CN101279999B - Method for modifying hirudin by polyethyleneglycol assisted by anion exchange column - Google Patents
Method for modifying hirudin by polyethyleneglycol assisted by anion exchange column Download PDFInfo
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- CN101279999B CN101279999B CN2008100115546A CN200810011554A CN101279999B CN 101279999 B CN101279999 B CN 101279999B CN 2008100115546 A CN2008100115546 A CN 2008100115546A CN 200810011554 A CN200810011554 A CN 200810011554A CN 101279999 B CN101279999 B CN 101279999B
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Abstract
Disclosed is a method to assist polyethylene glycol(PEG) to modify hirudin with anion exchange column, belonging to protein modification technical field. The method is characterized in that ion exchange column is introduced to assit protein modification with PEG; the ph value for the reaction is 6-9 and the reaction time is 15-120min; the mol ration of hirudin to PEG is 1:3-1:9; the ion exchange column is a strong anion exchange column and the quaternary ammonium groups are the charged groups.The substrate in the column is sephadex or agarose, with average particle size of 30-90 micrometre. The method simplifies the reaction process and separation process in protein modification and correspondingly redues the consumption of sample in the process. The eluted hirudin can be recycled after being desalted. Compared with conventional liquid phase modification method, the method can produce hirudin with great activity in vitro, which is only modified by PEG.
Description
Technical field
The invention belongs to the protein modification technical field, relate to proteic polyoxyethylene glycol solid phase and modify, particularly a kind of method of utilizing the auxiliary polyethyleneglycol modified r-hirudin of anion-exchange column.
Background technology
It is a chemical reaction with neutral, unreactiveness, the polymer-modified protein drug of hydrophilic PEG that polyoxyethylene glycol (PEG) is modified.Since the seventies in last century, scientists is found some pharmaceutical proteins after PEG modifies, and its medicinal character is improved greatly, and like transformation period prolongation in the body, immunogenicity reduces or the like.
Some PEG-protein conjugate such as Adagen of having gone on the market now, Oncospar, PEG-Intron etc. are based on all that first-generation PEGization is technological to prepare, and it is characterized in that: the molecular weight of (1) modifier is generally all very low; (2) bifunctional polyoxyethylene glycol impurity causes crosslinked easily and reunites; (3) stability to hydrolysis of connecting key is poor; (4) selectivity is low, promptly obtains the different modified outcome of modification degree after the reaction, and has side reaction.For solving above-mentioned drawback, based on polyethyleneglycol modified dose of application that is obtaining more and more widely of s-generation PEGization technology.
The polyethyleneglycol modified of s-generation PEGization is target with the pointed decoration.All kinds of activated PEG of Nekta company exploitation make proteic directed modification become possibility, and main products has the PEG verivate to the amido modified all kinds of straight or brancheds of amino, sulfydryl and N end, and all kinds of PEG that have label.But also there is its limitation in this species specific modification, requires to be contained free sulfhydryl groups by modified protein like the modification of sulfydryl, and is in outside the avtive spot; Hold alpha-amino modification to N, need epsilon-amino protection Lys, and reaction conditions comparatively harsh (a kind of method of modifying protein alpha-amido by carbowax, CN1687106).
R-hirudin (hirudin) is as specific thrombin inhibitors, and the shortcoming of transformation period weak point has seriously restricted its clinical application in its body, therefore is necessary to adopt the PEG modifying method to improve its medicinal character.Analyzing the amino acid of r-hirudin forms; Not having free-SH in its molecule supplies corresponding PEG to modify; And terminal amino group is that its activity is essential, so can not select specificity to modify the PEG of terminal amino group, finally selects corresponding PEG specificity modifying hirudin Methionin (Lys).The experimental study in early stage shows that if 4 lysine residues are all modified by PEG on the r-hirudin, its biological activity will obviously reduce.Analyze the effect situation of r-hirudin and zymoplasm, find that 47 Lys are positioned at the groove of zymoplasm and r-hirudin effect, if 47 Lys are modified, its activity can be affected.
For the avtive spot of protection r-hirudin is not modified, affine protection is that (affine method is protected method, product and the purposes of avtive spot modifying protein to a kind of solid phase modification strategy commonly used, CN1453292).Though this method has the protection avtive spot, series of advantages such as high specificity, its shortcoming is a complex steps; Need find the aglucon that has affinity interaction with target protein; And some aglucon costs are high, improved the cost of medicine, limited its use range.
