CN112546672A - Use of non-porous chromatographic medium for virus purification - Google Patents
Use of non-porous chromatographic medium for virus purification Download PDFInfo
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- 239000012501 chromatography medium Substances 0.000 title claims abstract description 76
- 241000700605 Viruses Species 0.000 title claims abstract description 62
- 238000000746 purification Methods 0.000 title claims abstract description 37
- 239000002245 particle Substances 0.000 claims abstract description 43
- 239000002609 medium Substances 0.000 claims abstract description 14
- 238000013375 chromatographic separation Methods 0.000 claims description 7
- 238000011097 chromatography purification Methods 0.000 claims description 7
- 241000726445 Viroids Species 0.000 claims description 5
- 239000011159 matrix material Substances 0.000 claims description 5
- 229920005615 natural polymer Polymers 0.000 claims description 4
- 229920003053 polystyrene-divinylbenzene Polymers 0.000 claims description 4
- 229920001059 synthetic polymer Polymers 0.000 claims description 4
- 229920000936 Agarose Polymers 0.000 claims description 3
- 239000004952 Polyamide Substances 0.000 claims description 3
- 229920002678 cellulose Polymers 0.000 claims description 3
- 230000002209 hydrophobic effect Effects 0.000 claims description 3
- 238000005342 ion exchange Methods 0.000 claims description 3
- 229920002647 polyamide Polymers 0.000 claims description 3
- 229920000570 polyether Polymers 0.000 claims description 3
- 239000001913 cellulose Substances 0.000 claims description 2
- 229920000728 polyester Polymers 0.000 claims description 2
- 239000012535 impurity Substances 0.000 abstract description 17
- 238000001179 sorption measurement Methods 0.000 abstract description 13
- 238000010828 elution Methods 0.000 abstract description 10
- 238000000926 separation method Methods 0.000 abstract description 5
- 239000011148 porous material Substances 0.000 description 18
- 238000004587 chromatography analysis Methods 0.000 description 13
- 238000012856 packing Methods 0.000 description 13
- 239000011347 resin Substances 0.000 description 13
- 229920005989 resin Polymers 0.000 description 13
- 239000004005 microsphere Substances 0.000 description 10
- 238000005349 anion exchange Methods 0.000 description 8
- 238000002474 experimental method Methods 0.000 description 7
- 239000000945 filler Substances 0.000 description 7
- 238000011068 loading method Methods 0.000 description 7
- 239000000463 material Substances 0.000 description 7
- 238000002360 preparation method Methods 0.000 description 7
- 239000007787 solid Substances 0.000 description 7
- 244000309459 oncolytic virus Species 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 5
- 238000000034 method Methods 0.000 description 5
- 230000003612 virological effect Effects 0.000 description 5
- 229920000193 polymethacrylate Polymers 0.000 description 4
- 150000003242 quaternary ammonium salts Chemical group 0.000 description 4
- 238000005571 anion exchange chromatography Methods 0.000 description 3
- 230000007717 exclusion Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 239000004793 Polystyrene Substances 0.000 description 2
- 125000000129 anionic group Chemical group 0.000 description 2
- 235000010980 cellulose Nutrition 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000012527 feed solution Substances 0.000 description 2
- 229920002223 polystyrene Polymers 0.000 description 2
- 238000002305 strong-anion-exchange chromatography Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 229960005486 vaccine Drugs 0.000 description 2
- 230000000274 adsorptive effect Effects 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 238000002659 cell therapy Methods 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000004237 preparative chromatography Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
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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
- B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
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- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
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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
- C12N7/02—Recovery or purification
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- Virology (AREA)
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Abstract
The invention provides an application of a nonporous chromatographic medium in virus purification, wherein the particle size of the nonporous chromatographic medium is more than or equal to 10 microns, and the porosity of a nonporous layer plate medium is less than 20 percent. The nonporous chromatographic medium has low porosity and less adsorption of impurities in virus, so that the separation purity of a virus sample obtained after chromatographic elution is obviously improved, and the purification efficiency is greatly improved.
Description
Technical Field
The invention belongs to the field of purification, and particularly relates to an application of a non-porous chromatography medium in virus purification.
