CN1569892A - Multi-branched polyethylene glycol and protein or polypeptide combined products and their preparation method - Google Patents

Multi-branched polyethylene glycol and protein or polypeptide combined products and their preparation method Download PDF

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CN1569892A
CN1569892A CN 200410035191 CN200410035191A CN1569892A CN 1569892 A CN1569892 A CN 1569892A CN 200410035191 CN200410035191 CN 200410035191 CN 200410035191 A CN200410035191 A CN 200410035191A CN 1569892 A CN1569892 A CN 1569892A
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fork type
protein
arm fork
polypeptide
bond
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胡小剑
张映辉
温华欢
陈剑
魏晓慧
卢洁
周向军
黄骏廉
徐宇虹
徐敏
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New Biological Technology (shanghai) Co Ltd
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New Biological Technology (shanghai) Co Ltd
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Abstract

The invention discloses a multi-branched polyethylene glycol and protein or polypeptide combined products and their preparation method, wherein the multiple arm branched type polyethylene glycol formed from three functional group small molecular compound through corresponding chemical reactions is subject to coupled reaction with proteins or polypeptides.

Description

Bond of multi-arm fork type polyethylene glycol and protein or polypeptide and preparation method thereof
Technical field
The present invention relates to bond of a kind of polyethylene glycol (PEG) and protein or polypeptide and preparation method thereof, more particularly, relate to bond of a kind of multi-arm fork type PEG and protein or polypeptide and preparation method thereof.
Background technology
Various proteins and peptides natural and restructuring all have medical serviceability, but when treatment is used, there are many factors all to make it be easy to from body-internal-circulation, clear out: the enzymolysis of protease, the filtration of kidney and immune antigen-antibody reaction. At this wherein, the various enzymes in the filtration of kidney and the blood (the particularly hydrolysis of esterase) are too short main causes of protein medicaments time of staying in blood.
Protein or polypeptide are combined with polymer such as PEG, can overcome these problems. The people such as Davis are at United States Patent (USP) 4,179, disclose in 337 PEG and protein or polypeptide (such as enzyme and insulin) in conjunction with to obtain bond, and wherein the protein immunogenicity is less and kept most of physiologically active. The people such as Nakagawa are at United States Patent (USP) 4,791, disclose in 192 PEG is combined to reduce side effect and immunogenic method with insulin. The people such as Veronese (Applied Biochem and Biotech, 11,141-152,1985) disclose with the compound activated PEG of phenyl chloroformate to modify ribalgilase and peroxidase. PEG is that a kind of form is the polyethers that the ethylene glycol condensation forms, and good biocompatibility is arranged, and can be dissolved in well water and organic phase, and is nontoxic, and it is good to drain dynamics. At present PEG commonly used have line style with two kinds of the branch types of two arms, Monfardini and disclosed a kind of two arm type PEG (the Bioconjugate Chem. of J.Milton Harris such as Shearwater company, V6 (1), 62-69,1995) etc.
As everyone knows, structure, molecular weight, molecular weight distribution and the high molecular configuration of the performance of protein drug and polymer conjugate and used macromolecular material are relevant. At present general PEG and the bond of protein or polypeptide are because the limitation that its structure, configuration, solubility and apparent molecular weight exist, the physiological action of protein drug is not given full play to, still be easy in vivo be decomposed by various enzymes and removed by glomerular filtration.
Summary of the invention
The purpose of this invention is to provide bond of a kind of multi-arm fork type PEG and protein or polypeptide and preparation method thereof, with pharmacology half-life, immunogenicity and the biologically active retention that more effectively improves protein drug.
In order to realize this purpose, the multi-arm fork type PEG that utilization of the present invention forms by trifunctional micromolecular compound and corresponding chemical reaction and protein or polypeptide carry out coupled reaction, buffer system, protein or polypeptide by choose reasonable control reaction and the molar ratio of multi-arm fork type PEG, reaction temperature, reaction time etc., and purify, thereby prepare fork type PEG protein or the polypeptide conjugates sterling of 3 arm to 8 arms. Specifically, the invention provides multi-arm fork type PEG and protein or the polypeptide conjugates that is shown below:
Figure A20041003519100081
Protein or polypeptide
Z=3-8 wherein;
M is 1-2, and protein or polypeptide all have more than one-NH2Group, the i.e. action site of multi-arm fork type PEG among the present invention. Each protein or polypeptide can be in conjunction with more than one multi-arm fork type PEG. M is preferably 1 among the present invention.
PEG ZBe multi-arm fork type PEG, preferred following 9 kinds of forms:
Figure A20041003519100091
Figure A20041003519100101
Wherein, R11、R 12、R 13、R 14、R 15、R 16、R 17、R 18It is the inertia group that seals single terminal hydroxyl of PEG; 10 straight chained alkyls that carbon is following, isopropyl or benzyl can both be used for sealing single terminal hydroxyl of PEG, can not change the character of PEG, wherein are preferably methyl;
R s1、R s2、R s3、R s4、R s5、R s6、R s7、R s8、R t1、R t2、R t3、R t4、R t5、R t6、 R t7、R t8、R v1、R v2、R v3、R v4、R v5、R v6、R v7、R v8、R m1、R m2、R m3、R m4、 R m5、R m6、R m7For-H or 4 low alkyl groups below the carbon; Be preferably methyl;
R k1、R k2、R k3、R k4、R k5、R k6、R k7、R k8The another one terminal hydroxyl that is PEG forms in activation process; Rn1、R n2、R n3、R n4、R n5、R n6、R n7、R n8、R n9、R n10It is the inside linking group of three functional groups. These groups can be the low-grade alkylidenes of 0-10 carbon, are preferably-CH2-、-CH 2CH 2-、-(CH 2) 3-、-CH(CH 3) 2-、-(CH 2) 4-、-CH(CH 3)(CH 2CH 3)-、 -C(CH 3) 2CH 2-、-CH(CH 3)CH 2CH 2-、-CH 2CH(CH 3)CH 2-；
W 1、W 2、W 3、W 4、W 5、W 6、W 7、W 8、W 11、W 12、W 13、W 14、W 15、W 16For being that PEG after the activation is combined formed group with three functional groups, can be-O-,-S-,-NH-,-NHCH2-、-NHCH 2CH 2-、-NH(CH 2) 3-、-NHCH(CH 3) 2-、-NH(CH 2) 4-、 -NHCH(CH 3)(CH 2CH 3)-、-NHC(CH 3) 2CH 2-、-NHCH(CH 3)CH 2CH 2-、 -NHCH 2CH(CH 3)CH 2-, be preferably-NH-and-NHCH2-；
X1, y1, z1, x2, y2, z2, x3, y3, z3, x4, y4, z4, x5, y5, z5, x6, y6, z6, x7, y7, z7, x8, y8 and z8 are the Any Digit combination, so that the molecular weight of multi-arm fork type PEG is in the daltonian scope of 4000-100000.
