CN100391979C - Mono methoxy polyethylene glycol-insulin complex substance and its preparation method - Google Patents
Mono methoxy polyethylene glycol-insulin complex substance and its preparation method Download PDFInfo
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- CN100391979C CN100391979C CNB2004100890508A CN200410089050A CN100391979C CN 100391979 C CN100391979 C CN 100391979C CN B2004100890508 A CNB2004100890508 A CN B2004100890508A CN 200410089050 A CN200410089050 A CN 200410089050A CN 100391979 C CN100391979 C CN 100391979C
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Abstract
The present invention relates to a single methoxy polyethylene glycol derivate, namely an mPEG modification insulin complex, and a preparation method thereof, which belongs to the technical fields of modification complexes of peptides or proteins and the preparation thereof. The complex is formed from insulin, namely INS proteins, and the single methoxy polyethylene glycol derivate; an amide linkage is formed by connecting the free aminogens of the INS proteins and the propionaldehyde groups with the activity of the single methoxy polyethylene glycol derivate, and the INS proteins and the single methoxy polyethylene glycol derivate are connected by the amide linkage; the molecular weight of the complex is from 10.8 to 25.8KD, wherein the free aminogens are epsilon-NH2 of lysine or alpha-NH2 of N-terminal, and the active groups are the propionaldehyde groups of mPEG-ALD. A crude product solution of the mPEG modification insulin complex is prepared from the insulin proteins containing the free aminogens and the single methoxy polyethylene glycol derivate containing the propionaldehyde groups, namely that the mPEG-ALD is processed in a modification reaction; a pure product of the mPEG modification insulin complex is obtained by an ion exchange chromatography, the collection of composition parts, centrifugal ultrafiltration and concentration, freezing and drying.
Description
Technical field
The present invention relates to a kind of mono methoxy polyethylene glycol-insulin complex substance and preparation method thereof, definitely say, relate to a kind of mono methoxy polyethylene glycol, promptly mPEG modifies insulin complex substance and preparation method thereof, belongs to the technical field of peptide or proteinic modification mixture and preparation thereof.
Background technology
Regular Insulin (INS) is the synthetic and excretory of beta Cell of islet, by A and two polypeptide chains of B by two disulfide linkage proteohormone that be formed by connecting, that contain 51 amino-acid residues.The relative molecular weight of the insulin monomer of biologically active is 5.8KD.Regular Insulin is (nineteen twenty-one) a kind of protein with hormonal action of finding the earliest, also be crystallization at first, determine amino acid residue sequence, artificial chemosynthesis and adopt genetic engineering technique to produce and put on market the protein that supplies clinical application in batches that (Li Yuan edits " genetically engineered drug ", Chemical Industry Press, in December, 2002 first version P182).Regular Insulin is to keep the normal and necessary hormone of existence of body metabolism, and it is mainly used in the particularly treatment of type i diabetes of diabetes, can regulate sugar, protein and metabolism of fat in addition and promote cell growth and differentiation.But Regular Insulin belongs to protein and peptide class medicine, is easy to by intravital protease hydrolysis, causes the unstable and bioavailability reduction of physicochemical property; Molecular weight is less, is 5.8KD, is caused in the body transformation period shorter easily by glomerular filtration.As the clinical protocol for the treatment of the type i diabetes people is: 0.5～1U/Kg, and every day three times, long-term injecting drug use has like this brought painful and inconvenient to patient; In addition, Regular Insulin is had immunogenicity again, the diabetics through for a long time, repeatedly, the high dosage insulin injection, can cause to produce antibody in the body, thereby can't bring into play the normal physiological function of Regular Insulin, serious even cause allergy and insulin resistant.Therefore press for the novel insulin product of development.
