CN111394321A - Ultra-large pore size chromatography medium for virus particle purification and application method thereof - Google Patents
Ultra-large pore size chromatography medium for virus particle purification and application method thereof Download PDFInfo
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- CN111394321A CN111394321A CN202010234686.6A CN202010234686A CN111394321A CN 111394321 A CN111394321 A CN 111394321A CN 202010234686 A CN202010234686 A CN 202010234686A CN 111394321 A CN111394321 A CN 111394321A
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- pore size
- large pore
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- 239000012501 chromatography medium Substances 0.000 title claims abstract description 54
- 239000002245 particle Substances 0.000 title claims abstract description 38
- 241000700605 Viruses Species 0.000 title claims abstract description 32
- 239000011148 porous material Substances 0.000 title claims abstract description 31
- 238000000034 method Methods 0.000 title claims abstract description 13
- 238000000746 purification Methods 0.000 title claims description 18
- 239000004005 microsphere Substances 0.000 claims abstract description 16
- 229920003053 polystyrene-divinylbenzene Polymers 0.000 claims abstract description 16
- 125000000524 functional group Chemical group 0.000 claims abstract description 8
- 239000000463 material Substances 0.000 claims abstract description 6
- 239000011521 glass Substances 0.000 claims description 15
- 239000007788 liquid Substances 0.000 claims description 8
- 239000003480 eluent Substances 0.000 claims description 7
- 239000000758 substrate Substances 0.000 claims description 7
- 230000003612 virological effect Effects 0.000 claims description 7
- 238000011068 loading method Methods 0.000 claims description 5
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 claims description 3
- 238000010828 elution Methods 0.000 claims description 3
- 238000004140 cleaning Methods 0.000 claims description 2
- 238000011049 filling Methods 0.000 claims description 2
- 230000005847 immunogenicity Effects 0.000 abstract description 4
- 230000000694 effects Effects 0.000 abstract description 3
- 238000013375 chromatographic separation Methods 0.000 abstract description 2
- 238000005516 engineering process Methods 0.000 abstract description 2
- 241000711798 Rabies lyssavirus Species 0.000 description 6
- 229960005486 vaccine Drugs 0.000 description 3
- 238000000432 density-gradient centrifugation Methods 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000012856 packing Methods 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 210000000605 viral structure Anatomy 0.000 description 2
- 229920000936 Agarose Polymers 0.000 description 1
- ZOIORXHNWRGPMV-UHFFFAOYSA-N acetic acid;zinc Chemical compound [Zn].CC(O)=O.CC(O)=O ZOIORXHNWRGPMV-UHFFFAOYSA-N 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- ILRRQNADMUWWFW-UHFFFAOYSA-K aluminium phosphate Chemical compound O1[Al]2OP1(=O)O2 ILRRQNADMUWWFW-UHFFFAOYSA-K 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- 238000011067 equilibration Methods 0.000 description 1
- 238000001641 gel filtration chromatography Methods 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000013341 scale-up Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000004246 zinc acetate Substances 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Virology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Immunology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to a chromatographic separation technology, in particular to an ultra-large pore size chromatographic medium for purifying virus particles and an application method thereof, which are characterized in that a base material of the ultra-large pore size chromatographic medium is a polystyrene-divinylbenzene microsphere, the pore diameter of the polystyrene-divinylbenzene microsphere is more than or equal to 4000 Å, the particle diameter is more than or equal to 60 mu m, an affinity functional group is modified on the surface of the base material, the carrying capacity of the chromatographic medium to the virus particles is higher, and after the chromatographic medium is adopted to purify the virus, the virus activity cannot be lost, and the immunogenicity is higher.
Description
Technical Field
The invention relates to a chromatographic separation technology, in particular to an ultra-large pore size chromatographic medium for purifying virus particles and an application method thereof.
Background
The virus is an important vaccine type, and the virus with good immunogenicity is selected, artificially cultured and inactivated by a physical or chemical method to prepare the vaccine. Traditionally, virus purification is mainly carried out by precipitation methods such as ammonium sulfate, zinc acetate, aluminum phosphate and the like and density gradient centrifugation. Although the precipitation method is simple, the resolution is low, and impurities such as host DNA cannot be removed effectively. While density gradient centrifugation has high resolution, the operation cycle is long, and expensive equipment needs to be repeatedly purchased in scale-up production. Both methods are therefore increasingly being replaced by chromatographic techniques in production. Gel filtration chromatography and ion exchange chromatography are most commonly used in the purification of vaccines. Due to the characteristics of complex virus structure, poor stability and easy inactivation, the pore size of the chromatography medium is required to be adapted to the volume of the virus particles, and is generally 10-20 times larger than the diameter of the virus particles.
At present, the traditional chromatography medium has a small pore size, generally less than 200nm, even less than 50nm, and the size of virus particles is generally more than 20nm, so that the traditional chromatography medium has only one percent or even lower protein loading compared with the loading of virus particles. Moreover, viruses are susceptible to structural changes, aggregation or disaggregation within the pore size of the narrow chromatographic medium, resulting in loss of activity and reduced immunogenicity.
