CN109836479A - The method that application simulation thermopnore isolates and purifies FMD inactivation of viruses antigen - Google Patents
The method that application simulation thermopnore isolates and purifies FMD inactivation of viruses antigen Download PDFInfo
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Abstract
The present invention relates to a kind of methods that application simulation thermopnore isolates and purifies FMD inactivation of viruses antigen, comprising the following steps: with the chromatography column of equilibrating buffer balance simulation thermopnore；By FMD inactivation of viruses antigen samples loading into simulation thermopnore chromatography column, the cleaning of antigen samples impurity and sample desorption elution can be carried out to chromatography column simultaneously during loading, the cleaning and regeneration and equilibrium liquid of chromatograph packing material balance, and sample after purification is collected in continuous desorption elution process.The present invention will simulate thermopnore isolating and purifying, it can be achieved that continuous saturation loading lock out operation, substantially increases applied sample amount applied to FMD inactivation of viruses antigen for the first time；And continuously isolated and purified by multiple monomer columns, serialization degree is high, substantially reduces and isolates and purifies the time, improve preparation efficiency, reduces operating cost.And the present invention specifically selects rigid filled, greatly improves its filler carrying capacity i.e. resistance to pressure, reduces the usage amount of chromatograph packing material, extends the service life of chromatographic column in simulation thermopnore.
Description
Technical field
The present invention relates to veterinary biologics manufacturing technology fields, and in particular to a kind of application simulation thermopnore isolates and purifies
The method of FMD inactivation of viruses antigen.
Background technique
Foot and mouth disease virus (FMD) is a kind of virus disease with economic destruction propagated between artiodactyls.Mouthful
Mainly complete virion related with stimulation body generation antibody and sensitized lymphocyte in aphtovirus vaccine sample,
Sedimentation coefficient is 146S, is considered having parallel relation with the efficiency of aftosa vaccine.Aftosa vaccine production is main in the world
Using suspension culture techniques, culture reaction is between 3000L-8000L, and subsequent purification technique is mainly using heavy including polyethylene glycol
It forms sediment, ultrafiltration removal of impurities, the methods of aqueous two-phase extraction.But these methods are difficult to obtain the aftosa vaccine antigen of high-purity.
Industry chromatography technique is gradually applied to the purifying of aftosa inactivation of viruses antigen in recent years, there is patent report in succession
Foot-and-mouth disease virus antigen (aftosa disclosed in Chinese invention patent CN102988970B is purified using the method for gel filtration
Purified vaccine and its preparation method and application), foot-and-mouth disease virus antigen (Chinese invention is purified using ion exchange layer analysis system
The method of ion-exchange chromatogram purification aftosa inactivation of viruses antigen disclosed in patent CN104818254A), using hydrophobic work
Foot-and-mouth disease virus antigen (hydrophobic interaction chromatography disclosed in Chinese invention patent CN104892734 A is purified with tomographic system
The method for purifying aftosa inactivation of viruses antigen).From the point of view of content in patent, its method pair for being all made of fixed bed chromatography at present
Foot-and-mouth disease virus antigen is purified, and purification step is balance -- loading -- cleaning -- and elution -- on-line cleaning -- balance, so
After continue single line circulation.Often have that applied sample amount is small, filler carrying capacity is low, one-line operation when purifying using above-mentioned chromatographic process
Lead to that purifying process time long sample loss is big, waste liquid yield is big and processing cost is high.
Simulation flowing bed technique is the purification technique to grow up from the sixties in last century, is initially only applied to stone
The separation of oiling chemical product, the nineties, France Seperex was applied to isolating and purifying for drug and field of fine chemical, in recent years
Gradually it is applied to carbohydrate, isomer, chipal compounds, bio-fermented liquid over year to isolate and purify, and aftosa suspends training
Isolating and purifying for inactivation of viruses antigen is supported, does not have also at present and simulation flowing bed technique is applied to isolating and purifying for viral sample,
It is mainly very high to the filler requirement in flowing bed body using this technology, need to find wear-resistant, high pressure resistant, acidproof alkali process
Filler, and the filler of the typical chromatographic methods of aftosa inactivation of viruses antigen purification is based on glucan or agarose matrix at present
Filler, partial size is generally 75um, pressure-bearing property is less than 0.3MPa, and prolonged soda acid processing is easy to cause falling off for functional group
Lead to problems such as purification effect poor, limits simulation flowing bed technique in the application in aftosa inactivation of viruses antigen purification field.
Summary of the invention
For the defects in the prior art, the present invention provides a kind of application simulation thermopnores to isolate and purify FMD inactivation disease
The method of malicious antigen.
The purpose of the present invention is what is be achieved through the following technical solutions:
The present invention provides a kind of method that application simulation thermopnore isolates and purifies FMD inactivation of viruses antigen, including it is following
Step:
S1, with the chromatography column of equilibrating buffer balance simulation thermopnore；
S2, FMD inactivation of viruses antigen samples loading is extremely simulated in thermopnore chromatography column, it can be simultaneously during loading
The cleaning of antigen samples impurity is carried out to chromatography column and sample desorption elution, the cleaning and regeneration and equilibrium liquid of chromatograph packing material are flat
Weighing apparatus, collects sample after purification in continuous desorption elution process.
