CN114054006A - Oligo-dT affinity chromatography filler - Google Patents
Oligo-dT affinity chromatography filler Download PDFInfo
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- CN114054006A CN114054006A CN202111362343.9A CN202111362343A CN114054006A CN 114054006 A CN114054006 A CN 114054006A CN 202111362343 A CN202111362343 A CN 202111362343A CN 114054006 A CN114054006 A CN 114054006A
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- oligo
- affinity chromatography
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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
- B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
- B01J20/286—Phases chemically bonded to a substrate, e.g. to silica or to polymers
Abstract
The invention provides Oligo-dT affinity chromatography packing, which is prepared by taking polymer microspheres with hydrophilic surfaces and polyhydroxy hydrophilic functional groups as base spheres and bonding dT nucleotide monomers to generate Oligo-dT ligands on the surfaces of polymers through hydroxyl on the surfaces of the polymer microspheres. Compared with the conventional Oligo-dT affinity filler, the invention has higher mRNA loading capacity and higher mRNA recovery rate, and also has better alkali resistance than the conventional Oligo-dT affinity filler. The preparation method is simple, does not need oligo-dT ligand synthesis and purification required by oligo-dT affinity filler production in the prior art, and can greatly reduce the production cost of the affinity filler, thereby reducing the production and purification cost of mRNA.
Description
Technical Field
The invention relates to the technical field of purification, in particular to Oligo-dT affinity chromatography packing.
Background
Oligo-dT affinity chromatography is widely used for purification In Vitro Transcription (IVT) manufacturing processes of mRNA, and this affinity chromatography method can effectively separate mRNA from other components of the transcription reaction process (e.g., enzymes and plasmid DNA, etc.). Oligo-dT affinity chromatography packing produced by the Prior art (Priorarts) is generally prepared by bonding Oligo-dT ligand to the surface of chromatography packing, and Oligo-dT ligand is generally prepared by a multi-step solid phase synthesis method. The solid phase synthesis method needs to use expensive solid phase synthesis base balls and solid phase synthesis linkers, functional modification is carried out after Oligo-dT ligand is separated from a solid phase carrier after synthesis, and complicated chromatographic purification and separation processes, so that the Oligo-dT is very expensive, the production cost of Oligo-dT affinity packing is very high, and the application of the affinity packing in a large-scale mRNA production and purification process is seriously hindered. In addition, the ligand bonding chemical coupling used by Oligo-dT affinity filler produced by the prior art is often not stable under strong alkali, so that ligand shedding of the affinity filler is easy to occur in regeneration and CIP processes (the CIP condition of the affinity filler is generally 0.1-0.5M NaOH), and the service life of the filler is seriously influenced.
Disclosure of Invention
The invention aims to provide a novel Oligo-dT affinity chromatography filler, which not only has good mRNA binding capacity, higher mRNA loading capacity and higher mRNA recovery rate, but also has better alkali resistance.
In order to achieve the purpose, the technical scheme of the invention is as follows: an Oligo-dT affinity chromatography filler is prepared by using polymer microspheres with polyhydroxy hydrophilic functional groups on the surfaces as base spheres and bonding pyrimidine (dT) nucleotide monomers to generate Oligo-dT ligands to the surfaces of polymers through hydroxyl groups on the surfaces of the polymer microspheres.
Preferably, the polymer microsphere used as the base sphere is PS-DVB, polymethacrylate, polyamide, polyether and cross-linked cellulose polymer resin.
Preferably, the polyhydroxy hydrophilic functional group on the surface of the polymer microsphere can be introduced during the preparation of the polymer microsphere, or can be obtained by surface chemical modification or surface hydrophilic coating.
Preferably, the degree of crosslinking of the polymeric microspheres is greater than 20%.
Preferably, the crosslinking degree of the polymer microsphere is between 45% and 65%.