Summary of the invention
First technical problem that the present invention will solve is to propose a kind of mono methoxy polyethylene glycol, be that mPEG modifies insulin complex substance, it is characterized in that, this mixture is made up of insulin protein and mono methoxy polyethylene glycol, described mPEG is the mPEG that contains active propionic aldehyde group, be mPEG-ALD, both are by the former free amine group: the ε-NH of Methionin
2Or the α-NH of N-terminal
2The amido linkage that connects to the latter's active propionic aldehyde group links together, and molecular weight is between 10.8～25.8KD.MPEG modifies insulin complex substance, and promptly mPEG-INS not only has the hypoglycemic biologic activity of natural insulin, and has the advantage that biologically stable height in the body, the interior long half time of body and immunogenicity reduce.
Second technical problem that the present invention will solve provides a kind of mono methoxy polyethylene glycol, and promptly mPEG modifies the preparation method of insulin complex substance.
The technical scheme that the present invention solves the problems of the technologies described above is to contain the insulin protein and the mono methoxy polyethylene glycol that contains the propionic aldehyde group of free amine group, be that mPEG-ALD carries out modification reaction, make mPEG and modify the insulin complex substance crude product solution, through ion exchange chromatography, collection mixture part, concentrated, the lyophilize of centrifugal ultrafiltration, get mPEG and modify the pure product of insulin complex substance.
Now describe technical scheme of the present invention in detail.A kind of mono methoxy polyethylene glycol, promptly mPEG modifies the preparation method of insulin complex substance, it is characterized in that operation steps is as follows:
The first step is that the 200mM boric acid-borate buffer solution of pH7.4 of 0.16% insulin solutions and 400 parts of weight is mixed with the concentration of 500 parts of weight;
Second step added the mPEG of 0.68～27.2 part of weight in the first step solution, mixing, and described mPEG is mPEG-ALD, molecular weight is between 5～20KD;
The concentration of the 3rd step 100 parts of weight of adding in the second step solution is 0.75% NaBH
3CN solution is mixed into reaction solution;
The 4th step placed the solution of previous step under 4～40 ℃ reacts 0.5～24h;
The 5th step was regulated the pH4.2 termination reaction with Glacial acetic acid, made mono methoxy polyethylene glycol, and promptly mPEG modifies the insulin complex substance crude product solution, gets 20 μ l reaction solutions and carries out SDS-PAGE electrophoretic examinations degree of modification;
The 6th step was diluted 10 times with the reaction solution that the 20mM acetate buffer solution of pH4.2 made for the 4th step, last CM Sepharose FF chromatography column, 20mM acetate buffer solution with pH4.0 that contains 0～1M NaCl or pH4.6 carries out gradient elution, flow velocity is 0.1～1ml/min, 280nm ultraviolet detection absorption peak;
The 7th step collected each the absorption peak elutriant that obtains to the 6th step and carries out the SDS-PAGE electrophoresis, checked that mPEG modifies the polymorphism of insulin complex substance;
The single-point modification absorption peak solution molecular weight cut-off that the 8th step collected for the 7th step is the Millipore Amicon Ultra-15 ultrafiltration pipe ultrafiltration and concentration of 1～5KD, lyophilize, obtain the mono methoxy polyethylene glycol that single-point is modified, be that mPEG modifies the pure product of insulin complex substance, molecular weight is between 10.8～25.8KD.
Insulin protein of the present invention is to derive from the Regular Insulin of pig or ox or be gene engineering product.The mPEG-ALD modifier is the mono methoxy polyethylene glycol that contains the propionic aldehyde group, and NEKTAR company is on sale.
The product that aforesaid method makes has the structure that the present invention proposes: this product is made up of insulin protein and mono methoxy polyethylene glycol, both link together by the former free amine group and the amido linkage that connects to of the latter's propionic aldehyde group, and molecular weight is between 10.8～25.8KD.
Advantage of the present invention is: the amido linkage that mPEG modifies in the insulin complex substance is very stable, makes mPEG modify the insulin complex substance stable in properties, is difficult for being hydrolyzed separation; Modifier mPEG-ALD is the linear nontoxic macromolecular compound of high-hydrophilic, after the insulin protein stable bond, can form barrier on the protein molecule surface of Regular Insulin, make mPEG modify insulin complex substance and be difficult for having been improved its biologically stable by intravital proteasome degradation; MPEG modifies insulin complex substance long half time in vivo, has improved the bioavailability that mPEG modifies insulin complex substance.