Preferably, the specific steps of the S2 are as follows:
A, it is detected by FMD inactivation of viruses antigen samples loading into simulation thermopnore chromatography column, during loading every
Root chromatography column efflux, when detecting FMD inactivation of viruses antigen efflux and loading in n-th chromatography column efflux
When the UV of sample absorbs consistent, then feed liquid is distributed to (n+1)th chromatography column, while to the color for being saturated adsorption antigen
It composes chromatographic column and carries out sample washing impurity and sample desorption acquisition FMD inactivation of viruses antigen purification sample；
B, when detecting FMD inactivation of viruses antigen efflux and loading sample in (n+1)th chromatography column efflux
When UV absorbs consistent, feed liquid is distributed to the n-th+2 chromatography columns, at the same to be saturated the chromatography column of adsorption antigen into
Row sample washs impurity and sample desorption obtains inactivation of viruses antigen purification sample, and cleaning and regeneration has eluted the chromatography finished
Column；
C, and so on, until after m+n column chromatography column resin filler regeneration ending, with the color after equilibrium liquid balance regeneration
Spectrum chromatographic column continues on for adsorbing FMD inactivation of viruses antigen samples；
D, the step of circulation A is to C, until all purifying finishes FMD inactivation of viruses antigen samples；
Wherein, the n >=1, n+m > n, n and m are integer, the chromatography column number that time feed liquid distribution enters headed by n；n+
M is the total chromatography column number of system.
Preferably, 6≤n+m≤24 take into account loading, wash miscellaneous, sample solution according to simulation thermopnore practical operation situation
Suction and column regeneration process, so chromatographic column number is at least selected as 6；In view of simulation thermopnore actual moving process in it is soft,
Hardware controls, so chromatographic column is at most selected as 24.
Preferably, the chromatograph packing material loaded in the simulation thermopnore chromatography column is with hydrophobic or ionic aglucon
High-strength polymer filler.
Preferably, described to have hydrophobic or ionic aglucon high-strength polymer filler are as follows: with acrylate, styrene
In base benzene, divinylbenzene, styrene, silica rigidity agarose, silica rigidity glucan or rigid fiber element
At least one is matrix, with hydroxypropyl, propyl, benzyl, isopropyl, phenyl, normal-butyl, tert-butyl, butylthio, amyl, octane
At least one of base, diethylamino ethyl, sulfonic group, quaternary ammonium, carboxymethyl, diethyl amino propyl are filling out for aglucon
Material.
Preferably, described to have hydrophobic or ionic aglucon high-strength polymer filler as with styrene and divinyl
The polymer of benzene is the filler of matrix.
It is highly preferred that described have hydrophobic or ionic aglucon high-strength polymer filler as with styrene and divinyl
The polymer of base benzene is the filler that matrix, tert-butyl or diethyl amino propyl are aglucon.
Preferably, the partial size with hydrophobic or ionic aglucon high-strength polymer filler is 3um~20um, hole
DiameterThe partial size is excessive, it will reduces the service life of filler.
Preferably, the length of the spectrum chromatographic column of the color is 10~60cm.
Preferably, using the filler of hydrophobic type aglucon, the equilibration buffer is pH7~9, conductivity 100~
Ammonium sulfate, sodium sulphate or the ammonium chloride buffer of 300ms/cm；Using the filler of ionic aglucon, the equilibration buffer is
PH7~9, ammonium sulfate, sodium sulphate or the ammonium chloride buffer of 5~300ms/cm of conductivity；
The washing impurity buffer is pH7~9, the ammonium sulfate of 5~300ms/cm of conductivity, ammonium chloride buffer or
Conductivity 5~300ms/cm sodium phosphate buffer；
The elution solution that the elution uses, using the filler of hydrophobic type aglucon, it is described elute the elution solution that uses for
PH7~9, ammonium sulfate, sodium sulphate or the ammonium chloride buffer of 5~300ms/cm of conductivity；Using the filler of ionic aglucon, institute
The elution solution that uses of elution is stated as pH7~9, ammonium sulfate, sodium sulphate or the chloride buffer of 100~300ms/cm of conductivity
Liquid.
The regeneration washing liquid that the cleaning and regeneration uses is molten for sodium hydroxide solution, ethanol solution, methanol solution, isopropanol
At least one of liquid, urea liquid, neutral interface surface activating agent.
Preferably, the concentration of the sodium hydroxide solution is 0.1-1.0mol/L；The concentration of methanol solution, ethanol solution
It is 10-40% (V/V)；The concentration of aqueous isopropanol is 10-30% (V/V)；The concentration of urea liquid is 3-8mol/L；It is neutral
The concentration of interface surface activating agent is 0.2-0.3% (V/V).
It is highly preferred that the neutral interface surface activating agent includes polysorbas20, Tween 80, polyethylene glycol octyl phenyl
At least one of ether-100, Brij-35-35.
Preferably, the simulation thermopnore of the use includes multi-pass rotary valve, conduit external structure, feed liquid distributor, color
Chromatographic column is composed, the simulation thermopnore of the use includes multi-pass rotary valve, feed liquid distributor, conduit external structure, chromatography
Column；The multi-pass rotary valve and feed liquid distributor are arranged above chromatography column, and the feed liquid distributor setting is revolved in multi-pass
Between rotary valve and chromatography column；The feed liquid distributor is connect with chromatography column；The conduit external structure is arranged in color
Compose the outside of chromatographic column.The multi-pass rotary valve can require to switch between chromatographic column according to technique, feed liquid distributor
Multi-pass rotary valve can be cooperated to distribute feed liquid to chromatographic column, conduit external structure can play fixed and sealing chromatographic column.
Preferably, the multi-pass rotary valve can carry out loading simultaneously, elution, regeneration, balance four mac functions
Rotation uses.
Preferably, the feed liquid distributor at least can carry out liquid conveying simultaneously for n root chromatogram column.
It is additionally provided with collet on the outside of the chromatography column, can satisfy the requirement of the technological operation under different temperatures.
Preferably, the chromatography technological temperature of the chromatography column is 2-15 DEG C.
The present invention also provides a kind of for isolating and purifying the simulation thermopnore of FMD inactivation of viruses antigen, including multi-pass rotation
Rotary valve, feed liquid distributor, conduit external structure, chromatography column；The multi-pass rotary valve and feed liquid distributor are arranged in chromatography
Above chromatographic column, the feed liquid distributor is arranged between multi-pass rotary valve and chromatography column；The feed liquid distributor and color
Compose chromatographic column connection；The outside of chromatography column is arranged in the conduit external structure.The multi-pass rotary valve and feed liquid distribution
Device is located at the top of device, is responsible for the switching of valve and the distribution of feed liquid；Device lower part be conduit external structure and chromatography column,
For fixing, sealing, adjusting the chromatographic purifying of chromatographic column and feed liquid.