Such as one of them eight arm fork type PEG-insulin bonder:
Figure A20041003519100131
Other multi-arms PEG protein or polypeptide conjugates have the structure similar with above-mentioned bond, the main difference according to concrete arm number and concrete medicine of concrete structure and different. Have any protein of physiologically active or polypeptide and all can be used for preparing bond with above-mentioned PEG, if above-mentioned protein or polypeptide contain at least one can be for the amino of condensation.
Multi-arm fork type PEG of the present invention compares with the bond of protein or polypeptide with two arm PEG with the bond of protein or polypeptide and the linearity of same molecular amount, one, because the hydrodynamic volume of multi-arm fork type PEG is large, when it after being combined in certain position of protein or polypeptide, because sterically hindered effect, other positions just are difficult to the multi-arm fork type PEG reaction with another molecule, thereby raising is selective to binding site, and the biologically active retention of such protein drug is higher; Its two because the existence of multi-arm structure so that multi-arm fork type PEG can more effectively stop large molecule or cell near protein or polypeptide surface, thereby further improves the time that bond circulates in vivo, reduce immunoreactive generation; Its three because the hydrodynamic volume of multi-arm fork type PEG is large, the kidney clearance rate of multi-arm fork type PEG of the present invention and protein or polypeptide conjugates reduces greatly, thereby has improved widely the interior action time of body of bond.
Description of drawings
MALDI-TOF (substance assistant laser desorpted/ionization massspectrum) spectrogram of three arm fork type PEG-granulocyte colony stimulating factors in Fig. 1 embodiments of the invention 2.
The SDS-PAGE of three arm fork type PEG-protein conjugates among Fig. 2 embodiments of the invention 1, the 4-11 (dodecyl sodium sulfate one polyacrylamide gel electrophoresis) figure.
The MALDI-TOF spectrogram of four arm fork type PEG-recombinant human interferon betas in Fig. 3 embodiments of the invention 20.
The interferon of unmodified, PEG-INTRON, three arm fork type PEG-interferon are with the concentration decline curve map of tail vein injection mode in the rat body in Fig. 4 embodiments of the invention 49.
Four arm fork type PEG-granulocyte colony stimulating factors are to the drug effect figure of mouse experiment in Fig. 5 embodiments of the invention 50.
The specific embodiment
Below, the invention will be further described by specific embodiment. These embodiment are just in order to describe, rather than are used for limiting scope of the present invention.
Embodiment 1The preparation of three arm fork type PEG (molecular weight is 13.3K, and molecular formula is as follows)-interferon alpha 2 b bonds
Lysine (439mg, 3mmol) is dissolved in the water of 20ml pH value 8.0-8.3, then in 3 hours to wherein dividing 6 batches evenly to add mPEG2CO2PhNO 2(wherein an arm is 5,400 for two arm mono methoxy PEG p-nitrophenyl phenolic esters, molecular weight 11,000, and another arm is 5,600,11g, 1mmol, wherein mono methoxy PEG can be abbreviated as mPEG), the NaOH with 0.2N comes the pH of maintenance system 8.3 simultaneously. After stirred overnight at room temperature, reactant is cooled to 0 ℃, and with the hydrochloric acid of 2N the pH value of system is adjusted to 3. From water, extract impurity with ether first, extract three times continuously with chloroform again, add absolute ether after extract is concentrated, get white precipitate, the precipitation of gained obtains the mono-substituted lysine of mPEG2 (mPEG2-mono-Lysine) behind twice recrystallization of ethanol.
In the anhydrous methylene chloride that is dissolved with the said goods (9.9g, 0.9mmol) (20ml), add triethylamine (TEA) until the pH value reaches 8. MPEGCO2PhNO 2(line style mPEG p-nitrophenyl phenolic ester, molecular weight 2300,2.415g, 1.05mmol) be minute 6 batches of even addings in the reactant liquor within 3 hours, use simultaneously the pH value of TEA maintenance system about 8. Reactant was cooled to room temperature after refluxing 72 hours, after concentrating, filtered, and used ether sedimentation, then used a small amount of ethyl alcohol recrystallization. Excessive mPEGCO2PhNO 2Na at pH9-102CO 3Stir in the buffer solution and be hydrolyzed after spending the night, solution is cooled to 0 ℃, and with the hydrochloric acid of 2N the pH value of system is adjusted to 3. Then by the p-nitrophenol in the extracted by ether solution. Extract three times continuously with chloroform, then extract drying, concentrated rear with the absolute ether precipitation is recrystallized with absolute ethyl alcohol again. Products therefrom is further purified with QAE Sephadex A50 post (5 * 80cm, the borate buffer solution of leacheate: pH8.9), gets mPEG3CO2H。
The mPEG3CO that present embodiment is prepared2H (7.98g, 0.6mmol) be dissolved in the anhydrous methylene chloride (20ml), be cooled to 0 ℃ after, to wherein adding N-maloyl imines (0.138g, 1.2mmol) and dicyclohexylcarbodiimide (DCC, 0.48g, 1.2mmol), after the stirred overnight at room temperature, filter, with the absolute ether precipitation, again through re-crystallizing in ethyl acetate, get pure products mPEG3CO after the concentrating filter liquor2The Su product.
With above-mentioned mPEG3CO2Su (100mg) joins interferon-' alpha ' 2b at ambient temperature, and (concentration is 5mg/ml, 4ml, buffering liquid is the 50mM phosphate buffer of pH7.0) in, react after 1 hour, add the glycine 100 μ l cessation reactions of 1M, obtain three arm fork type PEG-interferon alpha 2 b bond crude products.
Twice of TSK-GEL Super SW3000 molecular sieve column purifying of three arm fork type PEG-interferon alpha 2 b bond crude products. With loading behind the chromatographic column usefulness 50mM NaCl solution equilibria of pH7.5, loading is 2% of column volume, and 280nm detects, and collects first eluting peak, obtains three arm fork type PEG-interferon alpha 2 b bond sterlings.
Embodiment 2The preparation of three arm fork type PEG (molecular weight is 15K, and molecular formula is as follows)-granulocyte colony stimulating factor (G-CSF) bond
According to the method for embodiment 1, the mPEG2CO take molecular weight as 10K2PhNO 2With molecular weight be the mPEGCO of 5K2PhNO 2Be raw material, prepare the mPEG3CO that molecular weight is 15K2Su; The granulocyte colony stimulating factor of modifying through PEG prepares according to method described in the embodiment 1 with this reagent.