Summary of the invention
The objective of the invention is to seek a kind of albumen PEG modifying method, this method is quick, simple and direct, can obtain single, active higher modified product.A kind of method of utilizing the auxiliary PEG modifying hirudin of anion-exchange column is provided in the invention; This method not only can be utilized the active site of ion exchange column shielding r-hirudin; Improve the specificity that PEG modifies, and can simplify the separating step of modified outcome, shorten the running time.
The step that realizes the inventive method is following:
The r-hirudin that (A) will be dissolved among 20～50mM phosphate buffered saline buffer a is splined in same buffer equilibrated ion column, and extremely all samples are adsorbed on the Ion Exchange Medium, does not have till the eluate outflow.Wherein r-hirudin comprises natural extract r-hirudin, all kinds of varients of lepirudin 023 ludon, the concentration range 1～10mg/ml of r-hirudin; PH of buffer scope 6～9, ion exchange column medium are sephadex or agarose, and cation exchange groups is that season is amino, matrix average particle size range 30～90 μ m.
The activated PEG solution that (B) will be dissolved in a liquid pumps in the post of steps A, is full of in the post to complete soln, promptly stops flow velocity.Wherein PEG and r-hirudin mol ratio are 3: 1～9: 1, and between PEG molecular weight ranges 2kDa～35kDa, the mPEG activation method is succinimdyl carbonate activation method or carbonyl dimidazoles activation method.
(C) behind question response 15～120min,, do not flow out to there being eluate with the ion exchange column of a liquid rinse step B.With a liquid that contains 1～2M sodium-chlor or Repone K material in the step B pillar is carried out gradient elution then, type of elution is gradient or stepwise elution, elution time 30～120min.Collect elution fraction then.
(D) component of collecting among the step C is carried out electrophoresis (SDS-PAGE) analysis, dyeing process is that iodine dyes, and promptly is directed against the dyeing process of PEG.
(E) the mono-modified component of verifying among the step D is measured protein concentration with the Lowry method, zymoplasm titration measuring vigor calculates its external vigor that compares.During the mensuration vigor, being substrate with the fibrinogen proenzyme, adding r-hirudin, is titration end point when zymoplasm titration to fibrinogen proenzyme condenses, and the r-hirudin vigor characterizes with titrating zymoplasm vigor.
Effect of the present invention and benefit are integrating remark and separating step, shorten the running time, raise the efficiency.Compare with traditional liquid phase modification, can improve the unicity and the external activity of product.Compare with the solid phase modifying method that adopts affine strategy, the step that simplifies the operation need not to seek the aglucon with protein-interacting.
Embodiment
Be described in detail specific embodiment of the present invention below in conjunction with technical scheme.
Embodiment one:
4ml is dissolved in the r-hirudin solution among the 0.02M pH8 phosphate buffered saline buffer a; Its concentration is that 1.5mg/ml is splined on that (the microballon structure is 6% highly cross-linked agarose in the post with same buffer equilibrated Hitrap Q HP 5ml prepackage ion exchange column; The aperture is 34 μ m; Charged group season is amino), wait not have eluate to flow out, then 4ml is dissolved in the succinimide activation mPEG 5kDa (SC-PEG among the damping fluid a
5000) solution, be splined in the post, applied sample amount and r-hirudin mol ratio 9: 1, reaction 1h, reaction finishes the back wash-out.Adopt linear gradient 0～30%b liquid during wash-out, wherein b liquid is a liquid that contains 1M NaCl.Elution time 100min.Each eluate of manual collection, through each elution fraction of electrophoresis checking, the electrophoresis dying method is that iodine dyes.Staining fluid is that 0.1M contains 5%BaCl
2I/KI solution.Dyeing time 4min, bleaching time 12h.The mono-modified product that checking obtains is mPEG
5000-hirudin, its molecular weight are 12kDa.In the mensuration of living, determination of protein concentration adopts the Lowry method, modifies the after product vitality test and adopts the zymoplasm volumetry.External is 91% than retention rate alive.