Compared with prior art, the present invention have it is following the utility model has the advantages that
1, the present invention will simulate thermopnore isolating and purifying, it can be achieved that continuous full applied to FMD inactivation of viruses antigen for the first time
With loading lock out operation, applied sample amount is substantially increased；And continuously isolated and purified by multiple monomer columns, serialization degree is high, greatly
It shortens greatly and isolates and purifies the time, improve preparation efficiency, reduce operating cost.
2, the present invention specifically selects rigid filled, greatly improves its filler carrying capacity i.e. resistance to pressure, reduces chromatography and fill out
The usage amount of material extends the service life of chromatographic column in simulation thermopnore.
3, after isolating and purifying FMD inactivation of viruses antigen using the method for the present invention, purity may be up to 98%, active antigens recycling
Rate is 95%.
Detailed description of the invention
Upon reading the detailed description of non-limiting embodiments with reference to the following drawings, other feature of the invention,
Objects and advantages will become more apparent upon:
Fig. 1 is that simulation thermopnore of the invention purifies FMD inactivation of viruses antigen chromatographic column operation schematic diagram.
Specific embodiment
The present invention is described in detail combined with specific embodiments below.Following embodiment will be helpful to the technology of this field
Personnel further understand the present invention, but the invention is not limited in any way.It should be pointed out that the ordinary skill of this field
For personnel, without departing from the inventive concept of the premise, several changes and improvements can also be made.These belong to the present invention
Protection scope.
FMD inactivation of viruses is isolated and purified using simulation flowing bed technique collocation rigid media filler the present invention provides a kind of
The method of antigen, simulation mobility bed technique is that one kind can be used for a kind of companies such as chromatography, absorption, ion exchange, gradient elution
The chromatographic equipment main body of reforwarding row, by multiple monomer column combinations, efficient, cheap by simulation moving process realization is continuous
Industrial separation purifying.Pass through the mould for successively, periodically moving realization separation equipment of material liquid entrance position in the process of running
Quasi- movement, and material liquid entrance can arbitrarily replace position, operating pressure and stability of flow, and the outer dead volume of column is few, this company
Continuous formula mode can guarantee that production is carried out continuously, and filler can be regenerated in failure, and the uniformity that packing layer uses can be improved, more general
Logical preparing chromatograph in industry and its matter of other separation equipments consumption are low, and low energy consumption, and operating cost is lower.The simulation thermopnore includes more
Way rotary valve, feed liquid distributor, conduit external structure, chromatography column；The multi-pass rotary valve and the setting of feed liquid distributor exist
Above chromatography column, the feed liquid distributor is arranged between multi-pass rotary valve and chromatography column；The feed liquid distributor
It is connect with chromatography column；The outside of chromatography column is arranged in the conduit external structure.Its operation schematic diagram such as Fig. 1 institute
Show, each chromatography chromatographic column is in different processing statuses, to realize viral sample while carry out loading, desorption elution and layer
Analyse regeneration washing, the equilibrium liquid balancing run of column.Sample introduction can be recycled in the equipment running process, can reduce sample overload
Loss.
In order to overcome simulation thermopnore chromatography filler to wear, larger, system pressure is larger simultaneously, and general industry chromatography
Filler be not available due to matrix (such as glucan or agar carbohydrate) the problem of, the present invention filter out using with dredge
The microballoon of the high-strength polymer of water aglucon is as chromatograph packing material, such medium is smaller and uniform with partial size, pressure-resistant performance is strong
The features such as (pressure-resistant maximum can achieve 4Mpa), acid and alkali-resistance be good, high resolution is very suitable to as FMDV inactivation of viruses Antigens are raw
The industrial separation purification work of object macromolecular.Such filler has been used in the patent, since such packing material size is compared with small specific surface
Product increases so that the volume containing the sample of FMDV inactivation of viruses antigen is greatly improved compared with general industry chromatography, to reduce filler use
Amount.And incumbent firms are made it easier to since such filler resistance to acid and alkali is stronger, also more common chromatography is long for service life.
Embodiment 1
Present embodiments provide a kind of method that application simulation thermopnore isolates and purifies FMD inactivation of viruses antigen, including with
Lower step:
S1, with the chromatography column of equilibrating buffer balance simulation thermopnore；
S2, FMD inactivation of viruses antigen samples loading is extremely simulated in thermopnore chromatography column, it can be simultaneously during loading
The cleaning of antigen samples impurity is carried out to chromatography column and sample desorption elution, the cleaning and regeneration and equilibrium liquid of chromatograph packing material are flat
Weighing apparatus, collects sample after purification in continuous desorption elution process.
The specific steps of the S2 are as follows:
A, it is detected by FMD inactivation of viruses antigen samples loading into simulation thermopnore chromatography column, during loading every
Root chromatography column efflux, when detecting FMD inactivation of viruses antigen efflux and loading in n-th chromatography column efflux
When the UV of sample absorbs consistent, then feed liquid is distributed to (n+1)th chromatography column, while to the color for being saturated adsorption antigen
It composes chromatographic column and carries out sample washing impurity and sample desorption acquisition FMD inactivation of viruses antigen purification sample；
B, when detecting FMD inactivation of viruses antigen efflux and loading sample in (n+1)th chromatography column efflux
When UV absorbs consistent, feed liquid is distributed to the n-th+2 chromatography columns, at the same to be saturated the chromatography column of adsorption antigen into
Row sample washs impurity and sample desorption obtains inactivation of viruses antigen purification sample, and cleaning and regeneration has eluted the chromatography finished
Column；C, and so on, until after m+n column chromatography column resin filler regeneration ending, with the chromatography layer after equilibrium liquid balance regeneration
Analysis column continues on for adsorbing FMD inactivation of viruses antigen samples；
C, after the n-th column chromatography column resin filler regeneration ending, continue on for adsorbing FMD inactivation of viruses after balancing with equilibrium liquid
Antigen samples, circulation S2 operation is until the purifying of FMD inactivation of viruses antigen samples finishes；
The n=5, m=7.