Disclosure of Invention
The invention aims to solve the technical problem of providing an ultra-large pore size chromatography medium for purifying virus particles and an application method thereof, wherein the chromatography medium has higher virus particle loading capacity; after the chromatography medium is used for purifying the virus, the activity of the virus cannot be lost, and the immunogenicity is higher.
In order to solve the problems, the following technical scheme is provided:
the ultra-large-aperture chromatographic medium for purifying virus particles is characterized in that a base material of the ultra-large-aperture chromatographic medium is a polystyrene-divinylbenzene microsphere, the aperture of the polystyrene-divinylbenzene microsphere is more than or equal to 4000 Å, the particle size is more than or equal to 60 mu m, and the surface of the base material is modified with an affinity functional group.
Wherein the aperture of the polystyrene-divinylbenzene microsphere is 4000 Å -7000 Å.
The particle size of the polystyrene-divinylbenzene microsphere is 60-100 mu m.
The affinity functional group is a sulfate.
The purification yield of the ultra-large-aperture chromatography medium is more than or equal to 70 percent.
The surface of the substrate needs to be modified by a hydrophilic layer.
The application method of the ultra-large aperture chromatography medium for purifying virus particles is characterized by comprising the following steps: firstly, filling an ultra-large-aperture chromatography medium into a glass column; then, balancing the inside of the glass column by using a balancing liquid; then, loading a sample containing virus particles on an ultra-large-aperture chromatographic medium in a glass column; then, cleaning the ultra-large pore size chromatographic medium in the glass column by using the balance liquid again; and finally, eluting the ultra-large pore size chromatography medium in the glass column by using an eluent, and collecting an elution component, namely the purified virus particles.
By adopting the scheme, the method has the following advantages:
the ultra-large-aperture chromatographic medium has the advantages that the substrate is the polystyrene-divinylbenzene microsphere, the aperture of the hydrophilic polystyrene-divinylbenzene microsphere is not less than 4000 Å, the particle size is not less than 60 mu m, and the surface of the substrate is modified with the affinity functional group.
Detailed Description
The present invention will be described in further detail below.
The ultra-large aperture chromatography medium for purifying virus particles is characterized in that a substrate of the ultra-large aperture chromatography medium is a polystyrene-divinylbenzene microsphere, the aperture of the polystyrene-divinylbenzene microsphere is not less than 4000 Å, the particle size is not less than 60 mu m, the surface of the substrate is modified with an affinity functional group, the aperture of the polystyrene-divinylbenzene microsphere is 4000 Å -7000 Å, the particle size of the polystyrene-divinylbenzene microsphere is 60-100 mu m, the affinity functional group is sulfate, the purification yield of the ultra-large aperture chromatography medium is not less than 70%, and the surface of the substrate is required to be modified with a hydrophilic layer.
The application method of the ultra-large-aperture chromatographic medium for purifying the virus particles comprises the following steps: first, an ultra-large pore chromatography medium is loaded into a glass column. Subsequently, the inside of the glass column was equilibrated with an equilibration solution. The sample containing the viral particles is then loaded onto an ultra-large pore chromatography medium within a glass column. Then, the ultra-large pore size chromatography medium in the glass column is washed again with the equilibrium solution. And finally, eluting the ultra-large pore size chromatography medium in the glass column by using an eluent, and collecting an elution component, namely the purified virus particles.
The ultra-large-aperture chromatographic medium has the advantages that the aperture of the ultra-large-aperture chromatographic medium is obviously larger than that of the conventional chromatographic medium, the defects of the conventional chromatographic medium are effectively overcome, the influence of slow diffusion mass transfer speed is weakened by convection mass transfer in the hole, the mass transfer resistance is reduced, the mass transfer speed is improved, meanwhile, the effective surface area for adsorbing viruses is increased, and the problem of unstable structure of virus particles in the purification process is also reduced.
The following table shows the results of the test for rabies virus purification using ultra large pore size (635 nm) chromatography media for virus particle purification according to the invention
| Serial number | Volume ml | gpG titer UI/ml | Total gpG UI |
| Sample (I) | 30 | 5.82 | 174.6 |
| Flow-through liquid | 30 | 0 | 0 |
| Eluent | 3 | 42.7 | 128.1 |
As can be seen from the upper surface, the rabies virus is purified by using an ultra-large-aperture chromatographic medium, no target virus component exists in the flow-through liquid, the gpG content in the eluent/gpG content in the sample is calculated, and the recovery rate reaches 73.3%.
The following table shows the results of the test for rabies virus purification using a conventional pore size (30 nm) chromatography medium (cellulose affinity packing)
| Serial number | Volume ml | gpG titer UI/ml | Total gpG UI |
| Sample (I) | 30 | 5.82 | 174.6 |
| Flow-through liquid | 30 | 2.1 | 63 |
| Eluent | 5 | 14.9 | 74.5 |
As can be seen from the above surface, the yield of the purified rabies virus using the chromatography medium with the traditional 30nm pore is only 42.7%.