Embodiment two:
4ml is dissolved in the r-hirudin solution among the 0.02M pH 8 phosphate buffered saline buffer a; Concentration is 1.5mg/ml; Be splined on same buffer equilibrated Hitrap Q HP 5ml prepackage ion exchange column; Wait not have eluate to flow out, then 4ml is dissolved in the succinimide activatory mPEG 20kDa (SC-PEG among the damping fluid a
20000) solution, be splined in the post, applied sample amount and r-hirudin mol ratio 9: 1, reaction 1h, reaction finishes the back wash-out.Wash-out adopts linear gradient 0～30%b liquid, and wherein b liquid is a liquid that contains 1M NaCl.Elution time 60min.Each eluate of manual collection, through each elution fraction of electrophoresis checking, the electrophoresis dying method is that iodine dyes.Staining fluid is that 0.1M contains 5%BaCl
2I/KI solution.Dyeing time 4min, bleaching time 12h.The mono-modified product product of collecting is mPEG
20000-hirudin, its molecular weight are 27kDa.In the mensuration of living, determination of protein concentration adopts the Lowry method, modifies the after product vitality test and adopts the zymoplasm volumetry.MPEG
20000-hirudin is external to be 96% than retention rate alive.
Claims (3)
1. method of utilizing the auxiliary polyethyleneglycol modified r-hirudin of anion-exchange column, its characteristic may further comprise the steps:
(A) will be dissolved in concentration is 20～50mM; PH is that the r-hirudin among 6～9 the phosphate buffered saline buffer a is splined in same buffer equilibrated ion column; Be adsorbed in until whole samples on the Ion Exchange Medium of matrix average particle size range 30～90 μ m, do not have till the eluate outflow;
The molecular weight that (B) will be dissolved in a liquid is the activated polyethylene glycol solution of 2～35kDa, is splined in 3: 1～9: 1 in the post of steps A by polyoxyethylene glycol and r-hirudin mol ratio, is full of ion column to complete soln, stops to go up appearance;
(C) behind question response 15～120min,, do not flow out to there being eluate with the reactive ion post of a liquid rinse step B; A liquid with containing 1～2M sodium-chlor or Repone K carries out gradient elution to material in the step B pillar, and elution time 30～120min collects elution fraction.
2. a kind of method of utilizing the auxiliary polyethyleneglycol modified r-hirudin of anion-exchange column according to claim 1 is characterized in that the r-hirudin in the steps A comprises natural extract r-hirudin, all kinds of varients of lepirudin 023 ludon, its concentration range 1～10mg/ml; The ion column that uses be the reinforcing yin essence ion exchange column, charged group is that season is amino; The post mesostroma is sephadex or agarose.
3. a kind of method of utilizing the auxiliary polyethyleneglycol modified r-hirudin of anion-exchange column according to claim 1 is characterized in that the activation method of polyoxyethylene glycol is carbonyl dimidazoles method or succinimdyl carbonate method among the step B.
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Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102329395B (en) * | 2011-05-16 | 2013-06-12 | 温州医学院 | Method for preparing PEG (Polyethylene Glycol)ylation basic fibroblast growth factor |
| CN108395475B (en) * | 2018-03-29 | 2021-09-10 | 苏州至汇生物科技有限公司 | Hirudin separation and purification method based on affinity chromatography |
| CN113621046B (en) * | 2021-08-19 | 2022-05-27 | 重庆宸安生物制药有限公司 | Use and method of polymer anion exchange filler in preparation of liraglutide |
| CN114853872B (en) * | 2022-04-27 | 2023-11-17 | 山东新时代药业有限公司 | Preparation method of polyethylene glycol modified rhG-CSF |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1453292A (en) * | 2002-04-26 | 2003-11-05 | 中国人民解放军军事医学科学院放射医学研究所 | Chemical modification method of protein in its affinity protecting active region and its product and use |
| CN1687106A (en) * | 2005-03-25 | 2005-10-26 | 山东格兰百克生物制药有限公司 | Method of modifying protein alpha-amido by carbowax |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1453292A (en) * | 2002-04-26 | 2003-11-05 | 中国人民解放军军事医学科学院放射医学研究所 | Chemical modification method of protein in its affinity protecting active region and its product and use |
| CN1687106A (en) * | 2005-03-25 | 2005-10-26 | 山东格兰百克生物制药有限公司 | Method of modifying protein alpha-amido by carbowax |
Non-Patent Citations (3)
| Title |
|---|
| HOU,B.B.等.Design,preparation and in vitro bioactivity of mono-PEGylated recombinant hirudin.《Chinese Journal of Chemical Engineering》.2007,第15卷(第6期),775-780. * |
| 于爱平等.聚乙二醇修饰水蛭素的分离纯化与活性分析.《药物生物技术》.2004,第11卷(第5期),302-305. * |
| 侯蓓蓓.聚乙二醇单修饰重组水蛙素的研究.《中国优秀硕士学位论文全文数据库，工程科技I辑》.2008,(第1期),B016-99. * |
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