Background
With the rapid development of genetic engineering, vaccines, cell therapy and other fields, more and more large virus or virus-like particles need to be prepared or semi-prepared and purified. In addition to ultrafiltration, chromatographic separation and purification are the most common methods for obtaining high purity viral particle samples, including ion exchange chromatography, affinity chromatography, hydrophobic chromatography, and the like. The traditional prepared or semi-prepared chromatography medium filler is porous microspheres, and the porous microsphere material has large specific surface area and can provide higher adsorption capacity for common biomolecule separators. The prior art (prior art) chromatographic media all have porosities (porosities) greater than 20%,generally between 30% and 90%. The pore size of these chromatographic media is generally less than
(typically less than 100 nanometers) macroore or mesopore. The particle size of many virus particles is generally larger than 20 nm, and some of them exceed 100 nm. Viral particles cannot diffuse into the pores of the chromatographic medium due to size exclusion (see e.g. Journal of Chromatography a 2007, 1142: 2-12 and Journal of Chromatography a 2013, 1297: 96-105), so that only the outer surface of the medium microspheres is available for chromatographic adsorption of viral particles, and the surface area in the pores cannot adsorb viral particles due to size exclusion. However, the contaminant molecules in the virus sample are generally smaller than the virus and can diffuse into the pores of the chromatographic medium and be adsorbed therein. Because the surface area in the pores of the traditional chromatography medium is far larger than the surface area of the medium microspheres, the impurity adsorption is usually very obvious due to the existence of the surface area in the pores, the impurity content is higher during adsorption and elution, and the virus particle purification effect is not ideal.
Disclosure of Invention
In order to solve the problems, the invention discloses the application of a nonporous chromatographic medium in virus purification, the particle size of the nonporous chromatographic packing is more than 10 microns, the porosity of mesopore and macroore in the chromatographic packing is less than 20 percent, and impurities in virus are less adsorbed, so that the separation purity of a virus sample obtained after chromatographic elution is obviously improved.
In order to achieve the technical purpose, the invention adopts the technical scheme that the application of the nonporous chromatographic medium in virus purification is realized, the particle size of the nonporous chromatographic medium is more than or equal to 10 microns, and the porosity of the nonporous laminate medium is less than 20%. Porosity refers to the volume of pores in the media as a percentage of the total volume of the media microspheres.
Preferably, the particle size of the nonporous chromatographic medium is more than or equal to 15 microns.
Preferably, the porosity of the non-porous laminate media is less than 10%.
Preferably, the non-porous chromatographic medium is applied to the chromatographic separation and purification of viruses, viroid particles and similar virus-sized ultra-large biomolecules.
Preferably, the chromatographic functions of the non-porous chromatographic medium include ion exchange, affinity, and hydrophobicity.
Preferably, the matrix of the non-porous chromatographic medium can be a synthetic polymer or a natural polymer.
Preferably, the synthetic polymers include, but are not limited to, polyesters, polyethers, polystyrene-divinylbenzene, polyamides, and the natural polymers include, but are not limited to, agarose, cellulose.
As previously described in the background section, chromatography media packing materials currently used for the preparation or semi-preparation of purified virus or viroid particles are porous microspheres because of the large specific surface area of the porous microsphere material, which provides a high adsorptive loading for typical biomolecule isolates. However, the method has the defects that only the outer surface of the medium microsphere can be utilized when the virus particles are adsorbed by chromatography, and the surface area in the pores of the medium cannot adsorb the virus particles due to size exclusion. The impurity molecules in the virus sample are generally smaller than the virus, so that the impurity molecules can diffuse into the chromatography medium hole and then be adsorbed in the medium hole. Because the surface area in the pores of the porous chromatography medium is far larger than the surface area of the medium microspheres, impurity adsorption is often very remarkable due to the existence of the surface area in the pores, so that the impurity content is higher during adsorption and elution, and the virus particle purification effect is often not ideal.