Description of drawings
Fig. 1 is the SDS-PAGE electrophoretogram of the mPEG-INS that makes with the inventive method, wherein, the 1st, the INS contrast, the 2nd, protein molecular weight standard, the 3rd, molecular weight is mPEG-ALD and the INS reaction mixture electrophoretogram of 5KD, the polymorphism that shows modified outcome: both contained the single-point modified outcome, and also contained the multiple spot modified outcome.
Fig. 2 is the CM Sepharose FF chromatographic separation purifying figure of the mPEG-INS that makes with the inventive method, wherein, the 5th, penetrate the peak, 6 is first elution peaks, and 7 is second elution peaks, and 8 is the 3rd elution peaks.The molecular weight of mPEG in the mPEG-INS mixture is 5KD.Penetrating peak 5 is the mPEG-ALD that have neither part nor lot in modification reaction, and first elution peak 6 is mPEG-INS products that multiple spot is modified, and second elution peak 7 is mPEG-INS products that single-point is modified, and the 3rd elution peak 8 is INS of unmodified.
Fig. 3 be with Fig. 2 in the SDS-PAGE electrophoretogram of each elution peak purified product, wherein, the 9th, protein molecular weight standard, the 10th, the INS contrast, the 11st, mPEG-ALD and INS reaction mixture electrophoretogram, the 12nd, the mPEG-INS product that first elution peak, 6 multiple spots are modified among Fig. 2, the 13rd, the mPEG-INS product that second elution peak, 7 single-points are modified among Fig. 2, the 14th, the INS product of the 3rd elution peak 8 unmodifieds among Fig. 2.
Fig. 4 is the hypoglycemic activity figure as a result of mPEG-INS.The result shows among the figure: kept the hypoglycemic biologic activity of natural INS through the INS of PEGization, and prolong action time.
Embodiment
Now in conjunction with the accompanying drawings and embodiments content of the present invention is described further.All embodiment all operate according to above-mentioned preparation method.
The first step is that 200mM boric acid-borate buffer solution of pH7.4 of 0.16% insulin solutions and 0.6ml is mixed with the concentration of 0.75ml;
Second step added the 0.96mg molecular weight in the first step solution be the mPEG-ALD of 5KD, mixing;
The concentration of the 3rd step adding 0.15ml in the second step solution is 0.75% NaBH
3CN solution is mixed into reaction solution;
The reaction solution of the 4th step with the 3rd step gained places 4～40 ℃ of reaction 0.5～24h down;
The 5th step was regulated the pH4.2 termination reaction with Glacial acetic acid, made mPEG and modified the insulin complex substance crude product solution, got 20 μ l reaction solutions and carried out the SDS-PAGE electrophoresis, checked degree of modification, the results are shown in Figure 1;
The 6th step was diluted 10 times with the crude product solution that the 20mM acetate buffer solution of pH4.0 made for the 4th step, last CM Sepharose FF chromatography column, 20mM acetate buffer solution with the pH4.0 that contains 0～1M NaCl carries out gradient elution, flow velocity is 0.1～1ml/min, 280nm ultraviolet detection absorption peak the results are shown in Figure 2;
The 7th step collected each the absorption peak elutriant that obtains to the 6th step and carries out the SDS-PAGE electrophoresis, checked that mPEG modifies the polymorphism of insulin complex substance, the results are shown in Figure 3;
The single-point modification absorption peak solution molecular weight cut-off that the 8th step collected for the 7th step is the Millipore Amicon Ultra-15 ultrafiltration pipe ultrafiltration and concentration of 1～5KD, lyophilize, the mPEG that obtains the single-point modification of 0.17～0.21mg modifies the pure product of insulin complex substance, and molecular weight is 10.8KD.
