearly understand the present invention.
The following examples are merely illustrative of the present invention and are not intended to limit the scope of the present invention. All other embodiments obtained by a person skilled in the art based on the specific embodiments of the present invention without any inventive step are within the scope of the present invention.
In the examples of the present invention, all the raw material components are commercially available products well known to those skilled in the art, unless otherwise specified; in the examples of the present invention, unless otherwise specified, 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 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 in an alkaline condition, controlling the temperature to be 20-60 ℃, and the stirring speed to be 200-500 rpm/min, and reacting for 16-24 hours to obtain glycerol ether activated microspheres;
(2) vacuum-pumping, filtering and washing the obtained glycerol ether activated microspheres by using purified water with the volume being 10 times that of the glycerol ether activated microspheres, adding the glycerol ether activated microspheres into a new three-neck flask, adding purified water with the mass being about 1/5 of the glycerol ether activated microspheres, stirring, adding bromine water to ensure that the mass ratio of the glycerol ether activated microspheres to the bromine water is 1: 0.01-1: 0.2, controlling the temperature to be 20-60 ℃, and reacting for 10-60 min at the stirring speed of 200-500 rpm/min;
(3) then adding sodium formate, ensuring the mass ratio of the sodium formate to the glycerol ether activated microspheres to be 1: 0.01-0.2, controlling the temperature to be 20-60 ℃, stirring at the rotating speed of 200-500 rpm/min, and reacting for 10-60 min to obtain brominated microspheres;
(4) vacuum-pumping and filtering brominated microspheres by using 10 times of purified water, adding the cleaned microspheres into another new three-neck flask, adding purified water with the mass of about 1/5 of the brominated microspheres, stirring, adding n-octylamine to ensure that the mass ratio of the brominated microspheres to the n-octylamine is 1: 0.001-1: 0.2, simultaneously adjusting the pH to 9-11, controlling the temperature to be 20-60 ℃, stirring at the rotating speed of 200-500 rpm/min, reacting for 16-24 h, and vacuum-pumping and cleaning by using 10 times of purified water to obtain the virus exclusion 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 of Focurose microspheres, adding the Focurose microspheres into a 1000mL three-neck flask, adding 100g of pure water, and stirring;
(2) weighing 25g of allyl glycerol ether, adding into the three-neck flask, simultaneously adding 100g of sodium hydroxide, measuring the pH to be more than 7, controlling the temperature to be 30 ℃, stirring at the rotating speed of 300rpm/min, and stirring for 24 hours;
(3) carrying out vacuum filtration and cleaning on the microspheres by using 10 times of volume of purified water to obtain glycerol ether activated microspheres;
(4) weighing 500g of glycerol ether activated microspheres, adding into a 100mL three-neck flask, then adding 100g of pure water, and stirring;
(5) weighing 20g of bromine water, adding into the three-neck flask obtained in the step (4), controlling the temperature in a water bath at 30 ℃, and stirring for 30min at the stirring speed of 300 rpm/min;
(6) weighing 5g of sodium formate, adding the sodium formate into the three-neck flask obtained in the step (5), controlling the temperature in a water bath kettle at 30 ℃, and stirring for 30min at the stirring speed of 300 rpm/min;
(7) carrying out vacuum filtration on the cleaned microspheres by using 10 times of volume of purified water to obtain brominated microspheres;
(8) weighing 500g of brominated microspheres, adding the brominated microspheres into a 1000mL three-neck flask, adding 100g of pure water, and stirring;
(9) weighing 2g of n-octylamine, adding into the three-neck flask obtained in the step (8), adjusting the pH to 10.0, controlling the temperature in a water bath kettle at 30 ℃, and stirring for 24 hours at the stirring speed of 300 rpm/min;
(10) performing vacuum filtration and cleaning by using purified water with the volume 10 times that of the purified water, sampling and detecting the chloride ion load and the BSA protein load, wherein the results are shown in Table 1;
(11) and (5) packing the rabies virus sample, and verifying the protein removal rate and the virus recovery rate.