The invention uses non-porous chromatographic packing with particle size greater than 10 microns, and these medium packings have no significant mesopores or macropores, and the porosity of mesopores and macropores in the medium packings is less than 20%, preferably less than 10%. (according to the definition of the International Union of Pure and Applied Chemistry (IUPAC), pores with a diameter of less than 2 nm are called micropores; pores with a diameter of more than 50 nm are called macropores; pores with a diameter of between 2 and 50 nm are called mesopores or mesopores). Compared with the prior art, the nonporous chromatographic packing used by the invention has no holes which can only accommodate the impurities to enter but can not allow the virus particles to diffuse into, and the chromatographic adsorption quantity of the virus particles on the outer surface of the medium is not obviously changed, but the chromatographic adsorption quantity of the impurities in the sample is obviously reduced. Therefore, after the virus sample is chromatographically eluted by the non-porous chromatographic packing, the separation purity of the virus sample is obviously improved.
The particle size and pore state of the filler affect the chromatographic separation performance. The resolution is inversely proportional to the square root of the particle size of the filler, with smaller particle sizes giving better resolution. However, the back pressure of chromatography is inversely proportional to the square of the filler particle size, the particle size decreases and the back pressure increases exponentially with the square. The pore size and porosity of the filler have a significant impact on the chromatographic efficiency and loading. Generally, the larger the pore size and the lower the porosity, the better the mass transfer within the filler, the higher the resolution, but the lower the loading. Since analytical chromatographic separations have high resolution requirements, high back pressures and low loadings are generally acceptable. Analytical chromatographic packing can therefore employ small particle size (typically 2-10 microns) low porosity or even non-porous packing. For example, TSK-GEL Q-STAT columns from TOSOH and ProPac series analytical columns from ThermoFisher use non-porous packing materials having a particle size of less than 10 microns. However, chromatographic separation packing materials for the purpose of preparative purification (preparative purification) generally have a particle size of more than 10 μm because the pressure resistance of conventional preparative chromatography columns cannot withstand the high back pressure of chromatographic packing materials having a particle size of 10 μm or less. In addition, the preparation of purified chromatographic media has high requirements on the loading capacity, because the high loading capacity of the media can improve the production efficiency of the preparation and purification. For these reasons, the prior art chromatographic media are porous resins with particle sizes greater than 10 microns and containing large amounts of meso-pore and macroore, since these porous resins have a large pore surface area to provide high loading for chromatographic adsorption. Large particle size non-porous chromatographic purification media have not been previously available in the prior art (prior arts). The chromatographic medium used in the preparation and purification of ultra-large biomolecules such as viruses, vaccines and the like is a traditional porous chromatographic medium without exception in the prior art. The invention breaks the conventional inertial thinking, uses the nonporous preparative chromatographic medium to purify virus particles, and finds that the nonporous chromatographic medium can provide high-purity virus particles compared with the traditional porous chromatographic medium.
The chromatographic media of the present invention may employ conventional modes of chromatography including ion exchange, affinity, hydrophobic, and the like. The chromatography media matrix of the present invention may be of synthetic polymeric type (including polymethacrylates, polystyrenes, polyethers, polyamides, and the like) or of natural polymeric type (including agaroses, celluloses, and the like). Purification applications for which the chromatography media of the invention are suitable are various viral or viroid particles and similarly sized, very large biomolecules.
In this patent, chromatographic medium, chromatographic filler, microspheres, etc. all refer to spherical materials for chromatographic separation and purification.
The invention provides an application of a non-porous chromatography medium with the particle size of more than or equal to 10 microns and the porosity of less than 20 percent in virus purification, and the purification application objects are various virus or viroid particles and super-large biomolecules with similar sizes. Because the porosity of the nonporous chromatographic medium is low and the impurities in the virus are less adsorbed, the separation purity of the virus sample obtained after the nonporous chromatographic medium is chromatographically eluted is obviously improved, and the purification efficiency is greatly improved.
Drawings
FIG. 1 is the original image of the chromatography chart of the purification experiment of the porous chromatography medium. In the figure, 1 is a target virus peak, and 2 is an impurity peak.
FIG. 2 is the original drawing of the chromatography in the purification experiment using a non-porous chromatography medium, wherein 1 is the target virus peak and 2 is the impurity peak.
FIG. 3 is an as-received SEC analysis chromatogram of an oncolytic virus solution.
Figure 4 is a SEC analysis chromatogram of the virus peak collection component using a porous chromatographic gradient.