Prepared bond through the MALDI-TOF mass spectroscopy (the method for testing reference: Michael Grace, J.of interferon and cytokine research, 2001,1103-1115), measurement result is as shown in Figure 1. M/z 34641 peaks are single electric charge peak of bond among the figure; M/z 18966 peaks are single electric charge peak of G-CSF of trace extremely; M/z 17500 is the double charge peak of bond; Because G-CSF content is very low, so its double charge peak can not show in the drawings. As seen from the figure, three arm fork type PEG and G-CSF successfully link together. According to document (Michael Grace, J.of interferon and cytokine research, 2001,1103-1115) method, can reach a conclusion: the purity of this bond is very high, is more than 99%.
Embodiment 3The preparation of three arm fork type PEG (molecular weight is 90K, and molecular formula is as follows)-interferon alpha 2 b bonds
According to the method for embodiment 1, the mPEG2CO take molecular weight as 60K2PhNO 2With molecular weight be the mPEGCO of 30K2PhNO 2Be raw material, prepare the mPEG3CO that molecular weight is 90K2Su. The interferon of modifying through PEG prepares according to method described in the embodiment 1 with this reagent.
Embodiment 4-13
The bond for preparing among the embodiment The buffer system of used protein or polypeptide, reaction among the embodiment Other reactions steps and condition among the embodiment
Embodiment 4 three arm fork type PEG (with embodiment 1)-recombinant human interferon-alpha 1a bonds Recombined human interferon-alpha 1a (concentration is 6mg/ml, 3ml), buffering liquid is the 50mM phosphate buffer of pH7.5. With embodiment 1
Embodiment 5 three arm fork type PEG (with embodiment 1)-recombinant human interferon alpha 1 b bonds Recombined human interferon-alpha 1 b (concentration is 6mg/ml, 3ml), buffering liquid is the 50mM phosphate buffer of pH7.5. With embodiment 1
Embodiment 6 three arm fork type PEG (with embodiment 1)-recombinant human interferon alpha-2 bonds Recombinant human interferon alpha 2a (concentration is 6mg/ml, 3ml), buffering liquid is the 50mM phosphate buffer of pH7.5. With embodiment 1
Embodiment 7 three arm fork type PEG (with embodiment 1)-recombinant human interferon beta bonds Recombinant human interferon beta (concentration is 2mg/ml, 5ml), buffering liquid is the 50Mm phosphate buffer of pH7.5. With embodiment 1
Embodiment 8 three arm fork type PEG (with embodiment 1)-recombinant human interferon gamma bonds Recombinant human interferon gamma (concentration is 2mg/ml, 5ml), buffering liquid is the 50mM phosphate buffer of pH7.5. With embodiment 1
Embodiment 9 three arm fork type PEG (with embodiment 1)-recombinant human granulocyte colony stimulating factor bonds Recombinant human granulocyte colony stimulating factor (concentration is 6mg/ml, 3ml), buffering liquid is the 50mM phosphate buffer of pH7.5. With embodiment 1
Embodiment 10 3 arm fork type PEG (with embodiment 1)-recombinant humangranulocyte-macrophage colony stimulatory factor bonds Recombinant humangranulocyte-macrophage colony stimulatory factor (concentration is 6mg/ml, 3ml), buffering liquid is the 50mM phosphate buffer of pH7.5. With embodiment 1
Embodiment 11 3 arm fork type PEG (with embodiment 1)-recombinated interleukin-2 bonds Recombinated interleukin-2 (concentration is 3mg/ml, 5ml), buffering liquid is the 50mM phosphate buffer of pH8.5. With embodiment 1
Embodiment 12 3 arm fork type PEG are (with implementing Restructuring arginine deiminase (concentration is 2mg/ml, 5ml), buffering liquid is With embodiment 1
Example 1)-restructuring arginine deiminase bond The 50mM phosphate buffer of pH7.5.
Embodiment 13 3 arm fork type PEG (with embodiment 1)-rh-insulin's bonds Rh-insulin's (concentration is 2mg/ml, 5ml), buffering liquid is the 50mM phosphate buffer of pH7.5. With embodiment 1
And the product among embodiment 1, the 4-11 is carried out (the experimental technique reference: Wang Jiazheng of SDS-PAGE electrophoresis detection, the protein technical manual, science tech publishing house, front page), successful preparation with the proof bond, specifically see Fig. 2, wherein according to from left to right order, each band (lane) is followed successively by:
Three arm fork type PEG-recombinant human interferon beta bonds among lane1: the embodiment 7;
Three arm fork type PEG-recombinant human interferon-alpha 1a bonds among lane2: the embodiment 4;
Three arm fork type PEG-recombinant human interferon alpha-2 bonds among lane3: the embodiment 6;
Three arm fork type PEG-recombinant human interferon alpha 1 b bonds among lane4: the embodiment 5;
Three arm fork type PEG-recombinant human interferon alpha 2 b bonds among lane5: the embodiment 1;
Three arm fork type PEG-recombinant human granulocyte colony stimulating factor bonds among lane6: the embodiment 9;
Three arm fork type PEG-recombinant human interferon gamma bonds among lane7: the embodiment 8;
Three arm fork type PEG-recombinated interleukin-2 bonds among lane8: the embodiment 10;
Lane9: low-molecular-weight mark (marker), molecular weight is respectively 14k, 20k, 31k, 43k, 66k, 97k;
Three arm fork type PEG-recombinant humangranulocyte-macrophage colony stimulatory factor bonds among lane10: the embodiment 11;
As seen from the figure, above-mentioned bond all successfully prepares.
Embodiment 14The preparation of four arm fork type PEG (molecular weight 16K, molecular formula is as follows)-recombinant human granulocyte colony stimulating factor (G-CSF) bond
Figure A20041003519100181
It is in 8.0 the borate buffer solution, then to wherein adding mPEG2CO that FE-5 (365mg, 2mmol) is dissolved in 100ml pH value2Su (molecular weight 8,000,32g, 4mmol), the NaOH with 0.2N comes the pH of maintenance system 8.0 simultaneously. , after 24 hours reactant is extracted three times continuously with deionized water in stirring at room, be added dropwise in the absolute ether after extract is concentrated, obtain white precipitate, gained precipitates behind twice recrystallization of ethanol, obtains mPEG4CO2The H crude product. Crude product obtains pure mPEG4CO after DEAE Sephadex FF post separates2H。
With above-mentioned mPEG4CO 2H (9.6g, 0.6mmol) is dissolved in the anhydrous methylene chloride (20ml), be cooled to 0 ℃ after, prepare pure products mPEG4CO according to the method for embodiment 12Su。
With above-mentioned mPEG4CO 2Su (100mg) joins G-CSF at ambient temperature, and (concentration is 4mg/ml, 5ml, buffering liquid is the 50mM phosphate buffer of pH6.5) in, react the glycine 50 μ l cessation reactions that add 1M after 1 hour, obtain four arm fork type PEG-G-CSF bond crude products, and according to the Methods For Purification of embodiment 1.