Preferably, the polymer microspheres are large-aperture polymer microspheres with the aperture larger than that of the polymer microspheres
Preferably, the pore size of the polymeric microspheres is larger than
Preferably, the polymer microsphere is directly bonded with the dT monomer through the hydroxyl on the surface, and an oligo-dT ligand is generated on the surface of the polymer microsphere through a solid phase synthesis method and is cyclically bonded at least once to grow an oligo-dT chain.
Preferably, the bonding cycle number of dT nucleotide monomers on the surface of the polymer microsphere is not less than 10.
Preferably, the bonding cycle number of dT nucleotide monomers on the surface of the polymer microsphere is not less than 15.
Preferably, the number of dT nucleotide monomer bonding cycles on the surface of the polymer microsphere is 15-30, i.e. 15-30 nucleotide units (dT15-dT30) are bonded.
Preferably, the dT nucleotide monomer may have a 5 '-protected phosphodiester (phosphodiester) structure or a 5' -protected phosphoramidite (phosphoramidite) structure.
Wherein, if the dT nucleotide monomer is of phosphodiester type, each monomer bonding solid phase synthesis cycle has two steps of bonding and 5' -position deprotection; if the dT nucleotide monomer is of phosphoramidite type, each monomer bonding cycle comprises three steps of bonding, oxidation, and deprotection of the 5' -position.
The invention takes the macroporous polymer microsphere with high crosslinking degree and hydrophilic surface as the base sphere (support) for solid phase synthesis to bond and connect the pyrimidine nucleotide (dT) monomer to the filler polymer microsphere in a circulating and reciprocating way to prepare the polymer microsphere directly, does not need the synthesis, separation and purification of oligo-dT ligand, and also omits the coupling of oligo-dT and filler with low conversion efficiency. The oligo-dT affinity filler of the invention not only has good mRNA binding capacity, but also has higher mRNA loading capacity and higher mRNA recovery rate compared with the conventional oligo-dT affinity filler. In addition, this oligo-dT affinity filler also has better alkali resistance than conventional oligo-dT affinity fillers. The filler does not need oligo-dT ligand synthesis and purification required by oligo-dT affinity filler production in the prior art, has simple preparation method, and can greatly reduce the production cost of the affinity filler, thereby reducing the production and purification cost of mRNA.
Detailed Description
The technical solution of the present invention will be further described with reference to the following specific examples, but the present invention is not limited to these examples.
EXAMPLE 1 Synthesis of Supermacroporous PS-DVB Polymer microsphere oligo-dT25 affinity Filler
Ultra-large pore (aperture) with particle size of 50 microns
) After the PS-DVB microspheres are coated by polyhydroxy compounds in a hydrophilic mode, the PS-DVB microspheres are cleaned by ethanol and placed in an oven at the temperature of 60 ℃ for drying. And (3) filling the dried microsphere powder into a solid-phase synthesis column.
The bonding reaction of the dT nucleotide monomer was controlled using the ARKTA solid phase synthesizer program. The bonding reaction of 5 '-position protected dT nucleotide monomer DMT-dT-CE-Phosphoramidite (5' -O- (4,4 '-dimethoxyltrityl) -thymidine-3' -cyanoethylphospyramide) was carried out for 12 minutes in the presence of anhydrous acetonitrile as a solvent and ethylmercaptotetrazole as a condensing agent.
The oxidation step was carried out using 0.05M iodine solution as a reaction reagent and pyridine aqueous solution (90: 10) as a solvent for 5 minutes.
The reaction reagent for DMT deprotection was dichloroacetic acid, the solvent was dichloromethane, and the reaction time was 3 minutes. After bonding of 25 circulating monomers is completed, soaking the super-macroporous PS-DVB polymer microspheres in 20% ammonia water for 16 hours. Washing with water until pH is neutral to obtain the super-macroporous PS-DVB polymer microsphere oligo-dT25 affinity filler with code No. NM 0501.
Affinity binding capacity and elution recovery for two different sizes of mRNA were tested with the oligo-dT affinity filler NW 0501. The test method for affinity binding loading is a static load test. In comparison, two conventional synthetic Oligo-dT25 fillers, POROS Oligo-dT25 and NanoGel dT25, were used and tested in the same manner. Wherein the base sphere of NanoGel dT25 was identical to the super macroporous PS-DVB polymer microspheres used in this example.
