CN109535222A - A kind of method and application thereof improving ion-exchange chromatography endotoxin removal efficiency - Google Patents
A kind of method and application thereof improving ion-exchange chromatography endotoxin removal efficiency Download PDFInfo
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Abstract
The present invention relates to a kind of methods and application thereof for the endotoxin removal efficiency adsorbed on raising ion exchange column, described method includes following steps: (1) in the ion exchange column that will be loaded by removing depyrogenation that treated containing target product and endotoxic material liquid, the target product Electrostatic Absorption is on the ion exchange column；(2) it is eluted with apirogen water and/or buffer；(3) it is eluted with the buffer containing nonionisable substance；(4) it is eluted once again with apirogen water or buffer；(5) elution ionic exchange column obtains target product.Wherein, target product include in protein, nucleic acid and polysaccharide any one or at least two combination, nonionisable substance has both hydrophilic structure and hydrophobic structure, and can be with endotoxin Non-covalent binding.In the identical situation of other conditions, the endotoxin content in target product finally eluted is 10% or less the level of endotoxin handled in product obtained without the present invention.
Description
Technical field
The present invention relates to biopharmaceutical technology more particularly to a kind of raising ion-exchange chromatography endotoxin removal efficiency
Method and application thereof.
Background technique
The safety of biotech drug is that an important performance assessment criteria, major influence factors are removed in its development process
Except drug itself physicochemical property, downstream preparation process is to determine its key point.It is opened in biotech drug downstream process
In hair, the removal degree of impurity and the safety of its final preparation are closely related, and endotoxic contents level control among these is
One of technology overcome is most difficult in the exploitation of biotech drug downstream technique.The related request of international and national, which has, clearly advises
It is fixed, it is considered that not have the contents level of doses of lipopolysaccharide to should be less than 5EU.
Endotoxin, which refers to, can cause the big substance of body temperature raised one, and in biotech drug, endotoxin is most absolutely
What is referred to when number is lipopolysaccharides, also known as endotoxin.Lipopolysaccharides is gram-negative bacterial cell wall constituent.Biotechnology
In pharmaceutical synthesis design, the Escherichia coli in gramnegative bacterium are often chosen for the expression system of target product, i.e., source and
Expression system, such as recombination protein expression or nucleic acid/plasmid amplification.The maximum advantage of coli expression system is it
Growth rate is fast, and division cycle is short, and condition of culture is simple, and nutritional ingredient requires low, target product expression quantity height etc..
It include at present interferon (IFN), granular leukocyte colony by the recombination pharmaceutical protein that coli expression system synthesizes
Stimulating factor (G-CSF), tumor necrosis factor (TNF), cell factors and the asparagine such as hematopoietin (EPO)
The enzymes albumen such as enzyme, uricase.Compared to other recombinant expression systems, the rouge of Escherichia coli own cells wall ingredient high-content is more
Sugared ingredient requires downstream purification removal technique high.
Lipopolysaccharides is the compound of a kind of lipid and polysaccharide, and structure is made of three parts.Lipid A (Lipid A) is structure
At the glycolipid of activity of endotoxin, heteroglycan chain is connected to covalent bond, another two parts: first is that core polysaccharide；Another O specificity chain
(O-specific chain).Due to the particularity of its own structure, i.e., containing hydrophobic interaction and a variety of purification medias
Such as hydrophobic interaction chromatograph chromatography media (phenyl, butyl, Xinji) and anionic exchange medium (Q-Sepharose,
DEAE-Sepharose, POROS HQ etc.) high carrying capacity absorption occurs.Furthermore lipopolysaccharides can equally be adsorbed in cation exchange and be situated between
In matter, although its adsorption mechanism is not still fully apparent from.
In downstream preparation process exploitation, in order to remove lipopolysaccharide components, target product and its physicochemical property are usually utilized
On difference come realize separation, wherein ion exchange column is the most commonly used.Using anion-exchange chromatography, target product is usually with stream
The mode (Flow-Through) worn flows through chromatographic column, and lipopolysaccharides is usually adsorbed on anionic exchange medium, reaches separation
Effect.And in cation-exchange chromatography, target product is usually adsorbed in purification media (Bind-Elute), lipopolysaccharides
It is not easy to be adsorbed due to its negatively charged, final lipopolysaccharides is flowed through removal.However existing greatest problem is exactly not to be
All albumen all meets this condition, is applicable in such mode.It seemingly such as most recombinant proteins and liopopolysaccharides, can
Negative electrical charge is enough had, it can be in conjunction with anion-exchange chromatography medium；Same many recombinant proteins are also difficult to hand over cation
Chromatography media combination is changed, polysaccharide is the same therewith is spread by medium, is finally difficult to realize separate.What is more, nearest research
Although showing that binding mechanism is imperfectly understood, lipopolysaccharides and cation exchange medium also can adsorb therewith combination with high load amount,
New challenge is constituted with the mode that Bind-Elute mode removes lipopolysaccharides for cation exchange medium.