Embodiment 15The preparation of four arm fork type PEG (molecular weight 4K, molecular formula is as follows)-G-CSF bonds
According to the method for embodiment 14, the mPEG2CO take molecular weight as 2K2Su is that raw material prepares the mPEG4CO that molecular weight is 4K2Su; And according to prepare the G-CSF that modifies through this reagent by embodiment 14 methods.
Embodiment 16-24
The bond for preparing in the enforcement The buffer system of used protein or polypeptide, reaction among the embodiment Other raw materials, reactions steps and condition among the embodiment
Embodiment 16 4 arm fork type PEG (with embodiment 14)-recombinant humangranulocyte-macrophage colony stimulatory factor (GM-CSF) bond GM-CSF (concentration is 2mg/ml, 5ml), buffering liquid is the 50mM phosphate buffer of pH6.5. With embodiment 14
Embodiment 17 4 arm fork type PEG (with embodiment 14)-recombinant human interferon-alpha 1a bonds Recombined human interferon-alpha 1a (concentration is 6mg/ml, 3ml), buffering liquid is the 50mM phosphate buffer of pH7.5. With embodiment 14
Embodiment 18 4 arm fork type PEG (with embodiment 14)-recombinant human interferon alpha 1 b bonds Recombined human interferon-alpha 1 b (concentration is 6mg/ml, 3ml), buffering liquid is the 50mM phosphate buffer of pH7.5. With embodiment 14
Embodiment 19 4 arm fork type PEG (with embodiment 14)-recombinant human interferon alpha 2 b bonds Recombined human interferon-alpha 2b (concentration is 6mg/ml, 3ml), buffering liquid is the 50mM phosphate buffer of pH7.5. With embodiment 14
Embodiment 20 4 arm fork type PEG (with embodiment 14)-recombinant human interferon beta bonds Recombinant human interferon beta (concentration is 2mg/ml, 5ml), buffering liquid is the 50mM phosphate buffer of pH7.5. With embodiment 14
Embodiment 21 4 arm fork type PEG (with embodiment 14)-recombinant human interferon gamma bonds Recombinant human interferon gamma (concentration is 2mg/ml, 5ml), buffering liquid is the 50mM phosphate buffer of pH7.5. With embodiment 14
Embodiment 22 4 arm fork type PEG (with embodiment 14)-recombinated interleukin-2 bonds Recombinated interleukin-2 (concentration is 3mg/ml, 5ml), buffering liquid is the 50mM phosphate buffer of pH8.5. With embodiment 14
Embodiment 23 4 arm fork type PEG (with embodiment 14)-restructuring arginine deiminase bond Restructuring arginine deiminase (concentration is 2mg/ml, 5ml), buffering liquid is the 50mM phosphate buffer of pH7.5. With embodiment 14
Embodiment 24 4 arm fork type PEG (with embodiment 14)-rh-insulin's bonds Rh-insulin's (concentration is 2mg/ml, 5ml), buffering liquid is the 50mM phosphate buffer of pH7.5. With embodiment 14
Wherein four arm fork type PEG-recombinant human interferon beta bonds among the embodiment 20 are through the MALDI-TOF mass spectroscopy, the result as shown in Figure 3, wherein m/z 36652 is single electric charge peak of bond; M/z 20282 is single electric charge peak of a small amount of residual interferon beta; M/z 18277 is the double charge peak of bond; M/z 10135 is the double charge peak of a small amount of residual interferon beta. As seen from the figure, four arm fork type PEG and recombinant human interferon beta successfully link together. According to document (Michael Grace, J.of interferon and cytokine research, 2001,1103-1115) method, can reach a conclusion: the purity of this bond is very high, is approximately 98%.
Embodiment 25The preparation of four arm fork type PEG (molecular weight is 40K, and molecular formula is as follows)-streptokinase bonds
Prepare pure products mPEG3CO according to embodiment 1 method2PhNO 2(molecular weight 30K, single armed length is respectively 10K), and then prepare the mono-substituted lysine of mPEG3 (mPEG3-mono-lysine). In the anhydrous methylene chloride that is dissolved with mPEG3-mono-lysine (15g, 0.5mmol) (20ml), add TEA until the pH value reaches 8.0, mPEGCO2PhNO 2(molecular weight is 10K, 6g, 0.6mmol) be minute 6 batches of even addings in the reactant liquor within 3 hours, use simultaneously the pH value of TEA maintenance system about 8. Reactant was cooled to room temperature after refluxing 72 hours, and concentrated rear the filtration used ether sedimentation, then uses a small amount of ethyl alcohol recrystallization. Excessive mPEGCO2PhNO 2Na at pH9-102CO 3Stir in the buffer solution and be hydrolyzed after spending the night, solution is cooled to 0 ℃, and with the hydrochloric acid of 2N the pH of system is adjusted to 3. Then by the p-nitrophenol in the extracted by ether solution. Extract three times continuously with chloroform again, the extract drying, concentrated rear with the absolute ether precipitation, then be recrystallized with absolute ethyl alcohol. Products therefrom is further purified with QAE Sephadex A50 post (5 * 80cm, the borate buffer of leacheate: pH8.9), gets the mPEG4CO that molecular weight is 40K2H。
The mPEG4CO that present embodiment is prepared2H (20g, 0.5mmol) is dissolved in the anhydrous methylene chloride (20ml), be cooled to 0 ℃ after, to wherein adding N-maloyl imines (0.115g, 1.0mmol) and DCC (0.40,1.0mmol), get pure products mPEG4CO according to embodiment 1 method2Su。
The mPEG4CO that present embodiment is prepared2Su (100mg) joins streptokinase at ambient temperature, and (concentration is 5mg/ml, 4ml, buffering liquid is the 50mM phosphate buffer of pH7.0) in, react the glycine 30 μ l cessation reactions that add 1M after 1 hour, obtain four arms fork type-streptokinase bond crude product. Four arm fork type PEG-streptokinase bonds are according to the Methods For Purification of embodiment 1.
