CN107999025A - Affinity chromatography medium using 4- pyrimidine radicals -2- aniline as functional ligand - Google Patents
Affinity chromatography medium using 4- pyrimidine radicals -2- aniline as functional ligand Download PDFInfo
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- CN107999025A CN107999025A CN201711276403.9A CN201711276403A CN107999025A CN 107999025 A CN107999025 A CN 107999025A CN 201711276403 A CN201711276403 A CN 201711276403A CN 107999025 A CN107999025 A CN 107999025A
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- aniline
- chromatography
- pyrimidine radicals
- medium
- functional ligand
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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
- B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
- B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 - B01D15/36
- B01D15/3804—Affinity chromatography
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
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- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Organic Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Solid-Sorbent Or Filter-Aiding Compositions (AREA)
- Treatment Of Liquids With Adsorbents In General (AREA)
Abstract
This case is related to a kind of affinity chromatography medium using 4 pyrimidine radicals, 2 aniline as functional ligand, it is characterized in that, including chromatography substrate and aglucon, the chromatography substrate is macropore beta cyclodextrin microballoon, and aglucon is 4 pyrimidine radicals, 2 aniline being coupled after the activation of diglycol bisglycidyl ether.The new chromatography media that the present invention is developed, using 4 pyrimidine radicals, 2 aniline as functional ligand, has significant hydrophobic charge-induction chromatography separation characteristic, has very high adsorption capacity to antibody, can be used for the prepare with scale of antibody.
Description
Technical field
The present invention relates to a kind of using 4- pyrimidine radicals -2- aniline as the affinity chromatography medium of functional ligand, belong to biochemical industry
Protein chromatographic isolation technics in field.
Background technology
Antibody can be specifically bound with corresponding antigens, produce various immunological effects.With biotechnology fast development and
Antibody engineering technology is constantly broken through, and monoclonal antibody, polyclonal antibody and genetic engineering antibody have been widely used for biology
And medical domain.Antibody purposes mainly includes disease treatment, in-vitro diagnosis and detection, tumor-localizing imaging and as affine
Aglucon is used to isolate and purify.
Often purity requirement is higher for antibody product, while must also keep bioactivity, therefore traditional separation process is past
It is past to be difficult to meet the requirements.Albumin A or protein g affinity chromatography medium can specifically bind antibody, but protide affinity ligand is easy
Come off by proteasome degradation in feed liquid, polluted product, and reuse number is low, medium is expensive, and running cost is high, limit
Its large-scale application is made.Although and the methods of traditional ion-exchange chromatography and hydrophobic interaction chromatography can combine it is anti-
Body, but specific and selectivity is poor, separating step is more, and purification effect is limited.Therefore, exploitation is with the non-of antibody selectivity
Protide aglucon becomes research hotspot.
The content of the invention
For above-mentioned shortcoming, it is an object of the invention to provide one kind using 4- pyrimidine radicals -2- aniline as functional ligand
Affinity chromatography medium.
A kind of affinity chromatography medium using 4- pyrimidine radicals -2- aniline as functional ligand, wherein, including chromatography substrate and match somebody with somebody
Base, the chromatography substrate are macropore beta-cyclodextrin microballoon, and aglucon is even after the activation of diglycol bisglycidyl ether
4- pyrimidine radicals -2- the aniline of connection.
Preferably, the chromatography media using aminobenzimidazole as functional ligand, wherein, the chromatography media
Ligand density be 30-160 μm of ol/ml.
Preferably, the preparation method using 4- pyrimidine radicals -2- aniline as the affinity chromatography medium of functional ligand, its
In, include the following steps：
1) after chromatography substrate is drained, the percent by volume for adding 0.5-2 times of chromatography substrate quality is 30% dimethyl
The hydrogen of sulfoxide, the diglycol bisglycidyl ether of 0.8-1.6 times of chromatography substrate quality and 0.1-1 times of chromatography substrate quality
Sodium oxide molybdena, activate in 200rpm shaking tables at 40 DEG C 5-10 it is small when, filter, be washed with deionized to obtain epoxy activation chromatography base
Matter；
2) epoxy is activated into chromatography substrate and the 4- pyrimidine radicals -2- aniline and 0.5- of 0.4-0.8 times of chromatography substrate quality
1M sodium carbonate buffers mix, and when reaction 8-24 is small in 200rpm shaking tables at 50 DEG C, after reaction, are washed with deionized
Medium to after 4- pyrimidine radicals -2- aniline couplings.
4th, the preparation side using 4- pyrimidine radicals -2- aniline as the affinity chromatography medium of functional ligand as claimed in claim 3
Method, it is characterised in that the mass ratio of the diglycol bisglycidyl ether and chromatography substrate is 0.8-1.6:1.
Preferably, the preparation method using 4- pyrimidine radicals -2- aniline as the affinity chromatography medium of functional ligand, its
In, the pH of the sodium carbonate buffer is 11-13.