The following table shows the results of the rabies virus purification test using a chromatography medium (agarose affinity packing) of conventional pore size (50 nm)
| Serial number | Volume ml | gpG titer UI/ml | Total gpG UI |
| Sample (I) | 30 | 5.82 | 174.6 |
| Flow-through liquid | 30 | 2.52 | 75.6 |
| Eluent | 5 | 12.54 | 62.7 |
As can be seen from the above surface, the yield of the purified rabies virus using the chromatography medium with the traditional 50nm pore is only 35.9%.
Claims (7)
1. The ultra-large-aperture chromatographic medium for purifying virus particles is characterized in that a base material of the ultra-large-aperture chromatographic medium is a polystyrene-divinylbenzene microsphere, the aperture of the polystyrene-divinylbenzene microsphere is more than or equal to 4000 Å, the particle size is more than or equal to 60 mu m, and the surface of the base material is modified with an affinity functional group.
2. An ultra-large pore size chromatography medium for the purification of viral particles as claimed in claim 1, wherein the polystyrene-divinylbenzene microspheres have a pore size of 4000 Å to 7000 Å.
3. The ultra-large pore size chromatography medium for purification of viral particles of claim 1, wherein the particle size of the polystyrene-divinylbenzene microspheres is between 60 and 100 μm.
4. An ultra-large pore chromatography medium for the purification of viral particles according to claim 1, wherein said affinity functional group is a sulfate.
5. The ultra-large pore size chromatographic medium for the purification of viral particles according to claim 1, wherein the purification yield of the ultra-large pore size chromatographic medium is 70% or more.
6. An ultra-large pore size chromatographic medium for the purification of viral particles according to claim 1 wherein the surface of said substrate is modified with a hydrophilic layer.
7. The method of using an ultra-large pore size chromatography medium for the purification of viral particles according to any of claims 1 to 6, comprising the steps of:
firstly, filling an ultra-large-aperture chromatography medium into a glass column; then, balancing the inside of the glass column by using a balancing liquid; then, loading a sample containing virus particles on an ultra-large-aperture chromatographic medium in a glass column; then, cleaning the ultra-large pore size chromatographic medium in the glass column by using the balance liquid again; and finally, eluting the ultra-large pore size chromatography medium in the glass column by using an eluent, and collecting an elution component, namely the purified virus particles.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010234686.6A CN111394321A (en) | 2020-03-30 | 2020-03-30 | Ultra-large pore size chromatography medium for virus particle purification and application method thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010234686.6A CN111394321A (en) | 2020-03-30 | 2020-03-30 | Ultra-large pore size chromatography medium for virus particle purification and application method thereof |
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| CN111394321A true CN111394321A (en) | 2020-07-10 |
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| CN202010234686.6A Pending CN111394321A (en) | 2020-03-30 | 2020-03-30 | Ultra-large pore size chromatography medium for virus particle purification and application method thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113416235A (en) * | 2021-06-24 | 2021-09-21 | 苏州赛分科技有限公司 | Liquid chromatography for purifying and separating virus antigens |
| CN114058483A (en) * | 2021-11-27 | 2022-02-18 | 中科宝承生物医学科技有限公司 | Purification equipment for umbilical cord mesenchymal stem cells and operation method thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20120083593A1 (en) * | 2010-10-01 | 2012-04-05 | Shanghai Yongyou Bioscience Inc. | Separation and Purification of Stevioside and Rebaudioside A |
| WO2015109068A1 (en) * | 2014-01-16 | 2015-07-23 | W. R. Grace & Co.-Conn. | Affinity chromatography media and chromatography devices |
| CN105744952A (en) * | 2013-09-14 | 2016-07-06 | 巴拉特生物技术国际有限公司 | A viral vaccine and methods of manufacture thereof |
| CN108949702A (en) * | 2018-08-01 | 2018-12-07 | 苏州纳微科技股份有限公司 | Application of the ultra-large aperture chromatography media in purified virus particles |
-
2020
- 2020-03-30 CN CN202010234686.6A patent/CN111394321A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20120083593A1 (en) * | 2010-10-01 | 2012-04-05 | Shanghai Yongyou Bioscience Inc. | Separation and Purification of Stevioside and Rebaudioside A |
| CN105744952A (en) * | 2013-09-14 | 2016-07-06 | 巴拉特生物技术国际有限公司 | A viral vaccine and methods of manufacture thereof |
| WO2015109068A1 (en) * | 2014-01-16 | 2015-07-23 | W. R. Grace & Co.-Conn. | Affinity chromatography media and chromatography devices |
| CN108949702A (en) * | 2018-08-01 | 2018-12-07 | 苏州纳微科技股份有限公司 | Application of the ultra-large aperture chromatography media in purified virus particles |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113416235A (en) * | 2021-06-24 | 2021-09-21 | 苏州赛分科技有限公司 | Liquid chromatography for purifying and separating virus antigens |
| CN114058483A (en) * | 2021-11-27 | 2022-02-18 | 中科宝承生物医学科技有限公司 | Purification equipment for umbilical cord mesenchymal stem cells and operation method thereof |
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Application publication date: 20200710 |