The first step is that 200mM boric acid-borate buffer solution of pH7.4 of 0.16% insulin solutions and 0.6ml is mixed with the concentration of 0.75ml;
Second step added the 3.84mg molecular weight in the first step solution be the mPEG-ALD of 5KD, mixing;
The 3rd step to the 7th step is undertaken by cycle and taking corresponding operation among the embodiment 1;
The single-point modification absorption peak solution molecular weight cut-off that the 8th step collected for the 7th step is the Millipore Amicon Ultra-15 ultrafiltration pipe ultrafiltration and concentration of 1～5KD, lyophilize, the mPEG that obtains the single-point modification of 0.30～0.35mg modifies the pure product of insulin complex substance, and molecular weight is 10.8KD.
The first step is that 200mM boric acid-borate buffer solution of pH7.4 of 0.16% insulin solutions and 0.6ml is mixed with the concentration of 0.75ml;
Second step added the 9.6mg molecular weight in the first step solution be the mPEG-ALD of 5KD, mixing;
The 3rd step to the 7th step is undertaken by cycle and taking corresponding operation among the embodiment 1;
The single-point modification absorption peak solution molecular weight cut-off that the 8th step collected for the 7th step is the Millipore Amicon Ultra-15 ultrafiltration pipe ultrafiltration and concentration of 1～5KD, lyophilize, the mPEG that obtains the single-point modification of 0.27～0.31mg modifies the pure product of insulin complex substance, and molecular weight is 10.8KD.
Embodiment 4 mPEG modify the preparation method of insulin complex substance.In the present embodiment, mPEG is mPEG-ALD, and molecular weight is 20KD.
The first step is that 200mM boric acid-borate buffer solution of pH7.4 of 0.16% insulin solutions and 0.6ml is mixed with the concentration of 0.75ml;
Second step added the mPEG-ALD that the 9.6mg molecular weight is 20KD, mixing in the first step solution;
The concentration of the 3rd step adding 0.15ml in the second step solution is 0.75% NaBH
3CN solution is mixed into reaction solution;
The reaction solution of the 4th step with the 3rd step gained places 4～40 ℃ of reaction 0.5～24h down;
The 5th step was regulated the pH4.2 termination reaction with Glacial acetic acid, made mPEG and modified the insulin complex substance crude product solution, got 20 μ l reaction solutions and carried out the SDS-PAGE electrophoresis, checked degree of modification;
The 6th step was diluted 10 times with the crude product solution that the 20mM acetate buffer solution of pH4.6 made for the 4th step, last CM Sepharose FF chromatography column, 20mM acetate buffer solution with the pH4.6 that contains 0～1M NaCl carries out gradient elution, and flow velocity is 0.1～1ml/min, 280nm ultraviolet detection absorption peak;
The 7th step collected each the absorption peak elutriant that obtains to the 6th step and carries out the SDS-PAGE electrophoresis, checked that mPEG modifies the polymorphism of insulin complex substance;
The single-point modification absorption peak solution molecular weight cut-off that the 8th step collected for the 7th step is the Millipore Amicon Ultra-15 ultrafiltration pipe ultrafiltration and concentration of 1～5KD, lyophilize, the mPEG that obtains the single-point modification of 0.13～0.15mg modifies the pure product of insulin complex substance, and molecular weight is 25.8KD.
Embodiment 5mPEG modifies the preparation method of insulin complex substance.In the present embodiment, mPEG is mPEG-ALD, and molecular weight is 20KD.
The first step is that 200mM boric acid-borate buffer solution of pH7.4 of 0.16% insulin solutions and 0.6ml is mixed with the concentration of 0.75ml;
Second step added the 19.2mg molecular weight in the first step solution be the mPEG-ALD of 20KD, mixing;
The 3rd step to the 7th step is undertaken by cycle and taking corresponding operation among the embodiment 4;
The single-point modification absorption peak solution molecular weight cut-off that the 8th step collected for the 7th step is the Millipore Amicon Ultra-15 ultrafiltration pipe ultrafiltration and concentration of 1～5KD, lyophilize, the mPEG that obtains the single-point modification of 0.25～0.30mg modifies the pure product of insulin complex substance, and molecular weight is 25.8KD.