The beneficial effects of the invention are as follows：
(1) antibody adsorption capacity is big, and saturated adsorption capacity is strong up to 150mg/ml humid mediums, disposal ability；
(2) elution is convenient, and the pKa of 4- pyrimidine radicals -2- aniline aglucons is 4.6, by adjusting pH value of solution to 5 nearby can be real
Now elution completely, avoids peracid, crosses alkali or high salt etc. to protein structure generation harmful effect or loss of activity；
(3) ligand density is high, and electric charge inductive effect is strong, and aglucon, coupling efficiency are coupled by the primary amino radical site on aglucon
Height, and the amino sites are also protonated, it is possible to provide the electrostatic repulsion of auxiliary；
(4) medium character is stablized, and is activated using diglycol bisglycidyl ether and is coupled aglucon, gained medium
Aglucon is stablized, and cleaning and regeneration is convenient；
(5) the new chromatography media that the present invention is developed, using 4- pyrimidine radicals -2- aniline as functional ligand, has significant
Hydrophobic charge-induction chromatography separation characteristic, has very high adsorption capacity to antibody, can be used for the prepare with scale of antibody.
Embodiment
With reference to embodiment, the present invention is described in further detail, to make those skilled in the art with reference to specification
Word can be implemented according to this.
Embodiment 1
Take and drain macropore beta-cyclodextrin microballoon 10g, the percent by volume for adding 5g is 30% dimethyl sulfoxide (DMSO), the one of 8g
The sodium hydroxide of diglycol ethylene bisglycidyl ether and 1g, when activation 6 is small in 200rpm shaking tables at 40 DEG C, filters, spend from
Sub- water washing obtains epoxy activation chromatography substrate；Epoxy is activated into chromatography substrate and the 4- pyrimidine radicals -2- aniline and 0.6M of 5g
Sodium carbonate buffer mixes, and when the pH of sodium carbonate buffer is that reaction 10 is small in 200rpm shaking tables at 12,50 DEG C, reaction terminates
Afterwards, it is washed with deionized to obtain the medium after 4- pyrimidine radicals -2- aniline couplings, the ligand density of chromatography media is 60 μm of ol/
ml。
Embodiment 2
Take and drain macropore beta-cyclodextrin microballoon 10g, the percent by volume for adding 8g is 30% dimethyl sulfoxide (DMSO), 10g
The sodium hydroxide of diglycol bisglycidyl ether and 3g, when activation 7 is small in 200rpm shaking tables at 40 DEG C, filters, spends
Ion water washing obtains epoxy activation chromatography substrate；By epoxy activate chromatography substrate and 7g 4- pyrimidine radicals -2- aniline and
0.6M sodium carbonate buffers mix, when the pH of sodium carbonate buffer is that reaction 12 is small in 200rpm shaking tables at 12,50 DEG C, reaction knot
Shu Hou, is washed with deionized to obtain the medium after 4- pyrimidine radicals -2- aniline couplings, the ligand density of chromatography media is 100 μ
mol/ml。
Embodiment 3
Take and drain macropore beta-cyclodextrin microballoon 10g, the percent by volume for adding 10g is 30% dimethyl sulfoxide (DMSO), 12g
The sodium hydroxide of diglycol bisglycidyl ether and 6g, when activation 10 is small in 200rpm shaking tables at 40 DEG C, filters, spends
Ion water washing obtains epoxy activation chromatography substrate；By epoxy activate chromatography substrate and 8g 4- pyrimidine radicals -2- aniline and
0.6M sodium carbonate buffers mix, when the pH of sodium carbonate buffer is that reaction 20 is small in 200rpm shaking tables at 12,50 DEG C, reaction knot
Shu Hou, is washed with deionized to obtain the medium after 4- pyrimidine radicals -2- aniline couplings, the ligand density of chromatography media is 120 μ
mol/ml。
Although embodiment of the present invention is disclosed as above, it is not restricted in specification and embodiment listed
With it can be applied to various suitable the field of the invention completely, can be easily for those skilled in the art
Realize other modification, therefore under the universal limited without departing substantially from claim and equivalency range, it is of the invention and unlimited
In specific details.
Claims (5)
- A kind of 1. affinity chromatography medium using 4- pyrimidine radicals -2- aniline as functional ligand, it is characterised in that including chromatography substrate and Aglucon, the chromatography substrate are macropore beta-cyclodextrin microballoon, and aglucon is after the activation of diglycol bisglycidyl ether 4- pyrimidine radicals -2- the aniline of coupling.
- 2. the chromatography media using aminobenzimidazole as functional ligand as claimed in claim 1, it is characterised in that the layer The ligand density for analysing medium is 30-160 μm of ol/ml.