The chromatograph packing material loaded in the simulation thermopnore chromatography column is with the polymerization of styrene and divinylbenzene
The filler that object is matrix, tert-butyl is aglucon；The partial size of filler is 3um, aperture
The length of the spectrum chromatographic column of the color is 10cm.
The equilibration buffer is pH7~9, the ammonium sulphate buffer of 100~300ms/cm of conductivity；
The washing impurity buffer is pH7~9, the ammonium sulphate buffer of 100~300ms/cm of conductivity；
The elution solution used that elutes is pH7~9, the ammonium sulfate ammonium buffer of 5~300ms/cm of conductivity；
The sodium hydroxide solution that the regeneration washing liquid that the cleaning and regeneration uses is 0.1-1.0mol/L for concentration.
The chromatography column chromatography technological temperature is 2-15 DEG C.
The purity of FMD inactivation of viruses antigen samples after using the present embodiment method to isolate and purify is 98%, active antigens
The rate of recovery is 95%.It is continuously monitored, is compared 100 times as a result, CV value is less than 5% using the present embodiment method.Illustrate to use
Chromatographic column of the invention is greatly improved its service life.
Embodiment 2
Present embodiments provide a kind of method that application simulation thermopnore isolates and purifies FMD inactivation of viruses antigen, including with
Lower step:
S1, with the chromatography column of equilibrating buffer balance simulation thermopnore；
S2, FMD inactivation of viruses antigen samples loading is extremely simulated in thermopnore chromatography column, it can be simultaneously during loading
The cleaning of antigen samples impurity is carried out to chromatography column and sample desorption elution, the cleaning and regeneration and equilibrium liquid of chromatograph packing material are flat
Weighing apparatus, collects sample after purification in continuous desorption elution process.
The specific steps of the S2 are as follows:
A, it is detected by FMD inactivation of viruses antigen samples loading into simulation thermopnore chromatography column, during loading every
Root chromatography column efflux, when detecting FMD inactivation of viruses antigen efflux and loading in n-th chromatography column efflux
When the UV of sample absorbs consistent, then feed liquid is distributed to (n+1)th chromatography column, while to the color for being saturated adsorption antigen
It composes chromatographic column and carries out sample washing impurity and sample desorption acquisition FMD inactivation of viruses antigen purification sample；
B, when detecting FMD inactivation of viruses antigen efflux and loading sample in (n+1)th chromatography column efflux
When UV absorbs consistent, feed liquid is distributed to the n-th+2 chromatography columns, at the same to be saturated the chromatography column of adsorption antigen into
Row sample washs impurity and sample desorption obtains inactivation of viruses antigen purification sample, and cleaning and regeneration has eluted the chromatography finished
Column；C, and so on, until after m+n column chromatography column resin filler regeneration ending, with the chromatography layer after equilibrium liquid balance regeneration
Analysis column continues on for adsorbing FMD inactivation of viruses antigen samples；
C, after the n-th column chromatography column resin filler regeneration ending, as after all (n+m) root chromatogram columns after being balanced using equilibrium liquid
The 1st root chromatogram column continue on for adsorbing FMD inactivation of viruses antigen samples, circulation S2 operation is until FMD inactivation of viruses antigen sample
Product purifying finishes；
The n=2, m=4.
The chromatograph packing material loaded in the simulation thermopnore chromatography column is with the polymerization of styrene and divinylbenzene
The filler that object is matrix, tert-butyl is aglucon；The partial size of filler is 10um, aperture
The length of the spectrum chromatographic column of the color is 30cm.
The equilibration buffer is pH7~9, the sodium phosphate buffer of 100~300ms/cm of conductivity；
The washing impurity buffer is pH7~9, the sodium phosphate buffer of 100~300ms/cm of conductivity；
The elution solution used that elutes is pH7~9, the sodium phosphate buffer of 5~300ms/cm of conductivity；
The ethanol solution that the regeneration washing liquid that the cleaning and regeneration uses is 10-40% (V/V) for concentration.
The chromatography column chromatography technological temperature is 2-15 DEG C.
The purity of FMD inactivation of viruses antigen samples after using the present embodiment method to isolate and purify is 97%, active antigens
The rate of recovery is continuously monitored for 96% using the present embodiment method, is compared 80 times as a result, CV value is less than 5%.Illustrate using this
The chromatographic column of invention is greatly improved its service life.
Embodiment 3
Present embodiments provide a kind of method that application simulation thermopnore isolates and purifies FMD inactivation of viruses antigen, including with
Lower step:
S1, with the chromatography column of equilibrating buffer balance simulation thermopnore；
S2, FMD inactivation of viruses antigen samples loading is extremely simulated in thermopnore chromatography column, it can be simultaneously during loading
The cleaning of antigen samples impurity is carried out to chromatography column and sample desorption elution, the cleaning and regeneration and equilibrium liquid of chromatograph packing material are flat
Weighing apparatus, collects sample after purification in continuous desorption elution process.