Embodiment 26The preparation of four arm fork type PEG (molecular weight 100K, molecular formula is as follows) protein or polypeptide conjugates
According to the method for embodiment 25, the mPEG3CO take molecular weight as 70K2PhNO 2With molecular weight be the mPEGCO of 30K2PhNO 2Be raw material, prepare the mPEG4CO that molecular weight is 100K2Su. And prepare protein or the polypeptide of modifying through this reagent according to the method for embodiment 25.
Embodiment 27The preparation of five arm fork type PEG (molecular weight 23.3K, molecular formula is as follows)-IL-2 bonds
Interleukin 2 is called again il-22, note IL-2.
Prepare the mono-substituted lysine of mPEG3 (molecular weight 13.3K, brachium is respectively 5.6K, 5.4K, 2.3K, mPEG3-mono-lysine) according to embodiment 1 method. In the anhydrous methylene chloride that is dissolved with mPEG3-mono-lysine (2.66g, 0.2mmol) (20ml), add TEA until the pH value reaches 8.0, mPEG2CO2PhNO 2(molecular weight is 10K, 2.5g, 0.25mmol) be minute 6 batches of even addings in the reactant liquor within 3 hours, use simultaneously the pH value of TEA maintenance system about 8. Reactant was cooled to room temperature after refluxing 72 hours, after concentrating, filtered, and used ether sedimentation, then used a small amount of ethyl alcohol recrystallization. Excessive mPEG2CO2PhNO 2Na at pH9-102CO 3Stir in the buffer solution and be hydrolyzed after spending the night, solution is cooled to 0 ℃, and with the hydrochloric acid of 2N the pH of system is adjusted to 3. Then by the p-nitrophenol in the extracted by ether solution. Extract three times continuously with chloroform, then extract drying, concentrated rear with the absolute ether precipitation is recrystallized with absolute ethyl alcohol again. Products therefrom is further purified with QAE Sephadex A50 post (5 * 80cm, the borate buffer of leacheate: pH8.9), gets mPEG5CO2H。
At 0 ℃, (molecular weight is 23.3K to being dissolved with above-mentioned mPEG5COOH, 4.66g, add p-nitrophenol (0.167g, 1.2mmol) and DCC (0.48g in anhydrous methylene chloride 0.2mmol) (20ml), 1.2mmol), after the stirred overnight at room temperature, filter, precipitate with absolute ether after the concentrating filter liquor, through re-crystallizing in ethyl acetate, get pure products mPEG5COOPhNO again2。
With above-mentioned mPEG5CO2PhNO 2(concentration is 2mg/ml (120mg) to join at ambient temperature IL-2,10ml, buffering liquid is the 50mM phosphate buffer of pH8.0) in, react the glycine 50 μ l cessation reactions that add 1M after 1 hour, obtain five arm fork type PEG-IL-2 bond crude products. Five arm fork type PEG-IL-2 bonds are according to the Methods For Purification of embodiment 1.
Embodiment 28The preparation of five arm fork type PEG (molecular weight is 6K, and molecular formula is as follows) protein or polypeptide conjugates
According to the method for embodiment 27, the mPEG3CO take molecular weight as 4K2PhNO 2With molecular weight be the mPEG2CO of 2K2PhNO 2For raw material prepares the mPEG5CO that molecular weight is 6K2PhNO 2 And prepared protein or the polypeptide conjugates of modifying through this reagent by the method for embodiment 27.
Embodiment 29The preparation of five arm fork type PEG (molecular weight is 90K, and molecular formula is as follows) protein or polypeptide conjugates
According to the method for embodiment 27, the mPEG3CO take molecular weight as 60K2PhNO 2With molecular weight be the mPEG2CO of 30K2PhNO 2Be raw material, prepare the mPEG5CO that molecular weight is 90K2PhNO 2 And make protein or the polypeptide conjugates of modifying through this reagent according to embodiment 27 methods.
Embodiment 30Five arm fork type PEG (molecular weight is 25K, and molecular formula is as follows)-ser125The preparation of IL-2 bond
Figure A20041003519100242
According to embodiment 14, with mPEG2CO2H (molecular weight is 10K, and every arm molecular weight is 5K, 30g, 3mmol) prepares mPEG4CO2H。
According to embodiment 27, with the prepared mPEG4CO of present embodiment2H (26g, 1.3mmol) and mPEGCO2PhNO 2(molecular weight is 5K, 7.5g, 1.5mmol) prepares mPEG5PhNO2。
According to embodiment 27, mPEG5CO2PhNO 2(molecular weight is 25K, 120mg) and IL-2 (concentration is 2mg/ml, 10ml) prepare five arm fork type PEG-ser125IL-2 bond sterling.
Embodiment 31The preparation of five arm fork type PEG (molecular weight is 5K, and molecular formula is as follows) protein or polypeptide conjugates
Figure A20041003519100251
According to the method for embodiment 30, the mPEG4CO take molecular weight as 4K2PhNO 2With molecular weight be the mPEGCO of 1K2PhNO 2Be raw material, prepare the mPEG5PhNO that molecular weight is 5K2 And made protein or the polypeptide conjugates of modifying through this reagent by the method for embodiment 30.
Embodiment 32The preparation of five arm fork type PEG (molecular weight is 100K, and molecular formula is as follows) protein or polypeptide conjugates
Figure A20041003519100252
MPEG4CO take molecular weight as 80K2PhNO 2With molecular weight be the mPEGCO of 20K2PhNO 2Be raw material, prepare the mPEG5PhNO that molecular weight is 100K2 And made protein or the polypeptide conjugates of modifying through this reagent by the method for embodiment 30.
Embodiment 33The preparation of six arm fork type PEG (molecular weight 24K, molecular formula is as follows)-HRBC's auxin (EPO) bond:
According to embodiment 1, with mPEG2CO2PhNO 2(molecular weight 10K, wherein an arm is 5K, another arm is 5K, 10g, 1mmol), and mPEGCO2PhNO 2(molecular weight 2K, 2.1g, 1.05mmol) prepares mPEG3CO2Su (molecular weight 12K).
According to embodiment 14, with the prepared mPEG3CO of present embodiment2Su (10.8g, 0.9mmol) obtains mPEG6CO2Su (molecular weight is 24K).
With above-mentioned mPEG6CO2Su (240mg) joins EPO at ambient temperature, and (concentration is 2mg/ml, 10ml, buffering liquid is the 50mM phosphate buffer of pH8.0) in, react the glycine 30 μ l cessation reactions that add 1M after 1 hour, obtain the crude product of six arm fork type PEG (molecular weight is 24K)-EPO bond. The six arm fork type PEG-EPO bonds Methods For Purification of embodiment 1.