The embodiment also provides a method for separating and purifying a rabies virus sample by using the virus exclusion composite type chromatographic medium, which comprises the following specific steps:
a. pre-packing 1mL of pre-packed column;
b. buffer solution:
balance liquid: 20mM PB, 0.15M NaCl, pH 7.35;
eluent: 30% isopropanol, 1M NaOH;
the solution is filtered through a 0.45um water phase filter membrane after the solution preparation is finished.
c. Sample pretreatment
The original sample was measured for pH 7.31(25 ℃ C.), the conductance was 12.45ms/cm and the sample was filtered through a 0.45 μm filter before loading.
d. And (3) purification process: the flow rate of the sample was 0.33mL/min during the purification.
e. And detecting the protein removal rate and the virus recovery rate, wherein 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 purification map of the rabies virus sample 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 of Focurose microspheres, adding the Focurose microspheres into a 1000mL three-neck flask, adding 100g of pure water, and stirring;
(2) weighing 100g of allyl glycerol ether, adding into the three-neck flask, simultaneously adding 100g of sodium hydroxide, measuring the pH to be more than 7, controlling the temperature to be 30 ℃, and stirring for 24 hours at the stirring speed of 300 rpm;
(3) carrying out vacuum filtration and cleaning on the microspheres by using 10 times of volume of purified water to obtain glycerol ether activated microspheres;
(4) weighing 500g of glycerol ether activated microspheres, adding into a 100mL three-neck flask, then adding 100g of pure water, and stirring;
(5) weighing 20g of bromine water, adding into the three-neck flask obtained in the step (4), controlling the temperature in a water bath at 30 ℃, and stirring for 30min at the stirring speed of 300 rpm/min;
(6) weighing 5g of sodium formate, adding the sodium formate into the three-neck flask obtained in the step (5), controlling the temperature in a water bath kettle at 30 ℃, and stirring for 30min at the stirring speed of 300 rpm/min;
(7) carrying out vacuum filtration on the cleaned microspheres by using 10 times of volume of purified water to obtain brominated microspheres;
(8) weighing 500g of brominated microspheres, adding the brominated microspheres into a 1000mL three-neck flask, adding 100g of pure water, and stirring;
(9) weighing 10g of n-octylamine, adding into the three-neck flask obtained in the step (8), adjusting the pH to 9-11, controlling the temperature in a water bath kettle at 30 ℃, and stirring at a rotation speed of 300rpm/min for 24 h;
(10) the microspheres were washed with 10 times the volume of purified water by vacuum filtration, and the chloride ion loading and the BSA protein loading were sampled and detected, the results are shown in Table 1;
(11) and (5) packing the rabies virus sample, and verifying the protein removal rate and the virus recovery rate.
The embodiment also provides a method for separating and purifying a rabies virus sample by using the virus exclusion composite type chromatographic medium, which comprises the following specific steps:
a. pre-packing 1mL of pre-packed column;
b. buffer solution:
balance liquid: 20mM PB, 0.15M NaCl, pH 7.35;
eluent: 30% isopropanol, 1M NaOH;
the solution is filtered through a 0.45um water phase filter membrane after the solution preparation is finished.
c. Sample pretreatment
The original sample was measured for pH 7.31(25 ℃ C.), the conductance was 12.45ms/cm and the sample was filtered through a 0.45 μm filter before loading.
d. And (3) purification process: the flow rate of the sample was 0.33mL/min during the purification.
e. And detecting the protein removal rate and the virus recovery rate, wherein 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 purification map of the rabies virus sample 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 of Focurose microspheres, adding the Focurose microspheres into a 1000mL three-neck flask, adding 100g of pure water, and stirring;
(2) weighing 25g of allyl glycerol ether, adding into the three-neck flask, simultaneously adding 100g of sodium hydroxide, measuring the pH to be more than 7, controlling the temperature to be 30 ℃, and stirring for 24 hours at the stirring speed of 300 rpm;
(3) carrying out vacuum filtration and cleaning on the microspheres by using 10 times of volume of purified water to obtain glycerol ether activated microspheres;
(4) weighing 500g of glycerol ether activated microspheres, adding into a 100mL three-neck flask, then adding 100g of pure water, and stirring;
(5) weighing 20g of bromine water, adding into the three-neck flask obtained in the step (4), controlling the temperature in a water bath at 30 ℃, and stirring for 30min at the stirring speed of 300 rpm/min;
(6) weighing 5g of sodium formate, adding the sodium formate into the three-neck flask obtained in the step (5), controlling the temperature in a water bath kettle at 30 ℃, and stirring for 30min at the stirring speed of 300 rpm/min;
(7) carrying out vacuum filtration on the cleaned microspheres by using 10 times of volume of purified water to obtain brominated microspheres;
(8) weighing 500g of brominated microspheres, adding the brominated microspheres into a 1000mL three-neck flask, adding 100g of pure water, and stirring;
(9) weighing 10g of n-octylamine, adding into the three-neck flask obtained in the step (8), adjusting the pH to 9-11, controlling the temperature in a water bath kettle at 30 ℃, and stirring at a rotation speed of 300rpm/min for 24 h;
(10) the microspheres were washed with 10 times the volume of purified water by vacuum filtration, and the chloride ion loading and the BSA protein loading were sampled and detected, the results are shown in Table 1;
(11) and (5) packing the rabies virus sample, and verifying the protein removal rate and the virus recovery rate.