The specific steps of the S2 are as follows:
A, it is detected by FMD inactivation of viruses antigen samples loading into simulation thermopnore chromatography column, during loading every
Root chromatography column efflux, when detecting FMD inactivation of viruses antigen efflux and loading in n-th chromatography column efflux
When the UV of sample absorbs consistent, then feed liquid is distributed to (n+1)th chromatography column, while to the color for being saturated adsorption antigen
It composes chromatographic column and carries out sample washing impurity and sample desorption acquisition FMD inactivation of viruses antigen purification sample；
B, when detecting FMD inactivation of viruses antigen efflux and loading sample in (n+1)th chromatography column efflux
When UV absorbs consistent, feed liquid is distributed to the n-th+2 chromatography columns, at the same to be saturated the chromatography column of adsorption antigen into
Row sample washs impurity and sample desorption obtains inactivation of viruses antigen purification sample, and cleaning and regeneration has eluted the chromatography finished
Column；C, and so on, until after m+n column chromatography column resin filler regeneration ending, with the chromatography layer after equilibrium liquid balance regeneration
Analysis column continues on for adsorbing FMD inactivation of viruses antigen samples；
C, after the n-th column chromatography column resin filler regeneration ending, as after all (n+m) root chromatogram columns after being balanced using equilibrium liquid
The 1st root chromatogram column continue on for adsorbing FMD inactivation of viruses antigen samples, circulation S2 operation is until FMD inactivation of viruses antigen sample
Product purifying finishes；
The n=5, m=7.
The chromatograph packing material loaded in the simulation thermopnore chromatography column is with the polymerization of styrene and divinylbenzene
The filler that object is matrix, tert-butyl is aglucon；The partial size of filler is 20um, aperture
The length of the spectrum chromatographic column of the color is 60cm.
The equilibration buffer is pH7~9, the ammonium chloride buffer of 100~300ms/cm of conductivity；
The washing impurity buffer is pH7~9, the ammonium chloride buffer of 100~300ms/cm of conductivity；
The elution solution used that elutes is pH7~9, the ammonium chloride buffer of 5~300ms/cm of conductivity；
The polysorbas20 that the regeneration washing liquid that the cleaning and regeneration uses is 0.2-0.3% (V/V) for concentration.
The chromatography column chromatography technological temperature is 2-15 DEG C.
The purity of FMD inactivation of viruses antigen samples after using the present embodiment method to isolate and purify is 95%, active antigens
The rate of recovery is continuously monitored for 98% using the present embodiment method, is compared 50 times as a result, CV value is less than 5%.Illustrate using this
The chromatographic column of invention is greatly improved its service life.
Embodiment 4
Present embodiments provide a kind of method that application simulation thermopnore isolates and purifies FMD inactivation of viruses antigen, including with
Lower step:
S1, with the chromatography column of equilibrating buffer balance simulation thermopnore；
S2, FMD inactivation of viruses antigen samples loading is extremely simulated in thermopnore chromatography column, it can be simultaneously during loading
The cleaning of antigen samples impurity is carried out to chromatography column and sample desorption elution, the cleaning and regeneration and equilibrium liquid of chromatograph packing material are flat
Weighing apparatus, collects sample after purification in continuous desorption elution process.
The specific steps of the S2 are as follows:
A, it is detected by FMD inactivation of viruses antigen samples loading into simulation thermopnore chromatography column, during loading every
Root chromatography column efflux, when detecting FMD inactivation of viruses antigen efflux and loading in n-th chromatography column efflux
When the UV of sample absorbs consistent, then feed liquid is distributed to (n+1)th chromatography column, while to the color for being saturated adsorption antigen
It composes chromatographic column and carries out sample washing impurity and sample desorption acquisition FMD inactivation of viruses antigen purification sample；
B, when detecting FMD inactivation of viruses antigen efflux and loading sample in (n+1)th chromatography column efflux
When UV absorbs consistent, feed liquid is distributed to the n-th+2 chromatography columns, at the same to be saturated the chromatography column of adsorption antigen into
Row sample washs impurity and sample desorption obtains inactivation of viruses antigen purification sample, and cleaning and regeneration has eluted the chromatography finished
Column；C, and so on, until after m+n column chromatography column resin filler regeneration ending, with the chromatography layer after equilibrium liquid balance regeneration
Analysis column continues on for adsorbing FMD inactivation of viruses antigen samples；
C, after the n-th column chromatography column resin filler regeneration ending, as after all (n+m) root chromatogram columns after being balanced using equilibrium liquid
The 1st root chromatogram column continue on for adsorbing FMD inactivation of viruses antigen samples, circulation S2 operation is until FMD inactivation of viruses antigen sample
Product purifying finishes；
The n=7, m=11.
The chromatograph packing material loaded in the simulation thermopnore chromatography column is with the polymerization of styrene and divinylbenzene
Object is matrix, the filler that diethylamino ethyl is aglucon；The partial size of filler is 20um, aperture
The length of the spectrum chromatographic column of the color is 10cm.
The equilibration buffer is pH7~9, the sodium phosphate buffer of 5~300ms/cm of conductivity；
The washing impurity buffer is pH7~9, the sodium phosphate buffer of 5~300ms/cm of conductivity；
The elution solution used that elutes is pH7~9, the sodium phosphate buffer of 100~300ms/cm of conductivity；
The polysorbas20 that the regeneration washing liquid that the cleaning and regeneration uses is 0.2-0.3% (V/V) for concentration.
The chromatography column chromatography technological temperature is 2-15 DEG C.
For the purity of FMD inactivation of viruses antigen samples after using the present embodiment method to isolate and purify for 96.5%, activity is anti-
The former rate of recovery is continuously monitored for 97% using the present embodiment method, is compared 50 times as a result, CV value is less than 5%.Illustrate to use
Chromatographic column of the invention is greatly improved its service life.
Embodiment 5
Present embodiments provide a kind of method that application simulation thermopnore isolates and purifies FMD inactivation of viruses antigen, including with
Lower step:
S1, with the chromatography column of equilibrating buffer balance simulation thermopnore；
S2, FMD inactivation of viruses antigen samples loading is extremely simulated in thermopnore chromatography column, it can be simultaneously during loading
The cleaning of antigen samples impurity is carried out to chromatography column and sample desorption elution, the cleaning and regeneration and equilibrium liquid of chromatograph packing material are flat
Weighing apparatus, collects sample after purification in continuous desorption elution process.
