CN107823915A - A kind of alkali resistance affinity chromatography medium - Google Patents
A kind of alkali resistance affinity chromatography medium Download PDFInfo
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- CN107823915A CN107823915A CN201711039770.7A CN201711039770A CN107823915A CN 107823915 A CN107823915 A CN 107823915A CN 201711039770 A CN201711039770 A CN 201711039770A CN 107823915 A CN107823915 A CN 107823915A
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- affinity chromatography
- chromatography medium
- polyamide
- straight
- aglucon
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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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- Analytical Chemistry (AREA)
- Organic Chemistry (AREA)
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- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Solid-Sorbent Or Filter-Aiding Compositions (AREA)
- Treatment Of Liquids With Adsorbents In General (AREA)
Abstract
This case is related to alkali resistance affinity chromatography medium, using polyamide microballoon as kernel, polyamide microsphere surface is crosslinked with straight-chain polysaccharide molecule, the exposed amide groups of hydroxyl and polyamide microsphere surface in straight-chain polysaccharide molecule simultaneously with pi-allyl 2,3 glycidyl ethers are bonded, the glycidyl ethers of pi-allyl 2,3 are also associated with aglucon, for isolating and purifying protein product；Wherein, pi-allyl 2,3 glycidyl ethers produce dual be bonded with being crosslinked with the polyamide kernel of straight-chain polysaccharide molecule in acid condition, after aglucon is connected, the stability of aglucon can be increased, in to affinity chromatography medium use and cleaning process, aglucon is difficult for drop-off or degraded, present invention gained affinity chromatography medium are improved to the alkaline tolerance degree of sodium hydroxide.
Description
Technical field
The present invention relates to a kind of chromatography media, and in particular to a kind of alkali resistance affinity chromatography medium.
Background technology
Affinity chromatography is a kind of acted on using reversible specific adsorption between feature aglucon and target molecule and reached
Separate the liquid chromatography of purpose, have selectivity is high, purification effect is obvious, operating condition is gentle, bioactivity conservation rate height,
The advantages that concentrated effect is good.Affinity chromatography is as a kind of important tools for purification, in large biological molecules such as protein, nucleic acid
Important function has been played in separation.With the development of modern biotechnology, the separation purifying technique of biological technology products is proposed
Higher requirement, the application field of affinity chromatography technology constantly extend, and are especially showed in the isolating and purifying of pharmaceutical protein
Go out huge superiority, become and isolate and purify one of most important method of bioactive molecule.
During isolating and purifying, raw material isolates and purifies to obtain satisfactory target production by affinity chromatography medium
Product, while the impurity in raw material can pollute chromatography media, so needing to clean chromatography media and sterilized.Hydroxide at present
Sodium is widely used and cleaned, sterilizes and preserves chromatography media and tomographic system, in order to save the time in actual industrial production,
The salt materials such as sodium chloride are added in sodium hydroxide, so as to which both cleaning and sterilization are combined.Existing affinity chromatography
Medium less stable in the basic conditions, carrying out aglucon usually occur when affinity chromatography medium cleans with sodium hydroxide coming off
Or the problem of degraded, the service life of chromatography media is shortened, increases and isolates and purifies cost.
The content of the invention
In view of the deficiencies of the prior art, it is an object of the invention to provide a kind of alkali resistance affinity chromatography medium.
Technical scheme is summarized as follows：
Using polyamide microballoon as kernel, polyamide microsphere surface is crosslinked with straight-chain polysaccharide molecule；The straight-chain polysaccharide molecule
In the exposed amide groups of hydroxyl and polyamide microsphere surface be bonded simultaneously with pi-allyl -2,3- glycidyl ethers；The pi-allyl-
2,3- glycidyl ethers are also associated with aglucon.
Preferably, the polyamide microballoon is realized and the crosslinking of straight-chain polysaccharide molecule under the conditions of 70~80 DEG C.
Preferably, the epoxy radicals of the hydroxyl in the straight-chain polysaccharide molecule and pi-allyl -2,3- glycidyl ethers is in acidity
Under the conditions of pass through epoxy ring-opening realize condensation；
Preferably, the hydroxyl generated after the epoxy ring-opening is with the exposed amide groups of polyamide microsphere surface in acidity
Under the conditions of be bonded.
Preferably, the acid condition is the sulfuric acid solution that mass fraction is 50%.