FIG. 5 is a SEC analytical chromatogram of the virus peak collection component using a non-porous chromatographic gradient.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention are further described below with reference to specific embodiments. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Furthermore, it should be understood that various changes or modifications can be made by those skilled in the art after reading the disclosure of the present invention, and such equivalents also fall within the scope of the invention.
Example 1 use of polystyrene anion exchange non-porous chromatography media with a particle size of 15 microns for virus purification
Preparation of chromatography medium: the nonporous chromatographic medium adopted in the embodiment is anion exchange nonporous chromatographic medium UniCore15Q, and is prepared by polystyrene-divinylbenzene nonporous resin with the particle size of 15 microns. The resin is solid and non-porous, and thus has zero porosity. After the nonporous solid resin is subjected to surface hydrophilization treatment, the surface of the nonporous solid resin is modified and bonded with trimethyl quaternary ammonium salt groups to obtain a nonporous anion exchange chromatography medium. In contrast to the non-porous chromatographic medium was Source 15Q from GE Healthcare. As with UniCore15Q of this example, Source 15Q is also a 15 micron anionic chromatography medium. The two mediums have the same matrix, are both polystyrene-divinylbenzene resin, have the same surface chemical function, are both quaternary ammonium salt type anion exchange, and mainly have the difference that the former is a non-porous solid chromatographic medium, while the latter is a traditional porous chromatographic medium, and has the porosity of more than 40 percent.
Virus purification application: the target of purification in this example was an oncolytic virus feed solution having a purity of 6.5% (FIG. 3 shows an original oncolytic virus SEC analysis chromatogram). The method of chromatographic purification is anion exchange adsorption followed by gradient elution (Bind-elute) mode. Table 1 shows the specific conditions of the chromatography method. After the eluate was collected in fractions, the collected fractions containing the virus target were analyzed for purity of the virus sample by SEC.
TABLE 1 anion exchange chromatography method Experimental conditions
FIG. 1 is an oncolytic virus purification chromatogram using conventional porous resin strong anion exchange chromatography medium Source 15Q, and FIG. 2 is an oncolytic virus purification chromatogram using the non-porous chromatography medium-resin strong anion exchange chromatography medium UniCore15Q of the present invention. When using conventional porous chromatography media Source 15Q, see FIG. 1, a very high eluting impurity peak (arrow represented by number 2 in FIG. 1) can be seen. When the non-porous homogeneous chromatographic medium UniCore15Q of the invention is used for carrying out the same chromatographic purification experiment, as shown in figure 2, the peak of elution impurities is very small (arrow represented by number 2 in figure 2), which shows that the non-porous chromatographic medium can obviously reduce the content of impurities when a virus sample is eluted.
SEC analysis of the virus samples collected after elution by chromatography revealed that the purity of the virus samples obtained by the conventional porous chromatography medium Source 15Q experiment was 54% (FIG. 4), while the purity of the virus samples obtained by the non-porous same type medium UniCore15Q experiment was nearly 90% (FIG. 5).
Example 2 use of polymethacrylate anion exchange non-porous chromatography media with a particle size of 30 microns for virus purification
Preparation of chromatography medium: the non-porous chromatographic medium used in this example was anion exchange non-porous chromatographic medium UniQ30-NP prepared from 30 μm polymethacrylate non-porous resin. The resin is solid and non-porous, and thus has zero porosity. The nonporous solid resin is subjected to surface hydrophilic modification treatment, and then is subjected to surface modification and bonding with a trimethyl quaternary ammonium salt group to obtain the anion exchange chromatography medium. In contrast to the non-porous chromatographic medium, UniQ-30S, manufactured by Suzhou Nami MicroTechnology. As with UniQ30-NP of this example, UniQ-30S is also a 30 micron anionic chromatography medium. The two mediums have the same matrix, are both high cross-linked polymethacrylate resin, have the same surface chemical function, are both quaternary ammonium salt type anion exchange, and mainly have the difference that the former is a non-porous solid chromatographic medium, while the latter is a traditional porous chromatographic medium, and has the porosity of more than 40 percent.