The specific steps of the S2 are as follows:
A, it is detected by FMD inactivation of viruses antigen samples loading into simulation thermopnore chromatography column, during loading every
Root chromatography column efflux, when detecting FMD inactivation of viruses antigen efflux and loading in n-th chromatography column efflux
When the UV of sample absorbs consistent, then feed liquid is distributed to (n+1)th chromatography column, while to the color for being saturated adsorption antigen
It composes chromatographic column and carries out sample washing impurity and sample desorption acquisition FMD inactivation of viruses antigen purification sample；
B, when detecting FMD inactivation of viruses antigen efflux and loading sample in (n+1)th chromatography column efflux
When UV absorbs consistent, feed liquid is distributed to the n-th+2 chromatography columns, at the same to be saturated the chromatography column of adsorption antigen into
Row sample washs impurity and sample desorption obtains inactivation of viruses antigen purification sample, and cleaning and regeneration has eluted the chromatography finished
Column；C, and so on, until after m+n column chromatography column resin filler regeneration ending, with the chromatography layer after equilibrium liquid balance regeneration
Analysis column continues on for adsorbing FMD inactivation of viruses antigen samples；
C, after the n-th column chromatography column resin filler regeneration ending, as after all (n+m) root chromatogram columns after being balanced using equilibrium liquid
The 1st root chromatogram column continue on for adsorbing FMD inactivation of viruses antigen samples, circulation S2 operation is until FMD inactivation of viruses antigen sample
Product purifying finishes；
The n=15, m=9.
The chromatograph packing material loaded in the simulation thermopnore chromatography column is with the polymerization of styrene and divinylbenzene
The filler that object is matrix, sulfonic group is aglucon；The partial size of filler is 10um, aperture
The length of the spectrum chromatographic column of the color is 30cm.
The equilibration buffer is pH7~9, the ammonium chloride buffer of 5~300ms/cm of conductivity；
The washing impurity buffer is pH7~9, the ammonium chloride buffer of 5~300ms/cm of conductivity；
The elution solution used that elutes is pH7~9, the ammonium chloride buffer of 100~300ms/cm of conductivity；
The polysorbas20 that the regeneration washing liquid that the cleaning and regeneration uses is 0.2-0.3% (V/V) for concentration.
The chromatography column chromatography technological temperature is 2-15 DEG C.
The purity of FMD inactivation of viruses antigen samples after using the present embodiment method to isolate and purify is 90%, active antigens
The rate of recovery is continuously monitored for 95% using the present embodiment method, is compared 50 times as a result, CV value is less than 5%.Illustrate using this
The chromatographic column of invention is greatly improved its service life.
Embodiment 6
Present embodiments provide a kind of method that application simulation thermopnore isolates and purifies FMD inactivation of viruses antigen, including with
Lower step:
S1, with the chromatography column of equilibrating buffer balance simulation thermopnore；
S2, FMD inactivation of viruses antigen samples loading is extremely simulated in thermopnore chromatography column, it can be simultaneously during loading
The cleaning of antigen samples impurity is carried out to chromatography column and sample desorption elution, the cleaning and regeneration and equilibrium liquid of chromatograph packing material are flat
Weighing apparatus, collects sample after purification in continuous desorption elution process.
The specific steps of the S2 are as follows:
A, it is detected by FMD inactivation of viruses antigen samples loading into simulation thermopnore chromatography column, during loading every
Root chromatography column efflux, when detecting FMD inactivation of viruses antigen efflux and loading in n-th chromatography column efflux
When the UV of sample absorbs consistent, then feed liquid is distributed to (n+1)th chromatography column, while to the color for being saturated adsorption antigen
It composes chromatographic column and carries out sample washing impurity and sample desorption acquisition FMD inactivation of viruses antigen purification sample；
B, when detecting FMD inactivation of viruses antigen efflux and loading sample in (n+1)th chromatography column efflux
When UV absorbs consistent, feed liquid is distributed to the n-th+2 chromatography columns, at the same to be saturated the chromatography column of adsorption antigen into
Row sample washs impurity and sample desorption obtains inactivation of viruses antigen purification sample, and cleaning and regeneration has eluted the chromatography finished
Column；C, and so on, until after m+n column chromatography column resin filler regeneration ending, with the chromatography layer after equilibrium liquid balance regeneration
Analysis column continues on for adsorbing FMD inactivation of viruses antigen samples；
C, after the n-th column chromatography column resin filler regeneration ending, as after all (n+m) root chromatogram columns after being balanced using equilibrium liquid
The 1st root chromatogram column continue on for adsorbing FMD inactivation of viruses antigen samples, circulation S2 operation is until FMD inactivation of viruses antigen sample
Product purifying finishes；
The n=5, m=7.
The chromatograph packing material loaded in the simulation thermopnore chromatography column is with the polymerization of styrene and divinylbenzene
The filler that object is matrix, diethyl amino propyl is aglucon；The partial size of filler is 20um, aperture
The length of the spectrum chromatographic column of the color is 60cm.
The equilibration buffer is pH7~9, the ammonium sulphate buffer of 5~300ms/cm of conductivity；
The washing impurity buffer is pH7~9, the ammonium sulphate buffer of 5~300ms/cm of conductivity；
The elution solution used that elutes is pH7~9, the ammonium sulphate buffer of 100~300ms/cm of conductivity；
The polysorbas20 that the regeneration washing liquid that the cleaning and regeneration uses is 0.2-0.3% (V/V) for concentration.
The chromatography column chromatography technological temperature is 2-15 DEG C.
The purity of FMD inactivation of viruses antigen samples after using the present embodiment method to isolate and purify is 95%, active antigens
The rate of recovery is continuously monitored for 95% using the present embodiment method, is compared 50 times as a result, CV value is less than 5%.Illustrate using this
The chromatographic column of invention is greatly improved its service life.