Embodiment 34The preparation of six arm fork type PEG (molecular weight is 24K, and molecular formula is as follows)-EPO bonds
According to embodiment 14, with mPEG2CO2H (molecular weight is 8K, 40g, 5mmol) prepares mPEG4CO2H. Continuation according to embodiment 27 with the prepared mPEG4CO of present embodiment2H (molecular weight 16K, 32g, 2mmol) and mPEG2CO2PhNO 2(molecular weight is 8K, 17.6g, 2.2mmol) prepares mPEG6PhNO2 According to the preparation of embodiment 33 methods and the six arm fork type PEG-EPO bonds of purifying.
Embodiment 35The preparation of seven arm fork type PEG (molecular weight 20K, molecular formula is as follows)-tumor necrosis factor α bonds
Figure A20041003519100271
TNF (tumor necrosis factor, TNF) is can cause the tumor tissues hemorrhagic necrosis and have many-sided cell factor.
According to embodiment 1, with mPEG2CO2PhNO 2(molecular weight 8K, 8g, 1mmol) and mPEGCO2PhNO 2(molecular weight 2K, 2.1g, 1.05mmol) prepares mPEG3-mono-lysine (molecular weight is 10K).
According to embodiment 14, with mPEG2CO2Su (molecular weight 5K, 20g, 4mmol) prepares mPEG4CO2PhNO 2(molecular weight is 10K).
Continuation is according to embodiment 27, take mPEG3-mono-lysine (molecular weight is as 10K, 2g, 0.2mmol) and mPEG4CO2PhNO 2(molecular weight is 10K, 2.5g, 0.25mmol) prepares mPEG7CO 2PhNO 2(molecular weight 20K).
With above-mentioned mPEG7CO 2PhNO 2(molecular weight is 20K, (concentration is 2mg/ml 240mg) to join at ambient temperature TNF α, 10ml, buffering liquid is the 50mM phosphate buffer of pH8.0) in, react the glycine 30 μ l cessation reactions that add 1M after 1 hour, obtain seven arm fork type PEG-TNF alpha conjugates crude products. The seven arm fork type PEG-TNF alpha conjugates Methods For Purification of embodiment 1.
Embodiment 36The preparation of seven arm fork type PEG (with embodiment 35)-TNF β bonds
According to the method for embodiment 35, with mPEG7CO 2PhNO 2(200mg) prepare seven arm fork type PEG-TNF β bonds with TNF β (concentration is 4mg/ml, and 5ml, buffering liquid are the 50mM phosphate buffer of pH8.0).
Embodiment 37The preparation of eight arm fork type PEG (molecular weight 40K, molecular formula is as follows)-insulin bonders
Method according to embodiment 14, preparation mPEG4COSu (molecular weight is 20K) is take prepared mPEG4COSu as raw material, with FE-5 (365mg, 2mmol) being dissolved in 200ml pH value is in 8.0 the borate buffer solution, then to wherein adding mPEG4CO2Su (80g, 4mmol), the NaOH with 0.2N comes the pH of maintenance system 8.0 simultaneously. , after 24 hours reactant is extracted three times continuously with deionized water in stirring at room, be added dropwise in the absolute ether after extract is concentrated, obtain white precipitate, gained precipitates behind twice recrystallization of ethanol, obtains mPEG8CO2The H crude product. Crude product obtains pure mPEG8CO after DEAE Sephadex FF post separates2H. With above-mentioned mPEG8CO2H (24g, 0.6mmol) is dissolved in the anhydrous methylene chloride (40ml), prepares mPEG8CO according to the method for embodiment 12Su。
With above-mentioned mPEG8CO2Su (100mg) joins insulin at ambient temperature, and (concentration is 6mg/ml, 1ml, buffering liquid is the 50mM phosphate buffer of pH6.5) in, react the glycine 50 μ l cessation reactions that add 1M after 1 hour, obtain eight arm fork type PEG-insulin bonder crude products. Eight arm fork type PEG-insulin bonders are purified with the method for embodiment 1.
Embodiment 38The preparation of eight arm fork type PEG (molecular weight 6K, molecular formula is as follows) protein or polypeptide conjugates
Figure A20041003519100291
According to the method for embodiment 37, the mPEG5CO take molecular weight as 4K2PhNO 2With molecular weight be the mPEG3CO of 2K2PhNO 2Be raw material, prepare the mPEG8CO that molecular weight is 6K2Su; And prepare protein or the polypeptide conjugates of modifying through this reagent according to the method for embodiment 37.
Embodiment 39The preparation of eight arm fork type PEG (molecular weight 100K, molecular formula is as follows) protein or polypeptide conjugates
Figure A20041003519100292
According to the method for embodiment 37, the mPEG5CO take molecular weight as 70K2PhNO 2With molecular weight be the mPEG3CO of 30K2PhNO 2Be raw material, prepare the mPEG8CO that molecular weight is 100K2Su. And prepare protein or the polypeptide conjugates of modifying through this reagent according to the method for embodiment 37.
Embodiment 40-48：
The bond for preparing among the embodiment The buffer system of used protein or polypeptide, reaction among the embodiment Other raw materials, reactions steps and condition among the embodiment
Embodiment 40 8 arm fork type PEG (with embodiment 37)-recombinant human interferon-alpha 1a bonds Recombined human interferon-alpha 1a (concentration is 6mg/ml, 3ml), buffering liquid is the 50mM phosphate buffer of pH7.5. With embodiment 37
Embodiment 41 8 arm fork type PEG (with embodiment 37)-recombinant human interferon alpha 1 b bonds Recombined human interferon-alpha 1 b (concentration is 6mg/ml, 3ml), buffering liquid is the 50mM phosphate buffer of pH7.5. With embodiment 37
Embodiment 42 8 arm fork type PEG (with embodiment 37)-recombinant human interferon alpha-2 bonds Recombinant human interferon alpha 2a (concentration is 6mg/ml, 3ml), buffering liquid is the 50mM phosphate buffer of pH7.5. With embodiment 37
Embodiment 43 8 arm fork type PEG (with embodiment 37)-recombinant human interferon alpha 2 b bonds Recombined human interferon-alpha 2b (concentration is 6mg/ml, 3ml), buffering liquid is the 50mM phosphate buffer of pH7.5. With embodiment 37
Embodiment 44 8 arm fork type PEG (with embodiment 37)-recombinant human interferon beta bonds Recombinant human interferon beta (concentration is 2mg/ml, 5ml), buffering liquid is the 50mM phosphate buffer of pH7.5. With embodiment 37
Embodiment 45 8 arm fork type PEG (with embodiment 37)-recombinant human interferon gamma bonds Recombinant human interferon gamma (concentration is 2mg/ml, 5ml), buffering liquid is the 50mM phosphate buffer of pH7.5. With embodiment 37
Embodiment 46 8 arm fork type PEG (with embodiment 37)-G-CSF bonds G-CSF (concentration is 6mg/ml, 3ml), buffering liquid is the 50mM phosphate buffer of pH7.5. With embodiment 37
Embodiment 47 8 arm fork type PEG (with embodiment 37)-IL2 bonds IL2 (concentration is 3mg/ml, 5ml), buffering liquid is the 50mM phosphate buffer of pH8.5. With embodiment 37
Embodiment 48 8 arm fork type PEG (with embodiment 37)-restructuring arginine deiminase bond Restructuring arginine deiminase (concentration is 2mg/ml, 5ml), buffering liquid is the 50mM phosphate buffer of pH7.5. With embodiment 37
Embodiment 49The experiment of the pharmacokinetics of multi-arm fork type PEG-protein or polypeptide conjugates here in the embodiment 1 three arm fork type PEG (molecular weight is as 13.3K) of preparation-interferon IFN α 2b bond as example, detected after multi-arm fork type PEG modifies, on medicine die-away time in vivo and the impact of attenuation curve.
