CN1687106A - Method of modifying protein alpha-amido by carbowax - Google Patents
Method of modifying protein alpha-amido by carbowax Download PDFInfo
- Publication number
- CN1687106A CN1687106A CN 200510042587 CN200510042587A CN1687106A CN 1687106 A CN1687106 A CN 1687106A CN 200510042587 CN200510042587 CN 200510042587 CN 200510042587 A CN200510042587 A CN 200510042587A CN 1687106 A CN1687106 A CN 1687106A
- Authority
- CN
- China
- Prior art keywords
- amido
- protein
- amino
- carbowax
- alpha
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Abstract
The present invention provides a method for modifying protein alpha-amino group by using polyethuylene glycol. Said method includes the following steps: utilizing amino protective agent to close lysine epsilon amino group capable of reacting with PEG in the protein, using activated PEG with dominant response with alpha amino group to make amino modification, then removing amino protective agent so as to obtain nitrogen end specific PEG modified protein derivative. The conversion rate of product obtained by said method is high, can be up to 90%-95%.
Description
Technical field:
The present invention relates to a kind of pointed decoration and remodeling method of modification and transformation, especially protein molecule of protein molecule.
Background technology:
Polypeptide, protein medicaments mainly are eliminated in vivo by effects such as degraded, drainage, receptor-mediated endocytosis, wherein molecular weight less than the polypeptide factor of 20 kDa in metabolic process easily by glomerular filtration, polypeptide factor is partly degraded by proteolytic enzyme wherein again and is discharged from urine by uriniferous tubules the time, thereby the transformation period is short.The removing transformation period of intravenous injection recombinant methionyl human G-CSF (rhG-CSF) is 1～2 hour, and subcutaneous injection then is 2～3 hours, needs use every day for keeping certain curative effect, has not only increased the painful of patient but also has easily caused a series of side reactions.
Chemically modified is an effective way that prolongs the protein medicaments transformation period, the modifier that wherein is most widely used is mono methoxy polyethylene glycol (methoxypoly ethyleneglycol, be called for short mPEG), next is polyose such as dextran, ficoll, starch etc., also can use homologous protein or artificial synthetic polypeptide class such as albumin, poly-L-Ala etc., long-chain fat acids and polyene belong to alkyl compound and also can be used as modifier in addition.
Polyoxyethylene glycol (being called for short PEG) is a kind of inertia, amphiphilic, uncharged long flexible chain high molecular polymer HO (CH
2CH
2O)
nCH
2CH
2OH, n are the number of polymerized unit, and the PEG molecular weight can increase to 50kDa by 1kDa with the increase of n), linear and two kinds of configurations of side chain are arranged, by the safety barrier of FDA approval as multiple medicine.PEG is connected with protein by covalent linkage, can modify with alpha-amino group (being positioned at the nitrogen latter end), ε amino (being positioned at lysine residue) reaction pair protein molecule in the protein molecule, by protein surface amino being modified to change polypeptide, protein medicaments distribution and pharmacology characteristic in vivo effectively.
PEG modifies and has been applied to the different proteic modifications of kind more than 40 at present, as porcine hemoglobin (BSA), granulocyte colony-stimulating factor (G-CSF), interleukin-2 (ILr2) etc.PEG modifies the back medicine generally can prolong several times to tens times or even hundreds of times plasma half-life, but most of proteic immunogenicity also decreases, and proteic biological activity also has in various degree and reduces.This may be because the PEG macromole forms one deck shell around protein molecular; having hindered immunocyte contacts with proteic; protected albumen, covered the proteolytic enzyme recognition site and avoided the generation of proteasome degradation, but simultaneously proteic avtive spot has been affected.