Summary of the invention
In view of problems of the prior art, one of the objects of the present invention is to provide a kind of raising endotoxin removal effects
The method with universality of rate realizes that endotoxin is removed by specificity, and target product yield is unaffected.
For this purpose, the present invention adopts the following technical scheme:
In a first aspect, the present invention provides a kind of side of endotoxin removal efficiency for improving and adsorbing on ion exchange column
Method includes the following steps:
(1) it is 10mS/cm hereinafter, and by pH value tune that conductivity will be diluted to containing target product and endotoxic material liquid
Section is is suitble to after target product is adsorbed in the pH value of Ion Exchange Medium, the ion exchange column that is loaded by removing depyrogenation that treated
In, by the target product Electrostatic Absorption on the ion exchange column；
(2) ion exchange column is eluted with apirogen water and/or buffer；
(3) ion exchange column is eluted with the buffer containing nonionisable substance again；
(4) ion exchange column is then eluted once again with apirogen water or buffer；
(5) ion exchange column is eluted, target product is obtained.
Wherein, the target product include in protein, nucleic acid and polysaccharide any one or at least two combination.
The nonionisable substance has both hydrophilic structure and hydrophobic structure, and can be with endotoxin Non-covalent binding.
By the way that target product to be adsorbed on ion-exchange chromatography media by electrostatic interaction, then using certain dense
The nonionisable substance of degree elutes chromatographic column, some non-ionic type substance, such as Triton X-100 due to its without
It is charge, it can hardly be by electrostatic interaction in conjunction with ion-exchange chromatography media and recombinant protein, but due to it
Equally contain water repellent region in self structure, therefore a degree of hydrophobic interaction can occur with lipopolysaccharides, and egg
The hydrophobic region of white matter is often wrapped in protein interior, and protein surface is mostly hydrophily, therefore such non-ionic surface is living
Property agent is similarly difficult in conjunction with protein.Based on this hydrophobic interaction principle, there is research in the nickel parent of protein at present
Nonionic surface active agent is used to carry out the elution of lipopolysaccharides in chromatography process, but it requires protein can be with medium
In conjunction with this is difficult to be applicable in for most of no affinity tag recombinant protein.In view of nonionisable substance is difficult to by quiet
Electric interactions and Ion Exchange Medium adsorb, and most albumen can adsorb thereon.Based in this physicochemical property
Protein can be adsorbed on ion-exchange chromatography media by difference by electrostatic interaction, then use non-ionic object
Matter elution, realize lipopolysaccharides by specificity elution removal and protein be still adsorbed in the principle on column realize target product with
Lipopolysaccharides efficiently separates.
Preferably, the protein includes the protein extracted in recombinant protein and/or natural goods.
Preferably, the protein includes cell factor albuminoid and/or enzyme albumen.
Preferably, the cell factor albuminoid include interferon, granulocyte colony stimulating factor, tumor necrosis factor and
Any one at least two combination in hematopoietin.
Preferably, the enzyme albumen includes asparaginase and/or uricase.
Preferably, the ion exchange column includes anion-exchange column and/or cation exchange column.
Preferably, step (1) described material liquid includes natural thallus, n cell, recombination to construct expression cellular lysate liquid
With any one at least two combination in the lysate of recombination to construct expression cell.
Preferably, the recombination to construct expression cellular lysate liquid includes Escherichia coli bacteria break supernatant liquid.
Preferably, the method for step (1) described loading includes that peristaltic pump is loaded into, in chromatography loading and tomographic system loading
Any one or at least two combination.