The test results are shown in Table 1, comparison of affinity loading of oligo-dT affinity filler NM0501 of the present invention and two conventional oligo-dT affinity fillers for two different mRNAs and elution recovery.
TABLE 1
Meanwhile, the alkali resistance of the three oligo-dT affinity fillers in Table 1 was tested in comparison. The change in static loading of the filler to mRNA was measured after 48 hours of immersion in 0.1M sodium hydroxide. The percent reduction in loading after sodium hydroxide soaking value reflects the resistance of the filler to 0.1M NaOH. The smaller the reduction percentage, the better the resistance of the filler to strong bases and the longer the life of the filler.
The results of the alkali resistance test are shown in Table 2, and the static loading changes of the oligo-dT filler NM0501 of the present invention are compared with those of two conventional oligo-dT affinity fillers after soaking in 0.1M NaOH for 48 hours.
TABLE 2
Therefore, the filler of the invention has higher affinity loading capacity and elution recovery rate, and simultaneously has better alkali resistance than the conventional oligo-dT affinity filler.
The preparation method is simple, does not need oligo-dT ligand synthesis and purification required by oligo-dT affinity filler production in the prior art, and can greatly reduce the production cost of the affinity filler, thereby reducing the production and purification cost of mRNA. Compared with the conventional oligo-dT affinity filler, the invention has higher mRNA loading capacity and higher mRNA recovery rate, and simultaneously has better alkali resistance than the conventional oligo-dT affinity filler.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various changes and modifications can be made without departing from the inventive concept of the present invention, and these changes and modifications are all within the scope of the present invention.
Claims (9)
1. An Oligo-dT affinity chromatography filler is characterized in that the Oligo-dT affinity chromatography filler is prepared by using polymer microspheres with polyhydroxy hydrophilic functional groups on the surfaces as base spheres and bonding dT nucleotide monomers to generate Oligo-dT ligands on the surfaces of polymers through hydroxyl groups on the surfaces of the polymer microspheres.
2. The Oligo-dT affinity chromatography packing according to claim 1, wherein the polymer microspheres as the base spheres are PS-DVB, polymethacrylate, polyamide, polyethers, cross-linked cellulose based polymer resins.
3. The Oligo-dT affinity chromatography packing according to claim 1, wherein the polyhydroxy hydrophilic functional group on the surface of the polymer microsphere is introduced during the preparation of the polymer microsphere, or is obtained by surface chemical modification or surface hydrophilic coating.
4. The Oligo-dT affinity chromatography packing according to claim 1, wherein the degree of crosslinking of the polymeric microspheres is greater than 20%.
5. The Oligo-dT affinity chromatography packing of claim 1, wherein the polymeric microspheres are large pore size polymeric microspheres with pore size larger than
6. The Oligo-dT affinity chromatography packing according to claim 1, wherein the polymer microspheres are directly bonded to dT nucleotide monomers through hydroxyl groups on the surface, Oligo-dT ligands are generated on the surfaces of the polymer microspheres by a solid phase synthesis method, and the Oligo-dT chains are grown by cyclic bonding at least once.
7. The Oligo-dT affinity chromatography packing of claim 6, wherein the dT nucleotide monomer bonding cycle number on the surface of the polymer microsphere is not less than 10.
8. The Oligo-dT affinity chromatography packing material according to claim 1, wherein the dT nucleotide monomer has a 5 '-protected phosphodiester structure or a 5' -protected phosphoramidite structure.
9. The Oligo-dT affinity chromatography packing according to claim 8, wherein if the dT nucleotide monomers are of phosphodiester type, each dT nucleotide monomer bonding solid phase synthesis cycle has two steps of bonding and 5' -position deprotection; if the dT nucleotide monomer is of the phosphoramidite type, each dT nucleotide monomer bonding cycle comprises three steps of bonding, oxidation, and deprotection of the 5' -position.
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2021
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