Method for preparing polyethylene glycol modified urate oxidase

A technology of uric acid oxidase and polyethylene glycol, applied in the direction of oxidoreductase, biochemical equipment and methods, enzymes, etc., can solve the problems of weakened efficacy of uricase, inability to use long-term treatment, and unresolved immunogenicity

Pending Publication Date: 2022-05-06
CHONGQING PEG BIO BIOTECH CO LTD +1
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  • Abstract
  • Description
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Problems solved by technology

In September 2010, the FDA approved the PEG-modified recombinant porcine uricase (Pegloticase) produced by Savient Company of the United States for the treatment of intractable gout, but because it did not solve the problem of immunogenicity, it has limited clinical application. 50% of patients are ineffective
However, the uricase derived from Aspergillus flavus is less than 40% homologous to the inferred human uricase (Lee C C, Wu X, Gibbs R A, Cook R G, Muzny D M, Caskey C T. Science. 1988.239: 1288-1291.) , the human body is prone to produce anti-uricase antibodies, the efficacy of Aspergillus flavus uricase rapidly weakens, and at the same time cause severe allergic reactions, so it cannot be used for long-term treatment

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  • Method for preparing polyethylene glycol modified urate oxidase
  • Method for preparing polyethylene glycol modified urate oxidase
  • Method for preparing polyethylene glycol modified urate oxidase

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preparation example Construction

[0120] In the preparation method of the polyethylene glycol-modified urate oxidase, various purification methods are used to obtain the high-purity polyethylene glycol-modified urate oxidase.

[0121] In another preferred embodiment, the purification of the modified sample includes but not limited to molecular sieve chromatography, ion exchange chromatography, hydrophobic chromatography, tangential flow ultrafiltration, or a combination. More preferred are molecular sieve chromatography and tangential flow ultrafiltration.

[0122] In another aspect of the present invention, the above polyethylene glycol-modified urate oxidase and its application are provided. The conjugate can achieve long-acting effect in vivo and significantly reduce the blood uric acid level, and can be used for the treatment of hyperuricemia and ventilation.

[0123] The polyethylene glycol urate oxidase is more suitable as a medicine for treating chronic hyperuricemia or gout and its composition. The m...

Embodiment 1

[0145] The preparation of embodiment 1 recombinant uric acid oxidase

[0146] 1.1 Construction of genes and expression plasmids for uricase expression

[0147] According to the codon usage bias data of E.coli, combined with factors such as codon bias and GC content, the cDNA sequence of uricase protein (code name: PHC) (SEQ ID NO: 1) was designed, the whole gene was synthesized, and named as pUC-57-PHC plasmid. Nde I and BamH I were used as the insertion site of the target gene, and the pET-30a plasmid was used as the expression vector (pET-30a-PHC).

[0148] 1.2 Transformation of expression plasmids into bacterial host cells

[0149] Using CaCl 2 The expression vector pET-30a-PHC was introduced into Escherichia coli BL21(DE3) by the method, and Kanamycin was screened for resistance, and high-expression clones were screened out, and the original seed bank strain (E3B) was preserved. These steps are in accordance with the molecular biology field Common methods are implement...

Embodiment 2

[0154] Embodiment 2 Preparation of pegylated oxidative uricase

[0155]Dissolve monomethoxy PEG derivatives of different molecular weight (500~20000Da), such as 5K molecular weight N-succinimide propionate PEG (5K-PEG-SPA) with 1~5mmol / L acid solution to 100~ 300mmol / L PEG solution, dissolved in 1:45~1:150 molar ratio (uric acid oxidase: 5K-PEG-SPA), adding carbonate concentration of uric acid oxidase dissolved in 0.1~0.3mol / L pH10 .0 carbonate buffer solution, make the coupling reaction between PEG and urate oxidase, the concentration of urate oxidase in the coupling reaction is 10mg / ml, and the coupling reaction needs to be stirred under the condition of 5~30℃ for 60 min or more until the degree of PEG conjugation no longer changes with time. After the reaction is complete, unmodified PEG and by-products are removed from the reaction by ultrafiltration and / or chromatography. A suitable molecular sieve chromatography medium can be selected to separate and remove the modifie...

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Abstract

The invention provides a method for preparing polyethylene glycol modified urate oxidase, at least 11 amino acid sites of the following amino acid sites of the polyethylene glycol modified urate oxidase have PEG modification, T1, K3, K4, K30, K35, K76, K79, K97, K112, K116, K120, K152, K179, K222, K231, K266, K272, K285, K291 and K293, the method comprises the following steps: coupling reaction is carried out on the urate oxidase and polyethylene glycol, the polyethylene glycol is provided in the form of an acidic solution, the coupling reaction is carried out in the form of an acidic solution, and the coupling reaction is carried out in the form of an acidic solution. The molar ratio of urate oxidase to polyethylene glycol is 1: (56-94), so as to obtain polyethylene glycol modified urate oxidase.

Description

technical field [0001] The present invention relates to the field of biomedicine, specifically, the present invention relates to a method for preparing polyethylene glycol-modified urate oxidase, more specifically, the present invention relates to a method for preparing polyethylene glycol-modified urate oxidase, reducing urate oxidase Methods of immunogenicity, peg-modified urate oxidase, drug combination, pharmaceutical use of peg-modified urate oxidase. Background technique [0002] Gout is a disease caused by purine metabolism disorder, its clinical feature is hyperuricemia, and tophi is formed due to the deposition of urate in the subcutaneous, joints and kidneys. Purine in the human body undergoes a series of changes, and the final product is uric acid. When the blood uric acid concentration exceeds 70mg / L, hyperuricemia can be caused. Among them, 5% to 12% of patients with hyperuricemia can develop gout. In blood or bursa fluid, when the concentration of sodium urat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/06A61K38/44A61K47/60A61P19/06A61P13/12A61P19/02A61P29/00A61P13/04A61P9/12A61P3/10A61P3/06A61P3/00A61P9/10
CPCC12N9/0048C12Y107/03003A61P19/06A61P13/12A61P19/02A61P29/00A61P13/04A61P9/12A61P3/10A61P3/06A61P3/00A61P9/10A61K38/00A61K38/44A61K47/60C12N9/96A61P13/00
Inventor 刘日勇王志明何云凤王彧付志成闫天文胡春兰苏国威谭长城丁旭朋杨辉王宏英丁琼汪倩文海燕范开
Owner CHONGQING PEG BIO BIOTECH CO LTD
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