Preferably, the conductivity of step (2) described buffer is 0~30mS/cm, such as 0mS/cm, 1mS/cm, 2mS/
cm、3mS/cm、4mS/cm、5mS/cm、6mS/cm、8mS/cm、10mS/cm、12mS/cm、15mS/cm、18mS/cm、20mS/
Cm, 22mS/cm, 25mS/cm, 28mS/cm or 30mS/cm etc., preferably 0~5mS/cm.
Preferably, in the buffer containing nonionisable substance described in step (3), the percentage by volume of nonionisable substance is
0.1~5%, for example, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%,
1.2%, 1.5%, 2%, 2.2%, 2.5%, 2.8%, 3%, 3.2%, 3.5%, 3.8%, 4%, 4.2%, 4.5%, 4.8%
Or 5% etc., preferably 0.1~1.0%.
Preferably, the obturator of step (3) buffer containing nonionisable substance and the ion exchange column
Long-pending volume ratio is (1~100): 1, such as 1:1,2:1,5:1,10:1,12:1,15:1,18:1,20:1,25:1,30:1,35:
1,40:1,45:1,50:1,55:1,60:1,65:1,70:1,75:1,80:1,85:1 or 90:1 etc., preferably (1~20): 1.
Preferably, the difference △ of the pH of buffer and step (2) described buffer containing nonionisable substance described in step (3)
PH meets | △ pH | it is less than or equal to 0.5, such as 0.5,0.45,0.4,0.3,0.2,0.1,0.05 or 0 etc., is preferably smaller than equal to
0.2。
Preferably, the conductivity of step (3) buffer and step (2) described buffer containing nonionisable substance it
Than for (0~10): 1, such as 0:1,0.2:1,0.5:1,0.6:1,0.7:1,0.8:1,0.9:1,1:1,1.1:1,1.2:1,
1.3:1,1.4:1,1.5:1,1.6:1,1.7:1,1.8:1,1.9:1,2:1,3:1,4:1,5:1,6:1,7:1,8:1,9:1 or
10:1 etc., preferably (0.5~2): 1.
Preferably, in the buffer containing nonionisable substance described in step (3), nonionisable substance includes that polyethylene glycol is pungent
Base phenyl ether (Triton X-100), polyethyleneglycol t-octyl phenyl ether (Triton X-114), polysorbate esters of gallic acid
(Tween-20/40/60/80) any one and in Span (Span-20/40/60/80) or at least two
Combination, wherein typical but non-limiting combination are as follows: Triton X-100 (Triton X-100) and polyethyleneglycol
The combination of t-octyl phenyl ether (Triton X-114), Triton X-100 (Triton X-100) and polysorbate acid
The combination of esters (Tween-20/40/60/80), polyethyleneglycol t-octyl phenyl ether (Triton X-114) and sorb alcohol ester
The combination of fat acid esters (Span-20/40/60/80), preferably Triton X-100 and/or Triton X-114.
Preferably, the eluent of step (5) described elution includes high concentration salt solutions, salt in the high concentration salt solutions
Mass-volume concentration be 0.1~2.0M, such as 0.1M, 0.2M, 0.5M, 0.6M, 0.8M, 0.9M, 1.0M, 1.2M, 1.3M,
1.5M, 1.8M, 1.9M or 2.0M etc., preferably 0.8~1.2M.
Preferably, in the high concentration salt solutions salt include in sodium chloride, ammonium sulfate and sodium sulphate any one or extremely
Few two kinds of combination, wherein typical but non-limiting combination includes: the combination of sodium chloride and ammonium sulfate, sodium chloride and sodium sulphate
Combination, the combination of ammonium sulfate and sodium sulphate, the combination of sodium chloride, ammonium sulfate and sodium sulphate.
Preferably, the mode of step (5) described elution includes linear gradient elution or step gradient elution.