Protein after the modification has some special nature: 1. the transformation period prolongs.Many protein drugs of being used widely at present in vivo the transformation period very short, want intermittent, regular administration during use, inconvenient and increased patient's misery.Molecular weight of albumen after the PEGization increases, and glomerular filtration reduces, and the sterically hindered effect of PEG molecule is slowed down albumen in vivo by proteasome degradation speed, and the transformation period increases in the medicine body thereby make, and drug effect is more lasting; 2. PEGization can reduce the pharmaceutical grade protein antigenicity, enlarges the range of application of medicine; 3. PEGization also may cause pharmaceutical grade protein to have some new living features, and such as thrombopoietic active the increasing of the interleukin-6 promotion of PEGization, and otherwise activity is constant substantially; 4. PEGization can influence some physics, the chemical property (wetting ability of PEG can increase solvability, and space steric effect reduces proteolysis etc.) of pharmaceutical grade protein.
At present, modify the PEG (in case a PEG molecule connects two protein molecules) that pharmaceutical protein adopts monomethylation usually,, introduce electrophilic group so that and amino nucleophilic group reaction earlier with its OH end activation.The method of PEGization modification is a lot, PEGization is to occur at random on any amino that may react in the early stage method, therefore the reaction product that obtains is the mixture of different molecular weight (the PEG number difference of connection), even also there is the isomery of decorating site in the product of same molecular amount.And, after being modified, some Methionin that is in protein active site or receptor binding site reduces activity of proteins greatly.
Olaf B Kinstler etc. notices different amino chemical reactivity differences in the protein: alpha-amino group is different with the pKa (dissociation constant) of ε amino, the pKa of alpha-amino group (being positioned at the nitrogen latter end) is 7.8, and the pKa of ε amino (being positioned at lysine residue) is 10.1.If carry out amido modifiedly with the PEG (mPEG-aldehyde) of aldehyde radicalization, the alpha-amino group that pKa is low has more the reaction advantage than ε amino, preferentially reacts, and can realize the strong response of protein nitrogen end.Therefore they carry out the modification of rhG-CSf with this activated PEG (mPEG-aldehyde) (pH5.0) under low pH with nitrogen end reaction advantage, and the modified outcome greater than 70% is a nitrogen terminal specific sex modification, has definite molecular weight and molecular structure.But still there is shortcoming in this method: 1. sluggish, and the cycle is long, generally needs 10～16 hours; 2. reaction just preferentially carry out PEGization at the nitrogen end, PEGization amino on the Methionin still can be carried out, and along with being increased by the PEGization proportion on the prolongation Methionin of time, because this method is just by the pH value that the reduces reaction system speed of response that slows down, thereby it is poor to widen two kinds of different amino speed of response, make reaction be convenient to control, but can not stop further PEGization amino on the Methionin.
The crosslinking reaction thing that PEG reaction conditions at present commonly used generates down is PEG-protein molecule (the molecular weight difference of non-homogeneous, the molecular structure difference, crosslinked PEG on the different lysine residue of protein molecule), run counter to the relevant principle of China's new drug evaluation, can't be applied to clinical, for develop can received homogeneous the PEGization protein molecule, so be necessary to seek the condition of a kind of PEG macromole and protein N terminal fixed point crosslinking reaction.
Summary of the invention:
In order to remedy the deficiencies in the prior art, develop can received homogeneous the PEGization protein molecule, the invention discloses the method for a kind of PEG macromole and protein N terminal fixed point crosslinking reaction, the method of the PEGization modification that can carry out fast, fix a point protein molecule as the small molecular weight of medicine, obtain the PEGization protein molecule that new drug enters clinical application that can be used as of homogeneous, before the recruit modified, its transformation period should prolong, external activity reduces should be less.
The inventor has carried out on the basis of comprehensive analysis in distributing position difference and chemical reactivity difference to amino; the mode that has proposed to adopt amino protecting agent will be in the amino sealing of Methionin that protein active site and receptor binding site be easy to react and selected for use polyoxyethylene glycol activated form with nitrogen end strong response to combine is carried out specificity modification technique scheme to the protein nitrogen end; can be in the shorter time (pH height; speed of response is fast), highly selective is finished the modification of proteinic nitrogen terminal specific PEGization.