Preferably, the straight-chain polysaccharide molecule by 10~50 same types or the polycondensation of different type monosaccharide molecule and
Into；The monosaccharide molecule includes five-carbon ring aldehydo sugar and six carbon aldoses.
Preferably, the polyamide microspherulite diameter is at 30~100 μm.
Preferably, the aglucon is determined by the material of purifying to be separated.
The beneficial effects of the invention are as follows：This case makes pi-allyl -2,3- by being improved to kernel in affinity chromatography medium
Glycidyl ethers in acid condition with polyamide kernel produce it is dual be bonded, after aglucon is connected, the stabilization of aglucon can be increased
Property, in affinity chromatography medium use and cleaning process, aglucon is difficult for drop-off or degrades, and especially present invention gained is affine
Chromatography media is improved to the alkaline tolerance degree of sodium hydroxide.
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.The invention provides a kind of alkali resistance affinity chromatography medium, pass through following embodiments and comparative example
It is specifically described.
Embodiment 1
Preparation process is as follows：
1) polyamide microballoon of the 20g particle diameters at 30~100 μm is immersed in 1% first containing 100g/L straight-chain polysaccharide molecules
In acid solution, stirring reaction 4~5 hours at 60~65 DEG C, with deionized water rinsing microballoon and drying；
2) the polyamide microballoon obtained to step 1) adds 15g pi-allyl -2,3- glycidyl ethers, 5g sodium sulphate, 100mL
50wt% sulfuric acid solutions, stirred 18 hours at 65~70 DEG C, it is micro- that polyamide that is net and being dried to obtain activation is washed with deionized water
Ball；
3) 15g albumin A aglucon, 100mL ethanol and 4g are added into the activation polyamide microballoon obtained by step 2)
NaOH, stirred 18 hours at 40~45 DEG C, vacuum pumps solvent after should terminating, and in triplicate, produces chromatography media.
Embodiment 2
Albumin A aglucon in step 3) in embodiment 1 is replaced with Protein G aglucon, remaining preparation process and the phase of embodiment 1
Together.
Comparative example 1
Formic acid solution is free of straight-chain polysaccharide molecule in the step 1) of embodiment 1, and remaining preparation process is same as Example 1.
Comparative example 2
3mol/L sodium hydroxide solution generations by the 50wt% sulfuric acid solutions of 100mL in the step 2) of embodiment 1 with 100mL
Replace, remaining preparation process is same as Example 1.
Comparative example 3
The commercially available affinity chromatography medium using albumin A as aglucon.
For the alkaline resistance properties of the affinity chromatography medium prepared by testing example 1~2 and comparative example 1~3, it is repeated 5 times
Chromatography assay, each chromatography assay are used with the 0.5mol/L sodium hydroxides and 0.15mol/L sodium chloride of 2 column volumes after terminating
Mixed solution rinses, then is balanced with the equilibrium liquid of 5 column volumes, compares the carrying capacity situation of change of 5 chromatography assays.Resisted with lgG
Body carries out carrying capacity test, table 1 is embodiment 1~2 and comparative example 1~3 as target protein with the affinity chromatography medium of preparation
For prepared affinity chromatography medium to the supported quantity of lgG antibody, supported quantity is more, illustrates that the absorption property of chromatography media is better,
With increasing for caustic times, supported quantity can keep constant, illustrate that chromatography media alkali resistance is preferable, and repeatability is good, and aglucon is not
It is easy to fall off or degraded, wherein Protein A ligand and Protein G aglucon to the selective combination of target protein lgG antibody, but
The combination of Protein G aglucon is stronger, so the supported quantity of embodiment 2 is than embodiment more than 1.
According to table 1 as can be seen that embodiment 1 and embodiment 2 are consolidated in the chromatography assay of 5 repetitions to target protein
Carrying capacity hardly changes, and illustrates that the flushing corrosive nature of the resistance to sodium hydroxide of affinity chromatography medium involved in the present invention is stronger, has
Repeatability well, aglucon is difficult for drop-off under alkaline washing condition or degrades, and has to the alkaline tolerance degree of sodium hydroxide
Improve.By comparative example 1, when in the absence of polysaccharide molecule, effective hydroxyl can not be provided and be used for bonded ligand, not only made
The supported quantity of chromatography media is reduced, and repeatability is bad, and medium supported quantity substantially reduces after being cleaned by sodium hydroxide solution, can
Can be that aglucon is degraded or come off caused.