Embodiment 7
Present embodiments provide a kind of method that application simulation thermopnore isolates and purifies FMD inactivation of viruses antigen, the step
Rapid substantially the same manner as Example 3, the difference is that only: the filler that the present embodiment uses is silica rigidity agar glycosyl
Material, the filler that tert-butyl is aglucon.
The purity of FMD inactivation of viruses antigen samples after using the present embodiment method to isolate and purify is 88%, active antigens
The rate of recovery is continuously monitored for 80% using the present embodiment method, is compared 40 times as a result, CV value is less than 5%.Illustrate using this
The chromatographic column of invention is greatly improved its service life.
Embodiment 8
Present embodiments provide a kind of method that application simulation thermopnore isolates and purifies FMD inactivation of viruses antigen, the step
Rapid substantially the same manner as Example 3, the difference is that only: the filler that the present embodiment uses is for acrylate base-material, tert-butyl
The filler of aglucon.
The purity of FMD inactivation of viruses antigen samples after using the present embodiment method to isolate and purify is 80%, active antigens
The rate of recovery is continuously monitored for 85% using the present embodiment method, is compared 45 times as a result, CV value is less than 5%.Illustrate using this
The chromatographic column of invention is greatly improved its service life.
Embodiment 9
Present embodiments provide a kind of method that application simulation thermopnore isolates and purifies FMD inactivation of viruses antigen, the step
Rapid substantially the same manner as Example 3, the difference is that only: the filler that the present embodiment uses is with styrene and divinylbenzene
The filler that polymer is matrix, normal-butyl is aglucon.
The purity of FMD inactivation of viruses antigen samples after using the present embodiment method to isolate and purify is 90%, active antigens
The rate of recovery is continuously monitored for 89% using the present embodiment method, is compared 50 times as a result, CV value is less than 5%.Illustrate using this
The chromatographic column of invention is greatly improved its service life.
Embodiment 10
Present embodiments provide a kind of method that application simulation thermopnore isolates and purifies FMD inactivation of viruses antigen, the step
Rapid substantially the same manner as Example 3, the difference is that only: the filler that the present embodiment uses is with styrene and divinylbenzene
The filler that polymer is matrix, phenyl is aglucon.
The purity of FMD inactivation of viruses antigen samples after using the present embodiment method to isolate and purify is 93%, active antigens
The rate of recovery is continuously monitored for 90% using the present embodiment method, is compared 60 times as a result, CV value is less than 5%.Illustrate using this
The chromatographic column of invention is greatly improved its service life.
Embodiment 11
Present embodiments provide a kind of method that application simulation thermopnore isolates and purifies FMD inactivation of viruses antigen, the step
Rapid substantially the same manner as Example 6, the difference is that only: the filler that the present embodiment uses is silica rigidity agar glycosyl
Material, the filler that diethylamino ethyl is aglucon.
The purity of FMD inactivation of viruses antigen samples after using the present embodiment method to isolate and purify is 80%, active antigens
The rate of recovery is continuously monitored for 75% using the present embodiment method, is compared 38 times as a result, CV value is less than 5%.Illustrate using this
The chromatographic column of invention is greatly improved its service life.
Embodiment 12
Present embodiments provide a kind of method that application simulation thermopnore isolates and purifies FMD inactivation of viruses antigen, the step
Rapid substantially the same manner as Example 6, the difference is that only: the filler that the present embodiment uses is acrylate base-material, diethyl amino
Base ethyl is the filler of aglucon.
The purity of FMD inactivation of viruses antigen samples after using the present embodiment method to isolate and purify is 86%, active antigens
The rate of recovery is continuously monitored for 80% using the present embodiment method, is compared 40 times as a result, CV value is less than 5%.Illustrate using this
The chromatographic column of invention is greatly improved its service life.
Embodiment 13
Present embodiments provide a kind of method that application simulation thermopnore isolates and purifies FMD inactivation of viruses antigen, the step
Rapid substantially the same manner as Example 6, the difference is that only: the filler that the present embodiment uses is with styrene and divinylbenzene
Polymer is matrix, the filler that diethylamino ethyl is aglucon.
The purity of FMD inactivation of viruses antigen samples after using the present embodiment method to isolate and purify is 90%, active antigens
The rate of recovery is continuously monitored for 88% using the present embodiment method, is compared 50 times as a result, CV value is less than 5%.Illustrate using this
The chromatographic column of invention is greatly improved its service life.
Embodiment 14
Present embodiments provide a kind of method that application simulation thermopnore isolates and purifies FMD inactivation of viruses antigen, the step
Rapid substantially the same manner as Example 6, the difference is that only: the filler that the present embodiment uses is with styrene and divinylbenzene
The filler that polymer is matrix, quaternary ammonium is aglucon.
The purity of FMD inactivation of viruses antigen samples after using the present embodiment method to isolate and purify is 87%, active antigens
The rate of recovery is continuously monitored for 85% using the present embodiment method, is compared 55 times as a result, CV value is less than 5%.Illustrate using this
The chromatographic column of invention is greatly improved its service life.
Comparative example 1
This comparative example provides a kind of method that application simulation thermopnore isolates and purifies FMD inactivation of viruses antigen, the side
Method is substantially the same manner as Example 4, the difference is that only: the chromatograph packing material that this comparative example uses are as follows: (no based on agarose matrix
Containing silica) filler, the length of column chromatography column is 30cm.
Through this comparative example method obtain isolate and purify after FMD inactivation of viruses antigen purity be 75%, active antigens return
Yield is 65%.It is continuously monitored, comparison 15 times as a result, CV value is greater than 5%.
Comparative example 2
This comparative example provides a kind of method using conventional chromatogram column separating purification FMD inactivation of viruses antigen, the color
It is identical as in embodiment 1 to compose filler.
Through this comparative example method obtain isolate and purify after FMD inactivation of viruses antigen purity be 80%, active antigens return
Yield is 75%.It is continuously monitored, comparison 25 times as a result, CV value is greater than 5%.