Technical solution of the present invention may further comprise the steps successively:
(A) provide a kind of amino protecting agent sealing to be in the ε amino of protein active site and receptor binding site;
(B) provide polyoxyethylene glycol after a kind of activation, untight protein nitrogen end alpha-amino group is modified;
(C) remove amino protecting agent;
(D) purify.
The protein that technical solution of the present invention is suitable for is not in the albumen of avtive spot or receptor binding site for its nitrogen end.
The amino protecting agent that technical solution of the present invention provided is dimethyl maleic anhydride (Dimethylmaleic anhydride; be called for short DMMAn); this protective material can protected protein matter the non-alpha-amino group that is easy to react; to be in the Methionin sealing that is beneficial to response location (protein active site or receptor binding site) most; make PEGization modification reaction thereafter more single-minded; needn't worry to occur non-alpha-amino group modifier; the product biological activity that obtains reduces less, and the PEGization modification reaction can carry out fast.
The consumption of amino protecting agent dimethyl maleic anhydride is 8～12 times (mol ratios) of protein alpha-amido and the amino sum of ε.
Above-mentioned sealing is in being reflected in the 0.1M phosphate buffered saline buffer (PH8.5) of ε amino of protein active site and receptor binding site and carries out.Changing protein buffer solution is in order to improve system PH, to satisfy the required condition of reaction for the 0.1M phosphate buffered saline buffer.
The used polyoxyethylene glycol activated form of technical solution of the present invention is mono methoxy polyethylene glycol (the monomethoxy PEG aldehyde of aldehyde radicalization; be called for short mPEG-aldehyde); the PEG of this activated form has the reaction advantage of protein nitrogen end; be used in combination with amino protecting agent, can realize specificity modification single-minded to protein nitrogen end alpha-amino group, fixed point.The consumption of the mono methoxy polyethylene glycol of aldehyde radicalization is 3～12 times (mol ratios) of protein alpha-amido.
The described PEGization modification reaction of technical solution of the present invention needs catalyst n aBH
3CN exists, its consumption and PEG equivalent (mol).
The described removal amino protecting agent of technical solution of the present invention is reaction mixture to be added 0.1M HCl transfer the mode of pH to implement.
Technical scheme concrete operations step of the present invention and condition are as follows:
A. changing protein buffer solution is 0.1M phosphate buffered saline buffer (pH8.5), adds 8～12
But, continue 30 minutes down at 0 ℃ doubly to the DMMAn of reacting ammonia radix amount.
But b. in above-mentioned reaction mixture, add 3～12 times of mPEG-aldehyde and NaBH to reacting ammonia radix amount
3CN reacted 0.5～1.5 hour.
C. regulate above-mentioned reaction mixture to pH5.5～6.5 with 0.1M HCl, continue 20～40 minutes down at 35～40 ℃.
D. reaction mixture is purified through cationic exchange gel SP Sepharose Big Beads and molecular sieve gel Superdex 75, removes free PEG, NaBH
3CN and unreacted albumen obtain pure polyethyleneglycol modified protein (mPEG-Protein).
The method of using modifying protein of the present invention can be used for preparing Pegylation recombinant methionyl human G-CSF (mPEG-rhG-CSF), Peg-Intron (mPEG-IFN), Pegylation tumour necrosis factor (mPEG-TNF).
Use the mPEG of the method preparation of modifying protein of the present invention
20000-rhG-CSF, molecular weight are that 39kD has increased 20kD, and its transformation period in vivo prolongs greatly, reaches more than 45 hours, and drug effect is more lasting, and its external activity is 5 * 10
7U/mg does not almost reduce
Product mPEG of the present invention
20000-rhG-CSF and rhG-CSFcssf compare:
| ??mPEG 20000-rhG-CSF | ????rhG-CSFcsf | |
| Molecular weight (kD) | ????39 | ????19 |
| External activity (U/mg) | ????5×10 7 | ????1.2×10 8 |
| Transformation period in the average body (hour) * | ????45.7 | ????3.5 |
| Iso-electric point | ????6.1 | ????6.0 |
*Compared with external like product by 100 μ g/kg dosage single subcutaneous injection in 24 hours after the lung cancer patient chemotherapy and use the inventive method reaction times short, the transformation efficiency height, the drug effect lasting period is long.