Table 1
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
Other modification is realized, therefore under the universal limited without departing substantially from claim and equivalency range, it is of the invention and unlimited
In specific details.
Claims (7)
- A kind of 1. alkali resistance affinity chromatography medium, it is characterised in that using polyamide microballoon as kernel, the crosslinking of polyamide microsphere surface There is straight-chain polysaccharide molecule；The exposed amide groups of hydroxyl and polyamide microsphere surface in the straight-chain polysaccharide molecule is simultaneously and allyl Base -2,3- glycidyl ethers are bonded；Pi-allyl -2,3- the glycidyl ethers are also associated with aglucon.
- 2. affinity chromatography medium according to claim 1, it is characterised in that the polyamide microballoon is in 70~80 DEG C of conditions Lower realization and the crosslinking of straight-chain polysaccharide molecule.
- 3. affinity chromatography medium according to claim 1, it is characterised in that hydroxyl and alkene in the straight-chain polysaccharide molecule The epoxy radicals of propyl group -2,3- glycidyl ethers is realized by epoxy ring-opening in acid condition and is condensed.
- 4. affinity chromatography medium according to claim 3, it is characterised in that the hydroxyl that is generated after the epoxy ring-opening with The exposed amide groups of polyamide microsphere surface is bonded in acid condition.
- 5. according to the affinity chromatography medium described in any one in claim 3 or 4, it is characterised in that the acid condition The sulfuric acid solution for being 50% for mass fraction.
- 6. affinity chromatography medium according to claim 1, it is characterised in that the straight-chain polysaccharide molecule is same by 10~50 Type or different type monosaccharide molecule polycondensation form；The monosaccharide molecule includes five-carbon ring aldehydo sugar and six carbon aldoses.
- 7. affinity chromatography medium according to claim 1, it is characterised in that the polyamide microspherulite diameter is in 30~100 μ m。
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711039770.7A CN107823915B (en) | 2017-10-31 | 2017-10-31 | Alkali-resistant affinity chromatography medium |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711039770.7A CN107823915B (en) | 2017-10-31 | 2017-10-31 | Alkali-resistant affinity chromatography medium |
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| CN107823915A true CN107823915A (en) | 2018-03-23 |
| CN107823915B CN107823915B (en) | 2020-03-06 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113692420A (en) * | 2019-04-08 | 2021-11-23 | 旭化成医疗株式会社 | Polyamide medium for purifying protein-containing solutions and method for producing same |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1820815A (en) * | 2006-01-26 | 2006-08-23 | 合肥工业大学 | Semi fixed coordinate chromatograph for separating and purifying |
| CN102234332A (en) * | 2010-04-26 | 2011-11-09 | 浙江海正药业股份有限公司 | Process for separating and purifying recombinant human serum albumin and fusion protein thereof |
| CN103028380A (en) * | 2006-01-06 | 2013-04-10 | Emd密理博公司 | Affinity chromatography matrices and methods of making and using the same |
| WO2013177118A2 (en) * | 2012-05-21 | 2013-11-28 | Abbvie Inc. | Novel purification of non-human antibodies using protein a affinity chromatography |
-
2017
- 2017-10-31 CN CN201711039770.7A patent/CN107823915B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103028380A (en) * | 2006-01-06 | 2013-04-10 | Emd密理博公司 | Affinity chromatography matrices and methods of making and using the same |
| CN1820815A (en) * | 2006-01-26 | 2006-08-23 | 合肥工业大学 | Semi fixed coordinate chromatograph for separating and purifying |
| CN102234332A (en) * | 2010-04-26 | 2011-11-09 | 浙江海正药业股份有限公司 | Process for separating and purifying recombinant human serum albumin and fusion protein thereof |
| WO2013177118A2 (en) * | 2012-05-21 | 2013-11-28 | Abbvie Inc. | Novel purification of non-human antibodies using protein a affinity chromatography |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113692420A (en) * | 2019-04-08 | 2021-11-23 | 旭化成医疗株式会社 | Polyamide medium for purifying protein-containing solutions and method for producing same |
| CN113692420B (en) * | 2019-04-08 | 2023-09-12 | 旭化成医疗株式会社 | Polyamide medium for purifying protein-containing solution and method for producing same |
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| Publication number | Publication date |
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| CN107823915B (en) | 2020-03-06 |
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