Specific embodiments of the present invention are described above.It is to be appreciated that the invention is not limited to above-mentioned
Particular implementation, those skilled in the art can make a variety of changes or modify within the scope of the claims, this not shadow
Ring substantive content of the invention.In the absence of conflict, the feature in embodiments herein and embodiment can any phase
Mutually combination.
Claims (10)
1. a kind of method that application simulation thermopnore isolates and purifies FMD inactivation of viruses antigen, which is characterized in that including following step
It is rapid:
S1, with the chromatography column of equilibrating buffer balance simulation thermopnore；
S2, FMD inactivation of viruses antigen samples loading is extremely simulated in thermopnore chromatography column, it can be simultaneously to color during loading
It composes chromatographic column progress antigen samples impurity cleaning and sample desorption elution, the cleaning and regeneration and equilibrium liquid of chromatograph packing material balances,
Sample after purification is collected in continuous desorption elution process.
2. the method that application simulation thermopnore according to claim 1 isolates and purifies FMD inactivation of viruses antigen, feature exist
In the specific steps of the S2 are as follows:
A, FMD inactivation of viruses antigen samples loading is detected into every color into simulation thermopnore chromatography column during loading
Chromatographic column effluent liquid is composed, when detecting FMD inactivation of viruses antigen efflux and loading sample in n-th chromatography column efflux
UV absorb it is consistent when, then feed liquid is distributed to (n+1)th chromatography column, while to the chromatography layer for being saturated adsorption antigen
It analyses column and carries out sample washing impurity and sample desorption acquisition FMD inactivation of viruses antigen purification sample；
B, when the UV suction for detecting FMD inactivation of viruses antigen efflux and loading sample in (n+1)th chromatography column efflux
When receiving consistent, feed liquid is distributed to the n-th+2 chromatography columns, while sample is carried out to the chromatography column for being saturated adsorption antigen
Product wash impurity and sample desorption obtains inactivation of viruses antigen purification sample, and cleaning and regeneration has eluted the chromatographic column finished；C,
And so on, until m+n column chromatography column resin filler regeneration ending after, with equilibrium liquid balance regeneration after chromatography column after
Continue for adsorbing FMD inactivation of viruses antigen samples；
D, the step of circulation A is to C, until all purifying finishes FMD inactivation of viruses antigen samples；
Wherein, the n >=1, n+m > n, n and m are integer, the chromatography column number that time feed liquid distribution enters headed by n；N+m is
The total chromatography column number of system.
3. the method that application simulation thermopnore according to claim 2 isolates and purifies FMD inactivation of viruses antigen, feature exist
In 6≤n+m≤24.
4. the method that application simulation thermopnore according to claim 1 isolates and purifies FMD inactivation of viruses antigen, feature exist
In the chromatograph packing material loaded in the simulation thermopnore chromatography column is with hydrophobic or ionic aglucon high-intensitive polymerization
Object filler.
5. the method that application simulation thermopnore according to claim 4 isolates and purifies FMD inactivation of viruses antigen, feature exist
In described to have hydrophobic or ionic aglucon high-strength polymer filler are as follows: with acrylate, styryl benzene, divinyl
At least one of base benzene, styrene, silica rigidity agarose, silica rigidity glucan or rigid fiber element are base
Matter, with hydroxypropyl, propyl, benzyl, isopropyl, phenyl, normal-butyl, tert-butyl, butylthio, amyl, octyl, diethylamino
At least one of ethyl, sulfonic group, quaternary ammonium, carboxymethyl, diethyl amino propyl are the filler of aglucon.
6. the method that application simulation thermopnore according to claim 4 isolates and purifies FMD inactivation of viruses antigen, feature exist
In described to have hydrophobic or ionic aglucon high-strength polymer filler to be with the polymer of styrene and divinylbenzene
The filler of matrix.
7. according to the method that the described in any item application simulation thermopnores of claim 4-6 isolate and purify FMD inactivation of viruses antigen,
It is characterized in that, the partial size with hydrophobic or ionic aglucon high-strength polymer filler is 3um~20um, aperture
8. the method that application simulation thermopnore according to claim 1 or 2 isolates and purifies FMD inactivation of viruses antigen, special
Sign is, using the filler of hydrophobic type aglucon, the equilibration buffer is pH7~9, the sulphur of 100~300ms/cm of conductivity
Sour ammonium, sodium sulphate or ammonium chloride buffer；Using the filler of ionic aglucon, the equilibration buffer is pH7~9, conductance
Ammonium sulfate, sodium sulphate or the ammonium chloride buffer of 5~300ms/cm of rate.
9. the method that application simulation thermopnore according to claim 1 or 2 isolates and purifies FMD inactivation of viruses antigen, special
Sign is, using the filler of hydrophobic type aglucon, the elution solution used that elutes is pH7~9,5~300ms/cm of conductivity
Ammonium sulfate, sodium sulphate or ammonium chloride buffer；Using the filler of ionic aglucon, it is described elute the elution solution that uses for
PH7~9, ammonium sulfate, sodium sulphate or the ammonium chloride buffer of 100~300ms/cm of conductivity.
10. the method that application simulation thermopnore according to claim 1 or 2 isolates and purifies FMD inactivation of viruses antigen, special
Sign is that the regeneration washing liquid that the cleaning and regeneration uses is molten for sodium hydroxide solution, ethanol solution, methanol solution, isopropanol
At least one of liquid, urea liquid, neutral interface surface activating agent.
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| CN110499287A (en) * | 2019-08-30 | 2019-11-26 | 博雅干细胞科技有限公司 | The method for simply preparing placenta mesenchyma stem cell excretion body |
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| CN110499287A (en) * | 2019-08-30 | 2019-11-26 | 博雅干细胞科技有限公司 | The method for simply preparing placenta mesenchyma stem cell excretion body |
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| CN115121035A (en) * | 2022-06-21 | 2022-09-30 | 永华化学股份有限公司 | Method for preparing ultra-dry reagent by using ISEP system |
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