Product mPEG of the present invention
20000-rhG-CSF and external like product compare:
| The present invention | External like product | |
| Reaction method | 1. amido protecting, 2. aldehyde radical PEG modifies, and 3. goes protection | Aldehyde radical PEG modifies |
| Reaction times | 0.5 hour (2.)+0.5, hour (1.)+1 hour (3.) | 16 hours |
| Temperature of reaction (℃) | 37 | ??4 |
| Reaction product | 95％mPEG 20000-rhG-CSF, 5%-rhG-CSF (unreacted, reusable) | ??71％mPEG 20000-rhG-CSF modifies mPEG more 28% 20000-rhG-CSF (molecular weight heterogeneity, this part in the end is a depleted), 1%rhG-CSF (unreacted, reusable) |
| Molecular weight product (kD) | 39 | ??39 |
| Product external activity (U/mg) | 5×10 7 | No data |
| Transformation period in the average body (hour) * | 45.7 | ??33.2 |
| Other physico-chemical property | Molecular structure, iso-electric point be basically identical all | |
*After the lung cancer patient chemotherapy 24 hours by 100 μ g/kg dosage single subcutaneous injection
Because the technical scheme that the present invention adopts amino protecting agent will be in the amino sealing of Methionin that protein active site and receptor binding site be easy to react and selects for use polyoxyethylene glycol activated form with nitrogen end strong response to combine, thereby aspect protein modification, obtained significant beneficial effect, the protein molecular that adopts aforesaid method to form is tested, obtain following result: 1. reactivity height, 90%～95%; 2. molecular weight and structure homogeneous are the protein alpha-amido N-terminal and connect a PEG molecule, and the none protein molecular connects the product of several PEG, also do not have other sites to connect the isomery of PEG; 3. external specific activity reduces seldom, remains unchanged substantially; 4. intracorporeal active experiment is obviously long-acting, and the ANC rising than rhG-CSF prolongs about 2.5 times in the drug effect in the normal mouse body, and ANC rising amplitude is obvious.
Embodiment:
Embodiment 1.
MPEG
20000The preparation technology of-rhG-CSF:
1. ultrafiltration and concentration GC solution, the exchange buffering system is 0.1M phosphate buffered saline buffer (pH8.5), obtains the GC solution 1000ml of concentration 5mg/ml.
2. add 1.66gDMMAn, it is 0 ℃ that ice bath keeps temperature of reaction system, keeps reaction 30min.
3. in reaction system, add 52.6g mPEG
20000-acetaldehyde, stirring and dissolving is warming up to 37 ℃, adds 0.16g NaBH
3CN reacted 1 hour.Regulate above-mentioned reaction mixture to pH6.0 with 0.1M HCl, 37 ℃ continue 30 minutes.
4. with sample on the reaction product, through the SPSepharose Big Beads ion column that 10mM sodium acetate buffer (pH4.0) balance is crossed, with 10mM sodium acetate buffer (pH4.0), 0～0.45M sodium-chlor linear gradient elution, 6 column volumes, the collection main peak is mPEG
20000-rhG-CSF.Desalt through Superdex 75 posts, and exchange buffering liquid is 10mM sodium acetate (pH4.0), promptly obtains mPEG
20000-rhG-CSF stoste.
45.7 hours 95% molecular weight 39kD transformation period of transformation efficiency
External activity 5 * 10
7U/mg.
Embodiment 2
MPEG
30000The preparation technology of-rhG-CSF:
1. ultrafiltration and concentration GC solution, the exchange buffering system is 0.1M phosphate buffered saline buffer (pH8.5), obtains the GC solution 1000ml of concentration 5mg/ml.