As currently preferred technical solution, described method includes following steps:
(1) it is 10mS/cm hereinafter, and by pH value tune that conductivity will be diluted to containing target product and endotoxic material liquid
It after section is is suitble to target product to be adsorbed in the pH value of Ion Exchange Medium, is loaded by peristaltic pump, chromatography is loaded into and tomographic system
In any one or at least two combination ion exchange column that is loaded by removing depyrogenation that treated, the target product
Electrostatic Absorption is on the ion exchange column；Wherein, the target product includes any one in protein, nucleic acid and polysaccharide
Or at least two combination；
(2) ion exchange column is eluted with the buffer that apirogen water and/or conductivity are 0~30mS/cm；
(3) ion exchange column is eluted with the buffer that percentage by volume containing nonionisable substance is 0.1~5% again；
The volume ratio of the packing volume of the buffer containing nonionisable substance and the ion exchange column is (1~100): 1；
The difference △ pH of the pH of described buffer and step (2) described buffer containing nonionisable substance meets | △ pH | it is less than or equal to
0.5, the ratio between conductivity is (0~10): 1；The nonionisable substance includes Triton X-100, polyethyleneglycol uncle
In octyl phenyl ether, polysorbate esters of gallic acid and Span any one or at least two combination；
(4) ion exchange column is then eluted once again with apirogen water or buffer；
(5) ion exchange column is eluted with the high concentration salt solutions that the mass-volume concentration of salt is 0.1~2.0M, obtained
Target product.
Second aspect, the present invention provides the purposes of method as described in relation to the first aspect, the method is used for biotechnology medicine
Object downstream preparation process and bio-pharmaceuticals purifying process.
Compared with prior art, the present invention at least has the following beneficial effects:
1. the method through the invention, in the identical situation of other conditions, the target product that finally elutes
In endotoxin content be 10% or less the level of endotoxin handled without the present invention in product obtained.
2. the method for the invention process is simple, easily operated, time-consuming shorter, relatively cheap, wide application.
Detailed description of the invention
Fig. 1 is the representative thin layer chromatography figure that 1 method of the embodiment of the present invention is applied to anion-exchange chromatography process；
Fig. 2 is the representative thin layer chromatography figure that 2 method of the embodiment of the present invention is applied to anion-exchange chromatography process；
Fig. 3 is Q-Sepharose in the embodiment of the present invention 1, and tri- kinds of anion of DEAE-Sepharose and POROS HQ are handed over
It is final to elute pyrogen content residual comparison diagram in protein liquid when changing chromatographic purifying recombination human ciliary neurotrophy factor albumen；
Fig. 4 is SP-Sepharose in the embodiment of the present invention 2, tri- kinds of cation exchanges of CM-Sepharose and POROS HS
It is final to elute pyrogen content residual comparison diagram in protein liquid when chromatographic purifying recombinant human tumor necrosis factor's albumen
Specific embodiment
To further illustrate the technical scheme of the present invention below with reference to the accompanying drawings and specific embodiments.But following reality
Example is only simple example of the invention, does not represent or limit the scope of the present invention, protection scope of the present invention
It is subject to claims.
Embodiment 1
It is eluted during anion-exchange chromatography purifying effects of recombinant ciliary neurotrophic factor using Triton X-100
Remove endotoxin:
(1) effects of recombinant ciliary neurotrophic factor is expressed: the Escherichia coli containing rhCNTF plasmid are fermented by 20-L, when
When OD600nm value reaches 6.0 or more, 1.0mM IPTG induction rhCNTF expression is added, harvests thallus after 4 hours；20mM
Harvest thallus is resuspended in 10% ratio (mass/volume ratio) in Tris-HCl buffer, after 3 circulation of high pressure homogenizer homogenate,
Centrifugation obtains bacteria break supernatant liquid.
(2) Q-Sepharose column chromatographs:
Step 1: the bacteria break supernatant liquid of appropriate amount is loaded into the Q- crossed through naoh treatment using protein chromatography system
After Sepharose column, 10 cylinders are eluted using sample-loading buffer (20mM Tris-HCl pH8.0, conductance 1.0mS/cm)
After product；Detector Detection wavelength is set as 260nm and 280nm；
Step 2: the 20mM Tris-HCl pH8.0 buffer elution containing 0.5% volume Triton X-100 is reused
The buffer of 20 column volumes；
Step 3: after reusing sample-loading buffer 20 column volumes of elution, 30%1.0M NaCl 20mM is finally used
Tris-HCl pH8.0 buffer is with the buffer solution for gradient elution target protein of 5 column volumes.
Under the same conditions simultaneously, the 20mM of loading same protein amount and elution same volume (50 column volumes)
Tris-HCl pH8.0 buffer and omit other steps for control, finally eluted with same type of elution.