Virus purification application: the target of purification in this example was an oncolytic virus feed solution having a purity of 6%. The chromatographic methods and conditions were the same as described in example 1, both in anion exchange adsorption followed by gradient elution mode. After the eluate was collected in fractions, the collected fractions containing the virus target were analyzed for purity of the virus sample by SEC. SEC analysis of virus samples collected after elution by chromatography revealed that the purity of the virus samples obtained by the experiment with the traditional porous chromatography medium UniQ-30S was 57%, while the purity of the virus samples obtained by the experiment with the non-porous chromatography medium UniQ30-NP was 91%.
The invention provides the application of the nonporous chromatographic medium with the particle size of more than or equal to 10 microns and the porosity of less than 20 percent in virus purification, and because the nonporous chromatographic medium has low porosity and less adsorption of impurities in viruses, the separation purity of a virus sample obtained after chromatographic elution is obviously improved, and the purification efficiency is greatly improved.
The invention can be implemented in other ways than the embodiments described above, and any obvious alternatives are within the scope of the invention without departing from the invention.
Claims (7)
1. The application of the nonporous chromatographic medium in virus purification is characterized in that the particle size of the nonporous chromatographic medium is more than or equal to 10 microns, and the porosity of the nonporous laminate medium is less than 20 percent.
2. Use of a non-porous chromatography medium according to claim 1 for virus purification, wherein the non-porous layer medium has a particle size of 15 μm or more.
3. Use of a non-porous chromatography media according to claim 1 for virus purification, wherein the porosity of the non-porous lamina media is less than 10%.
4. The use of a non-porous chromatographic medium according to claim 1 for the purification of viruses, viroid particles and virus-like sized very large biomolecules for chromatographic separation and purification.
5. Use of a non-porous chromatographic medium according to claim 1 for the purification of a virus, wherein the chromatographic functions of the non-porous chromatographic medium include ion exchange, affinity, hydrophobic.
6. Use of a non-porous chromatographic medium according to claim 1 for the purification of a virus, wherein the matrix of the non-porous chromatographic medium can be a synthetic polymer or a natural polymer.
7. Use of a non-porous chromatographic medium for the purification of viruses according to claim 5 wherein said synthetic polymers include but are not limited to polyesters, polyethers, polystyrene-divinylbenzene, polyamides and said natural polymers include but are not limited to agarose, cellulose.
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| CN201910920576.2A CN112546672A (en) | 2019-09-26 | 2019-09-26 | Use of non-porous chromatographic medium for virus purification |
| PCT/CN2019/120178 WO2021056788A1 (en) | 2019-09-26 | 2019-11-22 | Application of non-porous chromatography medium in virus purification |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101745369A (en) * | 2008-12-19 | 2010-06-23 | 中国科学院兰州化学物理研究所 | Method for preparing spherical silicagel filler for ultra performance liquid chromatography |
| CN105413233A (en) * | 2015-12-10 | 2016-03-23 | 中国医学科学院生物医学工程研究所 | Micro-column separation device |
| WO2018136666A1 (en) * | 2017-01-20 | 2018-07-26 | Dionex Corporation | Multimodal chromatographic media for protein separation |
| 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 |
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| JP2007099555A (en) * | 2005-10-03 | 2007-04-19 | Pentax Corp | Particle production method, particle and adsorption device |
| JP5339033B2 (en) * | 2008-05-14 | 2013-11-13 | 真鍋 征一 | A multilayer structure film having different fine particle capturing performance on the front and back surfaces and a manufacturing method thereof. |
| CN102515179A (en) * | 2012-01-09 | 2012-06-27 | 江南大学 | Silicon oxide microsphere for high performance liquid chromatography and preparation method of slicon oxide microsphere |
| CN103374143B (en) * | 2012-04-28 | 2015-08-19 | 中国科学院过程工程研究所 | A kind of super large pore polymer microsphere and preparation method thereof |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101745369A (en) * | 2008-12-19 | 2010-06-23 | 中国科学院兰州化学物理研究所 | Method for preparing spherical silicagel filler for ultra performance liquid chromatography |
| CN105413233A (en) * | 2015-12-10 | 2016-03-23 | 中国医学科学院生物医学工程研究所 | Micro-column separation device |
| WO2018136666A1 (en) * | 2017-01-20 | 2018-07-26 | Dionex Corporation | Multimodal chromatographic media for protein separation |
| 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 |
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