2. add 1.83gDMMAn, it is 0 ℃ that ice bath keeps temperature of reaction system, keeps reaction 30min.
3. in reaction system, add 86.8gmPEG
20000-acetaldehyde, stirring and dissolving is warming up to 39 ℃, adds 0.176g NaBH
3CN reacted 1.5 hours.Regulate above-mentioned reaction mixture to pH6.2 with 0.1M HCl, 35 ℃ continue 40 minutes.
4. with sample on the reaction product, through the SPSepharose Big Beads ion column that 10mM sodium acetate buffer (pH4.0) balance is crossed, with 10mM sodium acetate buffer (pH4.0), 0～0.6M sodium-chlor linear gradient elution, 10 column volumes, the collection main peak is mPEG
20000-rhG-CSF.Desalt through Superdex 75 posts, and exchange buffering liquid is 10mM sodium acetate (pH4.0), promptly obtains mPEG
20000-rhG-CSF stoste.
58 hours 90% molecular weight 49kD transformation period of transformation efficiency
External activity 1 * 10
7U/mg.
Embodiment 3.
MPEG
20000The preparation technology of-IFN α-2b (Interferon, rabbit):
1. with 0.1M phosphate buffered saline buffer (pH8.5) dissolving Interferon, rabbit IFN α-2b, obtain dense
IFN α-2b solution 1000ml of degree 2mg/ml.
2. it is 0 ℃ that above-mentioned solution ice bath keeps temperature, is slowly adding 0.664gDMMAn under the stirring condition, keeps reaction 30min after the dissolving.
3. above-mentioned reaction system is warming up to 37 ℃, is slowly adding 26.3gmPEG under the stirring condition
20000-acetaldehyde, the dissolving back adds 0.032g NaBH
3CN, the dissolving back keeps reaction 0.5 hour.Regulate above-mentioned reaction mixture to pH6.0 with 0.1M HCl, 37 ℃ continue 30 minutes.
4. reaction mixture is modified through electrophoresis (SDS-PAGE) and reversed-phase HPLC (C18) mensuration～91%IFN α-2b.
5. with 20mM sodium acetate buffer (pH4.0) balance SP Sepharose Big Beads ion column.Reaction product is gone up sample with after 100 times of 20mM sodium acetate buffer (pH4.0) dilutions, and with 0～45% 20mM sodium acetate buffer (pH4.0) 1M sodium-chlor linear gradient elution 6 column volumes, the collection main peak is mPEG
20000-IFN α-2b.Purify through Superdex 75 posts are smart, and exchange buffering liquid is 10mM sodium phosphate (pH7.0), promptly obtains mPEG
20000-IFN α-2b stoste.
58 hours 91% molecular weight 39kD transformation period of transformation efficiency
External activity 1 * 10
7U/mg purity 99%.
Claims (9)
1. the method for a modifying protein alpha-amido by carbowax is characterized in that adopting amino protecting agent will be in the amino sealing of Methionin that protein active site and receptor binding site be easy to react and the mode of selecting for use polyoxyethylene glycol activated form with nitrogen end strong response to combine is carried out specificity to the protein nitrogen end and modified;
It may further comprise the steps successively:
(A) provide a kind of amino protecting agent sealing to be in the ε amino of protein active site and receptor binding site;
(B) provide polyoxyethylene glycol after a kind of activation, untight protein nitrogen end alpha-amino group is modified;
(C) remove amino protecting agent.
(D) purify.
2. the method for a kind of modifying protein alpha-amido by carbowax as claimed in claim 1 is characterized in that the protein that is suitable for is not in the albumen of avtive spot or receptor binding site for its nitrogen end.
3. a kind of method of modifying protein alpha-amido by carbowax according to claim 1; it is characterized in that amino protecting agent is dimethyl maleic anhydride (Dimethylmaleicanhydride), consumption is 8～12 times (mol ratios) of protein alpha-amido and the amino sum of ε.