(3) DEAE-Sepharose column chromatographs:
Step 1: the bacteria break supernatant liquid of appropriate amount is loaded into the Q- crossed through naoh treatment using protein chromatography system
After Sepharose column, after eluting 10 column volumes using sample-loading buffer (20mM Tris-HCL pH8.0)；Detector detection
Wavelength is set as 260nm and 280nm；
Step 2: it reuses the 20mM Tris-HCl pH8.0 buffer containing 0.5%Triton X-100 and elutes 20
The buffer of column volume；
Step 3: after reusing sample-loading buffer 20 column volumes of elution, 30%1.0M NaCl20mM is finally used
Tris-HCl pH8.0 buffer is with the buffer solution for gradient elution target protein of 5 column volumes.
Under the same conditions simultaneously, the 20mM Tris- of loading same protein amount and elution same volume (50 column volumes)
HCl pH8.0 buffer compares the most, is finally eluted with same type of elution.
(4) POROS HQ column chromatographs:
Step 1: the ciliary of above-mentioned appropriate amount is recombinated into neurotrophic factor albumen bacteria break supernatant using protein chromatography system
After liquid is loaded into the POROS HQ column crossed through naoh treatment, eluted using sample-loading buffer (20mM Tris-HCL pH8.0)
After 10 column volumes；Detector Detection wavelength is set as 260nm and 280nm；
Step 2: it reuses the 20mM Tris-HCl pH8.0 buffer containing 0.5%Triton X-100 and elutes 20
The buffer of column volume；
Step 3: after reusing sample-loading buffer 20 column volumes of elution, 30%1.0M NaCl 20mM is finally used
Tris-HCl pH8.0 buffer is with the buffer solution for gradient elution target protein of 5 column volumes.
Under the same conditions simultaneously, the 20mM Tris- of loading same protein amount and elution same volume (50 column volumes)
HCl pH8.0 buffer and omit other steps for control, finally eluted with same type of elution.
(5) endotoxin assay:
Use reagents detection method, reagents λ=0.06EU and λ=0.125EU；Endotoxin standard；Endotoxin detection
Dedicated apirogen water；Purchased from Zhanjiang Andusi Biology Co., Ltd..Detection method: shop instruction is referred to.
Embodiment 2
Elution removal is carried out using Triton X-100 during cation-exchange chromatography purifying recombinant tumor necrosis factor
Endotoxin:
(1) recombinant tumor necrosis factor Escherichia coli system is expressed: the Escherichia coli containing TNF-α plasmid are sent out by 20-L
1.0mM IPTG induction target protein expression is added when OD600nm value reaches 5.5 in ferment, and induction harvested thallus after 4 hours；Make
The thallus of harvest is resuspended in 10% ratio (mass/volume ratio) with 20mM Tris-HCl pH8.0 buffer, through high-pressure homogeneous
Machine homogenate 3 circulation after, centrifugation obtain bacteria break supernatant liquid, using 10% phosphoric acid will be centrifuged resulting supernatant pH value adjust to
PH6.0 or so, centrifugation removal protein precipitation, harvests supernatant again.
(2) SP-Sepharose column chromatographs:
Step 1: the above-mentioned supernatant of appropriate amount is loaded into the SP- crossed through naoh treatment using protein chromatography system
After Sepharose column, sample-loading buffer (20mM Na is used2HPO4/KH2PO4, pH6.0, conductance 2.0mS/cm) and 10 columns of elution
After volume；Detector Detection wavelength is set as 260nm and 280nm；
Step 2: the 20mM Na containing 0.5%Triton X-100 is reused2HPO4/KH2PO4, the leaching of pH6.0 buffer
Wash the buffer of 20 column volumes；
Step 3: after reusing sample-loading buffer 20 column volumes of elution, 100%1.0M NaCl 20mM is finally used
Na2HPO4/KH2PO4, pH6.0 buffer is with the buffer solution for gradient elution target protein of 5 column volumes.
Under the same conditions simultaneously, the 20mM Tris- of loading same protein amount and elution same volume (50 column volumes)
HCl pH8.0 buffer and omit other steps for control, finally eluted with same type of elution.
(3) CM-Sepharose column chromatographs:
Step 1: the above-mentioned supernatant of appropriate amount is loaded into the CM- crossed through naoh treatment using protein chromatography system
After Sepharose column, sample-loading buffer (20mM Na is used2HPO4/KH2PO4, pH6.0) elution 10 column volumes after；Detector
Detection wavelength is set as 260nm and 280nm；
Step 2: the 20mM Na containing 0.5%Triton X-100 is reused2HPO4/KH2PO4, the leaching of pH6.0 buffer
Wash the buffer of 20 column volumes；
Step 3: after reusing sample-loading buffer 20 column volumes of elution, 100%1.0M NaCl 20mM is finally used
Na2HPO4/KH2PO4, pH6.0 buffer is with the buffer solution for gradient elution target protein of 5 column volumes.