The embodiment also provides a method for separating and purifying a rabies virus sample by using the virus exclusion composite type chromatographic medium, which comprises the following specific steps:
a. pre-packing 1mL of pre-packed column;
b. buffer solution:
balance liquid: 20mM PB, 0.15M NaCl, pH 7.35;
eluent: 30% isopropanol, 1M NaOH;
the solution is filtered through a 0.45um water phase filter membrane after the solution preparation is finished.
c. Sample pretreatment
The original sample was measured for pH 7.31(25 ℃ C.), the conductance was 12.45ms/cm and the sample was filtered through a 0.45 μm filter before loading.
d. And (3) purification process: the flow rate of the sample was 0.33mL/min during the purification.
e. And detecting the protein removal rate and the virus recovery rate, wherein 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 purification map of the rabies virus sample is shown in table 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 of Focurose microspheres, adding the Focurose microspheres into a 1000mL three-neck flask, adding 100g of pure water, and stirring;
(2) weighing 25g of allyl glycerol ether, adding into the three-neck flask, simultaneously adding 100g of sodium hydroxide, measuring the pH to be more than 7, controlling the temperature to be 30 ℃, and stirring for 24 hours at the stirring speed of 300 rpm;
(3) carrying out vacuum filtration and cleaning on the microspheres by using 10 times of volume of purified water to obtain glycerol ether activated microspheres;
(4) weighing 500g of glycerol ether activated microspheres, adding into a 100mL three-neck flask, then adding 100g of pure water, and stirring;
(5) weighing 20g of bromine water, adding into the three-neck flask obtained in the step (4), controlling the temperature in a water bath at 30 ℃, and stirring for 30min at the stirring speed of 300 rpm/min;
(6) weighing 5g of sodium formate, adding the sodium formate into the three-neck flask obtained in the step (5), controlling the temperature in a water bath kettle at 30 ℃, and stirring for 30min at the stirring speed of 300 rpm/min;
(7) carrying out vacuum filtration on the cleaned microspheres by using 10 times of volume of purified water to obtain brominated microspheres;
(8) weighing 500g of brominated microspheres, adding the brominated microspheres into a 1000mL three-neck flask, adding 100g of pure water, and stirring;
(9) weighing 10g N-benzyl-N-methylethanolamine, adding into the three-neck flask obtained in the step (8), adjusting the pH to 9-11, controlling the temperature in a water bath kettle at 3 ℃, and stirring at the rotation speed of 300rpm/min for 24 hours;
(10) the microspheres were washed with 10 times the volume of purified water by vacuum filtration, and the chloride ion loading and the BSA protein loading were sampled and detected, the results are shown in Table 1;
(11) and (5) packing the 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 a rabies virus sample by using the virus exclusion composite type chromatographic medium, which comprises the following steps:
a. pre-packing 1mL of pre-packed column;
b. buffer solution:
balance liquid: 20mM PB, 0.15M NaCl, pH 7.35;
eluent: 30% isopropanol, 1M NaOH;
the solution is filtered through a 0.45um water phase filter membrane after the solution preparation is finished.
c. Sample pretreatment
The original sample was measured for pH 7.31(25 ℃ C.), the conductance was 12.45ms/cm and the sample was filtered through a 0.45 μm filter before loading.
d. And (3) purification process: the flow rate of the sample was 0.33mL/min during the purification.
e. And detecting the protein removal rate and the virus recovery rate, wherein 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 purification map of the rabies virus sample is shown in table 4.