4. a kind of method of modifying protein alpha-amido by carbowax according to claim 1 is characterized in that sealing being reflected in the 0.1M phosphate buffered saline buffer of the ε amino that is in protein active site and receptor binding site, and 0 ℃ continues 30 minutes down.
5. a kind of method of modifying protein alpha-amido by carbowax according to claim 1, it is characterized in that used polyoxyethylene glycol activated form is the mono methoxy polyethylene glycol of aldehyde radicalization (Monomethoxy PEG aldehyde), consumption is 3～12 times (mol ratios) of protein alpha-amido.
6. a kind of method of modifying protein alpha-amido by carbowax according to claim 1 is characterized in that the Pegylation modification reaction needs the NaBH with polyoxyethylene glycol equivalent (mol)
3CN exists, and reacts 0.5～1.5 hour.
7. a kind of method of modifying protein alpha-amido by carbowax according to claim 1 is characterized in that transferring pH to 5.5～6.5,35～40 ℃ to continue to remove in 20～40 minutes amino protecting agent with 0.1M HCl reaction mixture of last step.
8. a kind of method of modifying protein alpha-amido by carbowax according to claim 1 is characterized in that can be used for preparing Pegylation recombinant methionyl human G-CSF (mPEG-rhG-CSF), Peg-Intron (mPEG-IFN), Pegylation tumour necrosis factor (mPEG-TNF).
9. adopt the product mPEG20000-rhG-CSF of the method preparation of the described a kind of modifying protein alpha-amido by carbowax of claim 1, its molecular weight 39kD, 45.7 hours transformation period, external activity 5 * 10
7U/mg; Transformation efficiency is 90～95%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2005100425873A CN1298734C (en) | 2005-03-25 | 2005-03-25 | Method of modifying protein alpha-amido by carbowax |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2005100425873A CN1298734C (en) | 2005-03-25 | 2005-03-25 | Method of modifying protein alpha-amido by carbowax |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1687106A true CN1687106A (en) | 2005-10-26 |
| CN1298734C CN1298734C (en) | 2007-02-07 |
Family
ID=35305148
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB2005100425873A Expired - Fee Related CN1298734C (en) | 2005-03-25 | 2005-03-25 | Method of modifying protein alpha-amido by carbowax |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1298734C (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102507824A (en) * | 2011-11-01 | 2012-06-20 | 北京三元基因工程有限公司 | Analysis method for modification sites of polyethylene glycol modified protein |
| CN102585012A (en) * | 2012-02-10 | 2012-07-18 | 中国农业大学 | Preparation method for grouper alpha interferon derivative and application |
| CN101279999B (en) * | 2008-05-21 | 2012-07-18 | 大连理工大学 | Method for modifying hirudin by polyethyleneglycol assisted by anion exchange column |
| CN102585011A (en) * | 2012-02-10 | 2012-07-18 | 中国农业大学 | Preparation method for dog alpha interferon derivative and application |
| CN101176791B (en) * | 2006-11-07 | 2013-01-09 | 中国药科大学 | Amino acid communicating with polyglycol as well as manufacturing method and usage thereof |
| CN105237762A (en) * | 2015-10-27 | 2016-01-13 | 深圳市健元医药科技有限公司 | Pegylated leuprorelin |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1321134C (en) * | 2000-11-23 | 2007-06-13 | 赵剑 | Hetergeneous product of bio-active protein and its preparing process |
| CN1375502A (en) * | 2001-10-25 | 2002-10-23 | 南京药科大学 | Polyglycol modified recombinant human interferon |
| CN1511848A (en) * | 2002-12-30 | 2004-07-14 | 北京三元基因工程有限公司 | Branched chain polyethylene glycol-integrated int3erferon composition and preparation |
| CN100486997C (en) * | 2003-08-13 | 2009-05-13 | 中国科学院过程工程研究所 | Efficent polyethylene glycol activating process |
-
2005
- 2005-03-25 CN CNB2005100425873A patent/CN1298734C/en not_active Expired - Fee Related