Under the same conditions simultaneously, the 20mM Tris- of loading same protein amount and elution same volume (50 column volumes)
HCl pH8.0 buffer and omit other steps for control, finally eluted with same type of elution.
(4) POROS HS column chromatographs:
Step 1: the above-mentioned supernatant loading of appropriate amount is crossed through naoh treatment using protein chromatography system
After POROS HS column, sample-loading buffer (20mM Na is used2HPO4/KH2PO4, pH6.0) elution 10 column volumes after；Detector
Detection wavelength is set as 260nm and 280nm；
Step 2: the 20mM Na containing 0.5%Triton X-100 is reused2HPO4/KH2PO4, the leaching of pH6.0 buffer
Wash the buffer of 20 column volumes；
Step 3: after reusing sample-loading buffer 20 column volumes of elution, 100%1.0M NaCl 20mM is finally used
Na2HPO4/KH2PO4, pH6.0 buffer is with the buffer solution for gradient elution target protein of 5 column volumes.
Under the same conditions simultaneously, the 20mM Tris- of loading same protein amount and elution same volume (50 column volumes)
HCl pH8.0 buffer and omit other steps for control, finally eluted with same type of elution.
(5) endotoxin assay:
Use reagents detection method, reagents λ=0.06EU and λ=0.125EU；Endotoxin standard；Endotoxin detection
Dedicated apirogen water；Purchased from Zhanjiang Andusi Biology Co., Ltd..Detection method: shop instruction is referred to.
Table 1 is Q-Sepharose in embodiment 1, tri- kinds of anion-exchange chromatographies of DEAE-Sepharose and POROS HQ
It is final to elute pyrogen content residual concentration statistical form in protein liquid when purification of recombinant human ciliary neurotrophic factor albumen, wherein
What normal elution indicated is corresponding above-mentioned check experiment.
Table 2 is Q-Sepharose in embodiment 2, tri- kinds of anion-exchange chromatographies of DEAE-Sepharose and POROS HQ
It is final to elute pyrogen content residual concentration statistical form in protein liquid when purification of recombinant human tumor necrosis factor albumen, wherein normally
What elution indicated is corresponding above-mentioned check experiment.
Table 1
Table 2
It can be seen that the method described through the invention from Tables 1 and 2, Fig. 1 and Fig. 2, Fig. 3 and Fig. 4, protein led to
It crosses electrostatic interaction to be adsorbed on ion-exchange chromatography media, then be eluted using nonionisable substance, in other conditions
In identical situation, the endotoxin content in target product finally eluted is well below without Electrostatic Absorption of the present invention
The level of endotoxin in product obtained is eluted with nonionic, or even not in an order of magnitude.
The Applicant declares that the present invention is explained by the above embodiments detailed process equipment and process flow of the invention,
But the present invention is not limited to the above detailed process equipment and process flow, that is, it is above-mentioned detailed not mean that the present invention must rely on
Process equipment and process flow could be implemented.It should be clear to those skilled in the art, any improvement in the present invention,
Addition, selection of concrete mode of equivalence replacement and auxiliary element to each raw material of product of the present invention etc., all fall within of the invention
Within protection scope and the open scope.
Claims (10)
1. a kind of method for improving ion-exchange chromatography endotoxin removal efficiency, which is characterized in that the method includes walking as follows
It is rapid:
(1) conductivity will be diluted to containing target product and endotoxic material liquid is 10mS/cm hereinafter, and being adjusted to pH value
After suitable target product is adsorbed in the pH value of Ion Exchange Medium, in the ion exchange column that is loaded by removing depyrogenation that treated,
The target product Electrostatic Absorption is on the ion exchange column；
(2) ion exchange column is eluted with apirogen water and/or buffer；
(3) ion exchange column is eluted with the buffer containing nonionisable substance again；
(4) ion exchange column is then eluted once again with apirogen water or buffer；
(5) ion exchange column is eluted, target product is obtained；
Wherein, the target product include in protein, nucleic acid and polysaccharide any one or at least two combination；
The nonionisable substance has both hydrophilic structure and hydrophobic structure, and can be with endotoxin Non-covalent binding.