With the negative control group of rhIFN α 2b of unmodified, PEG-INTRON (Schering-Plough Corporation company product, connect for molecular weight be the line style PEG of 12K) positive control group carries out pharmacokinetic studies. Experimental subjects is the rat of 150-170g, and tail vein injection, ID are 400 μ g/ml. Negative control group is injection in continuous 4 days; Test group and positive controls are the single dose injection. EUSA (ELISA) method detects, and detects used antibody and is anti--rhIFN α, and the pharmacokinetic analysis model is the two compartment model of weight w=1/CC.
Experiment finds, the half-life of the PEG3-IFN bond that obtains take three arm fork type PEG modified interferons is 4.02 times of IFN, is 202% of the PEG-INTRON of Shering-Plough company, and concrete outcome is as shown in table 1.
PEG3-IFN PEG-INTRON IFN
Half-life (h) 27.1397 13.385 6.7530
Table 1: the half-life comparison sheet of three kinds of materials
Experiment finds that also the concentration decline curve of medicine in the rat body also shown three arm PEG-IFN very significantly with respect to other two kinds of testers, and prolong circulation timei in vivo, and average blood concentration has improved, and concrete outcome is seen Fig. 4.
Embodiment 50The effect experiment of multi-arm fork type PEG-protein or polypeptide conjugates
Here respectively in embodiment 1 and the embodiment 14 the multi-arm fork type PEG-protein of preparation or polypeptide conjugates detected after multi-arm fork type PEG modifies as example, on the impact of the drug effect of protein or polypeptide.
1. the antiviral activity of three arm fork type PEG-interferon alpha 2 b bonds detects
Detect according to the antiviral activity of " cell factor research methodology " (Sun Weimin, People's Health Publisher, 1999) to rhIFN α 2b, three arm fork type PEG-IFN. The specific activity of finding the interferon of unmodified is 3.45 * 107IU/mg, the specific activity of the interferon of modifying through three arm fork type PEG is 4.0 * 106IU/mg, clearly, the antiviral activity of interferon IFN has approximately increased by 10 times after modifying.
2. four arm fork type PEG-G-CSF drug effects detect
Colony stimulating factor is to induce people's (or mouse) bone marrow cell or hematopoietic cell to be the factor of clonal growth in semi-solid agar system. What can promote in the colony stimulating factor that hematopoietic cell forms granular leukocyte colony is called granulocyte colony stimulating factor (G-CSF). For pharmacodynamics detection method in the animal body of G-CSF be: in Mice Body, after the injection, detect leukocytic increase in the mouse blood. To inject rear leukocytic increase as the detection index of validity.
Mouse mainline four arm PEG-G-CSF (note PEG4-G-CSF), dosage is 250 μ g/kg, the single dose injection; Simultaneously with the negative control group of G-CSF, with buffer solution control group as a setting, take two arm PEG (every brachium molecular weight is as 5K)-G-CSF (note PEG2-G-CSF, according to Phillippe, American J of hematology, the preparation of 2001,245-251 method) positive control group, injection in continuous four days, dosage is identical. After the injected in mice, eye socket is got blood, carries out white blood cell count(WBC). Last injection is designated as the 0th day, before getting the blood time and being respectively injection in the 0th day, and the 1st day, the 2nd day, the 3rd day, the 4th day, the 5th day, the 6th day, the 7th day. All mouse are at injection before measurement ding white ware cell quantity, take this quantity as radix; The data of measuring later on are the multiple that leucocyte increases: measure number/radix, from the white blood cell count(WBC) result, find the Half-life in vivo of PEG4-G-CSF greater than 7 days, and in vivo rapidly decay of G-CSF. The drug effect that experiment shows the relative PEG2-G-CSF of the drug effect of PEG4-G-CSF in Mice Body simultaneously be greatly improved (specifically see Fig. 5: four arm fork type PEG-granulocyte colony stimulating factors are to the drug effect figure of mouse).
So can find out from above two embodiment, three arm fork type PEG-interferon are compared with crude protein and single, double arm PEG-protein conjugate with four arm fork type PEG-G-CSF bonds, because the existence of multi-arm structure, hydrodynamic volume is large, on the one hand, the biologically active retention of protein drug is higher, on the other hand, concentration decline in vivo can ease up, and clearance rate reduces, prolong circulation timei in vivo, and drug effect improves very obvious; And, along with the arm number of multi-arm fork type PEG increases, its hydrodynamic volume further increases, and is apparent, other multi-arm fork type PEG and protein or polypeptide conjugates also can increase circulation timei, promote drug effect, can be used in the corresponding treatment of preparation and prophylactic medicine; And can utilize the treatment inert carrier that multi-arm fork type PEG of the present invention and protein or polypeptide conjugates are modified, prepare corresponding pharmaceutical composition, for the preparation of corresponding treatment and prophylactic medicine, these all are the knowledge of this area, here just repeat no more.

Claims (10)

1, the bond of a kind of multi-arm fork type polyethylene glycol and protein or polypeptide is characterized in that, the general formula of described bond is:
Figure A2004100351910002C1
Protein or polypeptide
Wherein:
M is 1 or 2;
PEGZ is the multi-arm fork type polyethylene glycol, and Z is the arm number, is 3-8.