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101176791B (en) * | 2006-11-07 | 2013-01-09 | 中国药科大学 | Amino acid communicating with polyglycol as well as manufacturing method and usage thereof |
| CN101279999B (en) * | 2008-05-21 | 2012-07-18 | 大连理工大学 | Method for modifying hirudin by polyethyleneglycol assisted by anion exchange column |
| CN102507824A (en) * | 2011-11-01 | 2012-06-20 | 北京三元基因工程有限公司 | Analysis method for modification sites of polyethylene glycol modified protein |
| CN102507824B (en) * | 2011-11-01 | 2013-10-09 | 北京三元基因工程有限公司 | Analysis method for modification sites of polyethylene glycol modified protein |
| CN102585012A (en) * | 2012-02-10 | 2012-07-18 | 中国农业大学 | Preparation method for grouper alpha interferon derivative and application |
| CN102585011A (en) * | 2012-02-10 | 2012-07-18 | 中国农业大学 | Preparation method for dog alpha interferon derivative and application |
| CN102585012B (en) * | 2012-02-10 | 2013-12-04 | 中国农业大学 | Preparation method for grouper alpha interferon derivative and application |
| CN105237762A (en) * | 2015-10-27 | 2016-01-13 | 深圳市健元医药科技有限公司 | Pegylated leuprorelin |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1298734C (en) | 2007-02-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN1298734C (en) | Method of modifying protein alpha-amido by carbowax | |
| US9238079B2 (en) | Pegylated interleukin-10 | |
| TWI232882B (en) | Poly-IFN-beta conjugates | |
| RU2485134C2 (en) | Polyethylene glycol modified interferon alpha 2b, producing and using preparation | |
| CN1638808A (en) | Coupling proteins to a modified polysaccharide | |
| EP2495258B1 (en) | Catechol polyethylene glycol derivative and protein or peptide conjugates, and method for preparing same | |
| EP2196475B1 (en) | INTERFERON ALPHA 2a MODIFIED BY POLYETHYLENE GLYCOL, ITS SYNTHESIS PROCESS AND APPLICATION | |
| EP2272875A1 (en) | Double-stranded polyethylene glycol modified growth hormone, preparation method and application thereof | |
| KR20110047169A (en) | Conjugates of catechol polyethyleneglycol derivatives with proteins or peptides and methods for their preparation | |
| RU2575796C2 (en) | Pegylated recombinant consensus interferon version conjugate and preparation method and use thereof | |
| RU2575796C9 (en) | Pegylated recombinant consensus interferon version conjugate and preparation method and use thereof | |
| CN1461762A (en) | Method of preparing branched polyethylene glycol | |
| JP5225393B2 (en) | Water-soluble polymer modified G-CSF complex | |
| CN1651463A (en) | Mono methoxy polyethylene glycol-insulin complex substance and its preparation method | |
| CN1289528C (en) | Recombined human interferon-alpha 1b compound and process for preparation | |
| CN1569892A (en) | Multi-branched polyethylene glycol and protein or polypeptide combined products and their preparation method | |
| KR100353392B1 (en) | Method of Preparing the PEG-Protein Composition Having Enhanced Biological Activity | |
| CN1338463A (en) | Method for improving stability of polypeptide in body and its application | |
| CN101711876A (en) | Polyethylene glycol modified recombinant human granulocyte colony-stimulating factor and preparation method thereof | |
| TWI293882B (en) | Polymeric modifiers and pharmaceutical compositions | |
| CN1680448A (en) | Macrogol-interferon, preparation and use thereof | |
| CN1544468A (en) | Polyethylene glycol-interferon connected with amido bond and its making method and uses | |
| CN1316436A (en) | Process for preparing water-soluble slow-releasing recombination protein | |
| CN1314708C (en) | Human alpha 1-thymic peptide composition and its preparation | |
| CN1544472A (en) | Branched polyethylene glycol-interferon and its making method and uses |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C17 | Cessation of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20070207 Termination date: 20140325 |