The first step is that 200mM boric acid-borate buffer solution of pH7.4 of 0.16% insulin solutions and 0.6ml is mixed with the concentration of 0.75ml;
Second step added the 19.2mg molecular weight in the first step solution be the mPEG-ALD of 20KD, mixing;
The 3rd step to the 7th step is undertaken by cycle and taking corresponding operation among the embodiment 4;
The single-point modification absorption peak solution molecular weight cut-off that the 8th step collected for the 7th step is the Millipore Amicon Ultra-15 ultrafiltration pipe ultrafiltration and concentration of 1～5KD, lyophilize, the mPEG that obtains the single-point modification of 0.25～0.30mg modifies the pure product of insulin complex substance, and molecular weight is 25.8KD.
The hypoglycemic activity of embodiment 7 mPEG-INS mixtures.In the present embodiment, the mPEG modifier that is used to modify is mPEG-ALD, and its molecular weight is 5KD.
Experiment material and method:
Male and healthy kunming mice (cleaning level, Fudan University in Shanghai medical college animal center provides);
The pH7.0 phosphoric acid buffer, INS
Blood glucose monitoring system (the newly upright medicine equipment company limited in Shanghai);
Male and healthy kunming mice overnight fasting is divided into 3 groups (n=6).The 1st group, the physiological saline control group; The 2nd group, the insulin administration group; The 3rd group, mPEG-INS administration group, described mPEG-INS is the preparation product of embodiment 1.Described mPEG-INS is the preparation product of embodiment 1.
The 1st group of abdominal injection 100 μ l pH7.0 phosphoric acid buffers; The Regular Insulin of the 2nd group of abdominal injection 0.33U; The mPEG-INS of the 3rd group of abdominal injection 0.33U.Note was zero moment at this moment.Carried out mouse tail vein respectively at 60,120,360,600 minutes and get blood 10 μ l, measure blood sugar concentration with blood glucose monitoring system.
The result as shown in Figure 4, shown in numerical value be the blood sugar mean value of 6 mouse of administration group and the ratio of control group.Compare with physiological saline group mouse, within after the administration 120 minutes, INS administration group and mPEG-INS administration group all can reduce mouse blood sugar, illustrate that the mPEG-INS mixture of preparing by the inventive method has kept the biologic activity of INS.After 360 minutes, the blood glucose value of INS group mouse and control group mice is as broad as long, prompting INS metabolism is intact, no longer has hypoglycemic activity, and the mouse blood sugar of mPEG-INS administration group is lower than control group, the hypoglycemic time lengthening of mPEG-INS mixture that explanation is prepared by the inventive method, thus blood sugar concentration controlled effectively.
Also embodiment 2 to embodiment 6 preparation-obtained products of used mPEG-INS in the present embodiment.They carry out determination of activity according to the method for present embodiment, have all obtained similar experimental result.
Claims (2)
1. mono methoxy polyethylene glycol, be that mPEG modifies insulin complex substance, it is characterized in that, this mixture is made up of insulin protein and mono methoxy polyethylene glycol, described mPEG is the mPEG that contains active propionic aldehyde group, be mPEG-ALD, both are by the former free amine group: the ε-NH of Methionin
2Or the α-NH of N-terminal
2The amido linkage that connects to the latter's active propionic aldehyde group links together, and molecular weight is between 10.8～25.8KD.