2, the bond of multi-arm fork type polyethylene glycol as claimed in claim 1 and protein or polypeptide is characterized in that, described PEGZ is one of following 9 kinds of forms:
Figure A2004100351910002C2
Figure A2004100351910005C1
Wherein:
R 11、R 12、R 13、R 14、R 15、R 16、R 17、R 18Be following straight chained alkyl, isopropyl or benzyls of 10 carbon;
R s1、R s2、R s3、R s4、R s5、R s6、R s7、R s8、R t1、R t2、R t3、R t4、R t5、R t6、 R t7、R t8、R v1、R v2、R v3、R v4、R v5、R v6、R v7、R v8、R m1、R m2、R m3、R m4、 R m5、R m6、R m7For-H or 4 low alkyl groups below the carbon;
R k1、R k2、R k3、R k4、R k5、R k6、R k7、R k8、R n1、R n2、R n3、R n4、R n5、R n6、 R n7、R n8、R n9、R n10It is the low-grade alkylidene of 0 to 10 carbon;
W 1、W 2、W 3、W 4、W 5、W 6、W 7、W 8、W 11、W 12、W 13、W 14、W 15、W 16For-O-,-S-,-NH-,-NHCH2-、-NHCH 2CH 2-、-NH(CH 2) 3-、-NHCH(CH 3) 2-、 -NH(CH 2) 4-、-NHCH(CH 3)(CH 2CH 3)-、-NHC(CH 3) 2CH 2-、 -NHCH(CH 3)CH 2CH 2-、-NHCH 2CH(CH 3)CH 2-；
X1, y1, z1, x2, y2, z2, x3, y3, z3, x4, y4, z4, x5, y5, z5, x6, y6, z6, x7, y7, z7, x8, y8 and z8 are the Any Digit combination, so that the molecular weight of multi-arm fork type PEG is in the daltonian scope of 4000-100000.
3, the bond of multi-arm fork type polyethylene glycol as claimed in claim 2 and protein or polypeptide is characterized in that, described R11、R 12、R 13、R 14、R 15、R 16、R 17、R 18Be methyl.
4, the bond of multi-arm fork type polyethylene glycol as claimed in claim 2 and protein or polypeptide is characterized in that, described Rs1、R s2、R s3、R s4、R s5、R s6、R s7、R s8、R t1、R t2、R t3、 R t4、R t5、R t6、R t7、R t8、R v1、R v2、R v3、R v4、R v5、R v6、R v7、R v8、R m1、R m2、 R m3、R m4、R m5、R m6、R m7For-H.
5, the bond of multi-arm fork type polyethylene glycol as claimed in claim 2 and protein or polypeptide is characterized in that, described Rk1、R k2、R k3、R k4、R k5、R k6、R k7、R k8、R n1、R n、R n3、 R n4、R n5、R n6、R n7、R n8、R n9、R n10For-CH2-、-CH 2CH 2-、-(CH 2) 3-、-CH(CH 3) 2-、 -(CH 2) 4-、-CH(CH 3)(CH 2CH 3)-、-C(CH 3) 2CH 2-、-CH(CH 3)CH 2CH 2-or-CH2CH(CH 3)CH 2-。
6, the bond of multi-arm fork type polyethylene glycol as claimed in claim 2 and protein or polypeptide is characterized in that, described W1、W 2、W 3、W 4、W 5、W 6、W 7、W 8、W 11、W 12、W 13、 W 14、W 15、W 16, for-NH-or-NHCH2-。
7, such as the bond of each described multi-arm fork type polyethylene glycol and protein or polypeptide in the claim 1～6, it is characterized in that described protein or polypeptide are recombinant human interferon alpha 2, recombinant interleukin, rh-erythropoietin, recombined human anti-tumor necrosis factor, Recombinant Human Colony Stimulating Factors, restructuring arginine deiminase, recombined streptokinase or rh-insulin.
8, the preparation method of the bond of a kind of multi-arm fork type polyethylene glycol claimed in claim 1 and protein or polypeptide is characterized in that may further comprise the steps:
With 3-8 arm fork type PEG first and protein or polypeptide carry out coupled reaction, again by the bond of purify out in the reactant mixture multi-arm fork type polyethylene glycol and protein or polypeptide.
9, the bond of multi-arm fork type polyethylene glycol claimed in claim 1 and protein or polypeptide for the preparation of the treatment or prophylactic medicine in application.
10, a kind of pharmaceutical composition, said composition comprise bond and the treatment inert carrier of multi-arm fork type polyethylene glycol claimed in claim 1 and protein or polypeptide.
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CN104906594A (en) * 2005-11-30 2015-09-16 遗传工程与生物技术中心 Four branched dendrimer-PEG for conjugation to proteins and peptides
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EP3315531A4 (en) * 2015-06-23 2018-06-20 Xiamen Sinopeg Biotech Co., Ltd. 8-arm polyethylene glycol derivative, manufacturing method and modified bio-related substance thereby
CN110339367A (en) * 2018-04-02 2019-10-18 北京键凯科技股份有限公司 A kind of cell-penetrating peptide-multi-arm polyethylene glycol-drug conjugates and its application with targeting
US11324827B2 (en) 2014-10-01 2022-05-10 Xiamen Sinopeg Biotech Co., Ltd. Multifunctionalized polyethylene glycol derivative and preparation method therefor

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CN104906594A (en) * 2005-11-30 2015-09-16 遗传工程与生物技术中心 Four branched dendrimer-PEG for conjugation to proteins and peptides
CN101497690B (en) * 2009-02-19 2011-08-31 复旦大学 Comb type polymeric compound with polyethyleneglycol side chain and main chain and preparation thereof
CN105007896A (en) * 2012-12-28 2015-10-28 雅培心血管系统公司 Delivery of biologic therapeutics
CN105007896B (en) * 2012-12-28 2019-04-09 雅培心血管系统公司 Therapeutic combination comprising antibody
US11324827B2 (en) 2014-10-01 2022-05-10 Xiamen Sinopeg Biotech Co., Ltd. Multifunctionalized polyethylene glycol derivative and preparation method therefor
EP3315531A4 (en) * 2015-06-23 2018-06-20 Xiamen Sinopeg Biotech Co., Ltd. 8-arm polyethylene glycol derivative, manufacturing method and modified bio-related substance thereby
US10434182B2 (en) 2015-06-23 2019-10-08 Xiamen Sinopeg Biotech Co., Ltd. Eight-arm polyethylene glycol derivative, production method therefor, and modified bio-related substance thereof
US10660969B2 (en) 2015-06-23 2020-05-26 Xiamen Sinopeg Biotech Co., Ltd. Eight-arm polyethylene glycol derivative, production method therefor, and modified bio-related substance thereof
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