And (4) analyzing results:
(1) the detection results of the chloride ion load and the BSA protein load of the virus exclusion complex type chromatography media prepared in examples 1-3 and comparative example 1 are shown in the following Table 1:
TABLE 1 chloride ion load and BSA protein load of the chromatography media prepared in the 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 rate of the virus exclusion complex chromatographic medium for separation and purification of rabies vaccine viruses prepared in examples 1-3 and comparative example 1 are shown in Table 3 below:
TABLE 2 BSA protein Standard Curve test results
The standard curve for the BSA protein was obtained: y 0.0004x +0.5348, R2＝0.9934。
TABLE 3 protein removal rates of rabies vaccine virus separated and purified by chromatography medium prepared in examples and comparative examples
Remarking: protein removal rate 1- [ M (flow-through)/M (stock solution) ]. 100%
(3) The virus recovery rate results of the virus exclusion composite chromatography media prepared in examples 1-3 and comparative example 1 for separating and purifying rabies vaccine virus are shown in the following table 4:
TABLE 4 virus recovery rates of separation and purification of rabies vaccine virus by chromatography media prepared in examples and comparative examples
Remarking: no standard substance is available, and the virus recovery rate is OD450(flow-through)/OD450(Stock solution) 100%
According to the results, hydroxyl of the specific exclusion limit microsphere and epoxy of allyl glycidyl ether are subjected to ring opening reaction to obtain microsphere containing allyl, simple substance bromine is subjected to addition reaction to obtain microsphere with halogenated hydrocarbon at the tail end, and finally the microsphere and n-octylamine are subjected to addition reaction to obtain virus exclusion composite chromatographic medium containing ionic group amino and hydrophobic group octyl, wherein the chromatographic medium is subjected to molecular sieve action, ionic action of amino group and hydrophobic action of octyl respectively, the protein removal rate can reach more than 46%, and the virus recovery rate can reach more than 87%.
It should be noted that the above examples are only for further illustration and description of the technical solution of the present invention, and are not intended to further 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 protection scope of the present invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A virus exclusion composite chromatographic medium is characterized by comprising 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 chromatographic medium is as follows:
the exclusion limit of the microspheres is 10 KD-1000 KD.
2. The virus exclusion composite chromatography media of claim 1, wherein the microspheres are selected from one or more of agarose microspheres, dextran microspheres, cellulose microspheres, polystyrene microspheres, polymethacrylate microspheres.
3. The virus exclusion complex chromatography medium of claim 2, wherein the microspheres are Focurose microspheres.
4. The method for preparing a virus exclusion complex chromatography medium according to any of claims 1 to 3, comprising the steps of:
s1, activating the microspheres with allyl glyceryl ether under an alkaline condition to obtain glyceryl ether activated microspheres;
s2, brominating the glycerol ether activated microspheres with bromine water to obtain brominated microspheres;
s3, carrying out addition reaction on the brominated microspheres and n-octylamine to obtain the product.
5. The method for preparing a virus exclusion composite chromatography medium according to claim 4, wherein the mass ratio of the microspheres to the allyl glycerol ether in step S1 is: 1: 0.01-1: 0.5.
6. The method for preparing a virus exclusion complex chromatography medium according to claim 4 or 5, wherein the activation conditions of step S1 are: the temperature is 20-60 ℃.
7. The method for preparing a virus exclusion composite chromatography medium according to claim 4, wherein the mass ratio of the glycerol ether activated microspheres to the bromine water in step S2 is as follows: 1: 0.01-1: 0.2, and the bromination conditions are as follows: the temperature is 20-60 ℃.
8. The method for preparing a virus exclusion composite chromatography medium according to claim 4, wherein the mass ratio of the brominated microspheres to n-octylamine in step S3 is: 1: 0.001-1: 0.2.
9. The method for preparing a virus exclusion complex chromatography medium according to claim 4 or 8, wherein the conditions of the addition reaction in step S3 are: the pH value is 9-11, and the temperature is 20-60 ℃.
10. Use of a virus exclusion complex chromatography medium according to any of claims 1 to 3 for virus purification.
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