2. the described mono methoxy polyethylene glycol of claim 1, promptly mPEG modifies the preparation method of insulin complex substance, it is characterized in that operation steps is as follows:
The first step is that the 200mM boric acid-borate buffer solution of pH7.4 of 0.16% insulin solutions and 400 parts of weight is mixed with the concentration of 500 parts of weight;
Second step added the mPEG of 0.68～27.2 part of weight in the first step solution, mixing, and described mPEG is mPEG-ALD, molecular weight is between 5～20KD;
The concentration of the 3rd step 100 parts of weight of adding in the second step solution is 0.75% NaBH
3CN solution is mixed into reaction solution;
The 4th step placed the solution of previous step under 4～40 ℃ reacts 0.5～24h;
The 5th step was regulated the pH4.2 termination reaction with Glacial acetic acid, made mono methoxy polyethylene glycol, and promptly mPEG modifies the insulin complex substance crude product solution, gets 20 μ l reaction solutions and carries out SDS-PAGE electrophoretic examinations degree of modification;
The 6th step was diluted 10 times with the reaction solution that the 20mM acetate buffer solution of pH4.2 made for the 4th step, last CM Sepharose FF chromatography column, 20mM acetate buffer solution with pH4.0 that contains 0～1M NaCl or pH4.6 carries out gradient elution, flow velocity is 0.1～1ml/min, 280nm ultraviolet detection absorption peak;
The 7th step collected each the absorption peak elutriant that obtains to the 6th step and carries out the SDS-PAGE electrophoresis, checked that mPEG modifies the polymorphism of insulin complex substance;
The single-point modification absorption peak solution molecular weight cut-off that the 8th step collected for the 7th step is the Millipore Amicon Ultra-15 ultrafiltration pipe ultrafiltration and concentration of 1～5KD, lyophilize, obtain the mono methoxy polyethylene glycol that single-point is modified, be that mPEG modifies the pure product of insulin complex substance, molecular weight is between 10.8～25.8KD.
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| CN101062408B (en) * | 2006-04-27 | 2010-12-08 | 深圳市隆阳生物科技有限公司 | Oral insulin compound medicine preparation and its preparing method |
| ITMI20090548A1 (en) * | 2009-04-06 | 2010-10-07 | Indena Spa | PROCESS OF REMOVAL OF PESTICIDES FROM GINKGO BILOBA EXTRACTS AND EXTRACTS OBTAINED FROM SUCH PROCESS |
| CN102453089B (en) * | 2010-10-25 | 2014-06-04 | 北京凯因科技股份有限公司 | Preparation and application of recombinant consensus interferon mutant polyethylene glycol conjugate |
| CN102614498B (en) * | 2011-01-28 | 2014-12-17 | 四川科伦药物研究有限公司 | Insulin nanoparticle and preparation method thereof |
| CN102675452B (en) * | 2011-03-17 | 2015-09-16 | 重庆富进生物医药有限公司 | Tool continues the conjugate of insulin human that is hypoglycemic and that combined by height and analogue |
| CN102293748B (en) * | 2011-07-25 | 2013-02-13 | 华南理工大学 | Oral PEGylated insulin pH-sensitive naonparticle and preparation method thereof |
| CN105085640B (en) * | 2015-07-03 | 2018-08-21 | 山东农业大学 | A kind of J subgroup avian leucosis virus immunosuppressive polypeptides based on PEG modifications |
| CN107446038A (en) * | 2016-06-01 | 2017-12-08 | 湖南华腾制药有限公司 | Polyethyleneglycol modified rh-insulin and preparation method thereof |
| CN106963941A (en) * | 2017-03-14 | 2017-07-21 | 沈阳大学 | A kind of oral insulin agent with enteron aisle mucus penetration capacity and preparation method thereof |
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| CN1519266A (en) * | 2003-01-22 | 2004-08-11 | 中国科学院过程工程研究所 | Protein finishing agent with long tail of methoxy carbowax as well as preparation method and application |
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| CN1519266A (en) * | 2003-01-22 | 2004-08-11 | 中国科学院过程工程研究所 | Protein finishing agent with long tail of methoxy carbowax as well as preparation method and application |
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| 药用蛋白质的化学修饰. 谢芳等.生命的化学，第22卷第6期. 2002 |
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