2. the method as described in claim 1, which is characterized in that the protein includes in recombinant protein and/or natural goods
The protein of extraction；
Preferably, the protein includes cell factor albuminoid and/or enzyme albumen；
Preferably, the cell factor albuminoid includes interferon, granulocyte colony stimulating factor, tumor necrosis factor and promotees red
Any one at least two combination in erythropoietin；
Preferably, the enzyme albumen includes asparaginase and/or uricase；
Preferably, the ion exchange column includes anion-exchange column and/or cation exchange column.
3. method according to claim 1 or 2, which is characterized in that step (1) described material liquid includes natural thallus, natural
Any one at least two group in the lysate of cell, recombination to construct expression cellular lysate liquid and recombination to construct expression cell
It closes；
Preferably, the recombination to construct expression cellular lysate liquid includes Escherichia coli bacteria break supernatant liquid.
4. method as claimed in any one of claims 1 to 3, which is characterized in that the method for step (1) described loading includes wriggling
Pump be loaded into, chromatography be loaded into and tomographic system be loaded into any one or at least two combination.
5. such as the described in any item methods of Claims 1 to 4, which is characterized in that the conductivity of step (2) described buffer is 0
~30mS/cm, preferably 0~5mS/cm.
6. method as claimed in any one of claims 1 to 5, which is characterized in that step (3) is described slow containing nonionisable substance
In fliud flushing, the percentage by volume of nonionisable substance is 0.1~5%, preferably 0.1~1.0%；
Preferably, step (3) buffer containing nonionisable substance and the packing volume of the ion exchange column
Volume ratio is (1~100): 1, preferably (1~20): 1.
7. method as described in any one of claims 1 to 6, which is characterized in that step (3) is described slow containing nonionisable substance
The difference △ pH of the pH of fliud flushing and step (2) described buffer meets | △ pH | it is less than or equal to 0.5, is preferably smaller than equal to 0.2；
Preferably, the ratio between the conductivity of buffer and step (2) described buffer containing nonionisable substance described in step (3) is
(0~10): 1, preferably (0.5~2): 1.
8. method as described in any one of claims 1 to 7, which is characterized in that step (3) is described slow containing nonionisable substance
In fliud flushing, nonionisable substance includes Triton X-100, polyethyleneglycol t-octyl phenyl ether, polysorbate acid esters
In class and Span any one or at least two combination, preferred Triton X-100 and/or poly-
Ethylene glycol list t-octyl phenyl ether.
9. method as described in any one of claims 1 to 6, which is characterized in that the eluent of step (5) described elution includes
High concentration salt solutions, the mass-volume concentration of salt is 0.1~2.0M, preferably 0.8~1.2M in the high concentration salt solutions；
Preferably, in the high concentration salt solutions salt include in sodium chloride, ammonium sulfate and sodium sulphate any one or at least two
The combination of kind；
Preferably, the mode of step (5) described elution includes linear gradient elution or step gradient elution.
10. such as the purposes of any one of claim 1~9 the method, which is characterized in that the method is used for biotech drug
Downstream preparation process and bio-pharmaceuticals purifying process.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113000039A (en) * | 2021-03-19 | 2021-06-22 | 北京石油化工学院 | Preparation method of chromatography medium for removing endotoxin in biological nanoparticles |
| CN114452444A (en) * | 2022-01-27 | 2022-05-10 | 陕西巨子生物技术有限公司 | Preparation method of low endotoxin collagen |
-
2017
- 2017-09-21 CN CN201710859565.9A patent/CN109535222A/en not_active Withdrawn
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113000039A (en) * | 2021-03-19 | 2021-06-22 | 北京石油化工学院 | Preparation method of chromatography medium for removing endotoxin in biological nanoparticles |
| CN113000039B (en) * | 2021-03-19 | 2023-07-18 | 北京石油化工学院 | Preparation method of chromatographic medium for removing endotoxin in biological nano-particles |
| CN114452444A (en) * | 2022-01-27 | 2022-05-10 | 陕西巨子生物技术有限公司 | Preparation method of low endotoxin collagen |
| WO2023143391A1 (en) * | 2022-01-27 | 2023-08-03 | 陕西巨子生物技术有限公司 | Method for preparing low-endotoxin collagen |
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