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PEGylated uricoxidase compound, preparing method, preparation and use thereof

A technology of uric acid oxidase and pegylation, which is applied in the direction of oxidoreductase, drug combination, and pharmaceutical formula, can solve the problems of poor uniformity, low immunogenicity, systemic allergic reactivity, and difficulty in separation and purification, and achieve uniformity Improvement of sex, low anaphylaxis, easy purification effect

Active Publication Date: 2008-11-12
刘国安 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] In order to solve the technical defects of the above-mentioned PEG-modified uric acid oxidase modified product with poor uniformity and difficulty in separation and purification, an object of the present invention is to provide a modified product with good uniformity, which is beneficial to industrial production, and has extremely low immunogenicity and systemic Hypersensitivity, able to repeated use of pegylated urate oxidase compound

Method used

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  • PEGylated uricoxidase compound, preparing method, preparation and use thereof
  • PEGylated uricoxidase compound, preparing method, preparation and use thereof
  • PEGylated uricoxidase compound, preparing method, preparation and use thereof

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Embodiment 1

[0041] Modification reaction between polyethylene glycol and uric acid oxidase

[0042] The uric acid oxidase described in the present invention is a recombinant Aspergillus flavus uric acid oxidase obtained from genetic engineering technology, the purity is greater than 95%, and it maintains relatively high enzyme activity.

[0043] Dissolve uric acid oxidase (UOX) in pH 6.0, 100mM phosphate buffer, add NaCNBH after dissolving according to the ratio of UOX:mPEG active ester=1:5 (w / w) 3 Make the final concentration 10mM, and react at room temperature for 24 hours.

Embodiment 2

[0045] Separation and purification of PEGylated urate oxidase drug

[0046] The product obtained from the modification reaction of polyethylene glycol and uric acid oxidase was subjected to preparative cation exchange chromatography on the AKTA-Primer protein chromatography system (product of Amersham Pharmacia). The cationic chromatography gel is SP-Sepharose Fast Flow, and the column bed volume is 50ml. The chromatographic column was equilibrated with 20mM PB, pH8.0 buffer solution for at least 5 column bed volumes to make the pH of the effluent liquid the same as the pH value of the buffer solution, and after the balance, adjust the pH to 8.0. / min) sample loading. After sample loading, continue to equilibrate with equilibration buffer until the UV absorption returns to the baseline, and then use 0-0.5M NaCl gradient elution. When the NaCl concentration is 0.2-0.3mol / L, the protein peak that appears is mainly a polyethylene glycol Alcohol molecules are linked to a UOX mol...

Embodiment 3

[0048] Modification reaction of polyethylene glycol and uric acid oxidase in different ratios

[0049] Dissolve urate oxidase in pH 6.0, 100mM phosphate buffer, add UOX to mPEG active ester at the ratio of 1:4 (w / w), 1:5, 1:6.5, 1:8 After dissolving, add NaCNBH3 to make the final concentration 10mM, react at room temperature for 16-24 hours, analyze the ratio of the modified product and the specific enzyme activity by SDS-PAGE, the results are shown in the table below (the enzyme specific activity of unmodified urate oxidase is 17.6EAU / mg).

[0050]

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Abstract

The invention relates to a PEGylation urate oxidase compound as well as the preparation method thereof and the preparation of PEGylation urate oxidase compound as well as the application thereof, particularly relates to a PEGylation urate oxidase compound which has extremely low immunogenicity and anaphylaxis, can be applied repetitively and has a long acting as well as the preparation method thereof and the preparation of PEGylationurate oxidasee compound as well as the application thereof. The invention adopts a modification method which has high selectivity to a N-terminal of a urate oxidase, which means that PEG active ester is connected with the N-terminal of the urate oxidase by covalence bond, the uniformity of products is greatly improved, the products are easy to be purified, the activity of the purified products can be better preserved and the immunogenicity of prototype protein of the urate oxidase can be reduced obviously. The PEGylation urate oxidase compound can prevent systemic anaphylaxis when applied, can be applied repetitively and has the efficacy of treating and preventing gout, reducing the level of serum uric acid, preventing tumor lysis syndrome and improving the renal function.

Description

technical field [0001] The present invention relates to a pegylated uric acid oxidase compound and its preparation method, preparation and application thereof, in particular to a pegylated uric acid oxidase compound with extremely low immunogenicity and allergies, capable of repeated medication, and long-acting effect. Uric acid oxidase compound and its preparation method as well as its preparation and application. Background technique [0002] Uric acid oxidase (uric acid oxidoreductase, EC 1.7.3.3, Urate Oxidase, Uricase) participates in the purine metabolism process of organisms. Purine is an important biologically active molecule in the body. The product of purine nucleotide decomposition, xanthine, produces uric acid under the action of xanthine oxidase, and uric acid can be further decomposed into allantoin, allantoic acid, CO 2 and NH 3 . Uric acid oxidase oxidizes uric acid to allantoin. Humans and some primates lack active urate oxidase, so uric acid is the end ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/02A61K38/44A61P19/06A61P35/00A61P13/12
Inventor 刘国安杨忠刘全海吴学军刘珉宇来庆勤
Owner 刘国安
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