- A kind of 3. preparation side using 4- pyrimidine radicals -2- aniline as the affinity chromatography medium of functional ligand as claimed in claim 1 Method, it is characterised in that include the following steps：1) after chromatography substrate is drained, add 0.5-2 times of chromatography substrate quality percent by volume be 30% dimethyl sulfoxide (DMSO), The diglycol bisglycidyl ether of 0.8-1.6 times of chromatography substrate quality and the hydroxide of 0.1-1 times of chromatography substrate quality Sodium, activate in 200rpm shaking tables at 40 DEG C 5-10 it is small when, filter, be washed with deionized to obtain epoxy activation chromatography substrate；2) epoxy is activated into chromatography substrate and the 4- pyrimidine radicals -2- aniline and 0.5-1M carbon of 0.4-0.8 times of chromatography substrate quality Sour sodium buffer solution mixes, and when reaction 8-24 is small in 200rpm shaking tables at 50 DEG C, after reaction, is washed with deionized to obtain 4- Medium after pyrimidine radicals -2- aniline couplings.
- 4. the preparation method using 4- pyrimidine radicals -2- aniline as the affinity chromatography medium of functional ligand as claimed in claim 3, its It is characterized in that, the mass ratio of the diglycol bisglycidyl ether and chromatography substrate is 0.8-1.6:1.
- 5. the preparation method using 4- pyrimidine radicals -2- aniline as the affinity chromatography medium of functional ligand as claimed in claim 3, its It is characterized in that, the pH of the sodium carbonate buffer is 11-13.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711276403.9A CN107999025A (en) | 2017-12-06 | 2017-12-06 | Affinity chromatography medium using 4- pyrimidine radicals -2- aniline as functional ligand |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711276403.9A CN107999025A (en) | 2017-12-06 | 2017-12-06 | Affinity chromatography medium using 4- pyrimidine radicals -2- aniline as functional ligand |
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| CN107999025A true CN107999025A (en) | 2018-05-08 |
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| CN201711276403.9A Withdrawn CN107999025A (en) | 2017-12-06 | 2017-12-06 | Affinity chromatography medium using 4- pyrimidine radicals -2- aniline as functional ligand |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108479738A (en) * | 2018-05-09 | 2018-09-04 | 大连理工大学 | A kind of hydrophobic chromatoghaphy medium, preparation method and applications |
| CN111420644A (en) * | 2020-03-27 | 2020-07-17 | 北京石油化工学院 | Preparation method of composite mixed-mode polymer chromatography medium |
Citations (6)
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|---|---|---|---|---|
| WO2004082801A1 (en) * | 2003-03-20 | 2004-09-30 | Amersham Biosciences Ab | Use of ph-responsive polymers |
| CN101003017A (en) * | 2006-01-20 | 2007-07-25 | 中国科学院过程工程研究所 | Agarose hydrophobic chromatoghaphy medium and its application in purifying yeast expression HBsAg |
| CN101036877A (en) * | 2007-01-25 | 2007-09-19 | 天津大学 | Hydrophobic charge chromatogram abduction medium by obtaining protein from fermentation supernatants liquid directly and the preparing method |
| US20100009867A1 (en) * | 2007-01-10 | 2010-01-14 | Ge Healthcare Bio-Sciences Ab | Multi-modal ion exchange chromatography resins |
| CN104096544A (en) * | 2014-05-13 | 2014-10-15 | 浙江大学 | Chromatographic medium using amino benzimidazole as function ligand and preparation method thereof |
| CN104624171A (en) * | 2014-12-30 | 2015-05-20 | 浙江大学 | Polymer grafting type hydrophobic charge-induced chromatography medium and preparation method thereof |
-
2017
- 2017-12-06 CN CN201711276403.9A patent/CN107999025A/en not_active Withdrawn
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004082801A1 (en) * | 2003-03-20 | 2004-09-30 | Amersham Biosciences Ab | Use of ph-responsive polymers |
| CN101003017A (en) * | 2006-01-20 | 2007-07-25 | 中国科学院过程工程研究所 | Agarose hydrophobic chromatoghaphy medium and its application in purifying yeast expression HBsAg |
| US20100009867A1 (en) * | 2007-01-10 | 2010-01-14 | Ge Healthcare Bio-Sciences Ab | Multi-modal ion exchange chromatography resins |
| CN101036877A (en) * | 2007-01-25 | 2007-09-19 | 天津大学 | Hydrophobic charge chromatogram abduction medium by obtaining protein from fermentation supernatants liquid directly and the preparing method |
| CN104096544A (en) * | 2014-05-13 | 2014-10-15 | 浙江大学 | Chromatographic medium using amino benzimidazole as function ligand and preparation method thereof |
| CN104624171A (en) * | 2014-12-30 | 2015-05-20 | 浙江大学 | Polymer grafting type hydrophobic charge-induced chromatography medium and preparation method thereof |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108479738A (en) * | 2018-05-09 | 2018-09-04 | 大连理工大学 | A kind of hydrophobic chromatoghaphy medium, preparation method and applications |
| CN108479738B (en) * | 2018-05-09 | 2020-11-13 | 大连理工大学 | Hydrophobic chromatography medium, preparation method and application thereof |
| CN111420644A (en) * | 2020-03-27 | 2020-07-17 | 北京石油化工学院 | Preparation method of composite mixed-mode polymer chromatography medium |
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Application publication date: 20180508 |
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