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Polyethylene glycol dog source urate oxidase analogue, and preparation method and applications thereof

A technology of uric acid oxidase and polyethylene glycol, applied in biochemical equipment and methods, oxidoreductase, enzymes, etc., can solve problems such as failure to effectively eliminate immune reactions

Active Publication Date: 2012-08-15
SHANDONG NEWTIME PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although PEG modification can reduce the immunogenicity of microbial urate oxidase, it still fails to effectively eliminate the immune response in the human body. The PEG modified recombinant microbial urate oxidase developed abroad (such as Phenix Inc. and Enzon Inc.) has terminated the long-term Clinical research on the treatment of chronic gout

Method used

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  • Polyethylene glycol dog source urate oxidase analogue, and preparation method and applications thereof
  • Polyethylene glycol dog source urate oxidase analogue, and preparation method and applications thereof
  • Polyethylene glycol dog source urate oxidase analogue, and preparation method and applications thereof

Examples

Experimental program
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Effect test

example 1

[0040] Example 1. Modification conditions of pegylated canine urate oxidase analogs

[0041] The canine urate oxidase analogue involved in this example is a mutant protein (SEQ ID NO: 5, polymorphic in the following examples) that contains a part of the canine and human urate oxidase amino acid sequences obtained by genetic engineering technology. The ethylene glycolated canine uric acid oxidase analogs all refer to the protein encoded by this sequence), and the SDS-PAGE purity and RP-HPLC purity are both higher than 95.0%.

[0042] Get Source15Q anion exchange chromatography stuffing material, pack into chromatography column, use eluent (2M NaCl 2 0.2mol / L Na 2 CO 3 -NaHCO 3 Buffer, pH=10.3) wash 5 column volumes and regenerate, use equilibrium solution (0.2mol / LNa 2 CO 3 -NaHCO 3 buffer, pH=10.3) equilibrated. Load the above-mentioned purified samples at a uniform speed, and then balance them with a balance solution until the conductance is stable. Gradient elution ...

example 2

[0046] Example 2. Separation and Purification of Pegylated Canine Urate Oxidase Analogs

[0047] The modified canine uric acid oxidase analog sample was dialyzed overnight with 20mM PB, pH8.0. After the dialysis, the sample was concentrated by ultrafiltration (50KD Saturous tangential flow module) and then applied to a Sephacryl S300 molecular sieve chromatography column. The equilibrium buffer was 20mM PB , pH 8.0, flow rate 7.0ml / min, detect the absorbance at 254nm and 280nm at the same time after loading the sample, the first peak is the peak of the target protein, the obtained PEGylated canine urate oxidase analog protein was subjected to RP- Purity after HPLC and SEC-HPLC detection is higher than 98.0% (attached image 3 ).

example 3

[0048] Example 3. In vivo immunogenicity determination of purified proteins with different modification ratios

[0049] After four different modification ratios of 1:40, 1:80, 1:120, 1:160, the modified canine uric acid oxidase analog protein was purified according to the method described in Example 2, and then injected into mice to detect its IgG antibody titer. 24 Kunming rats were randomly divided into 4 groups (n=6), and each group was injected with canine urate oxidase analogue protein with different modified ratios, and the injection dose was 1.0 mg / kg. Inject once every five days for 6 consecutive injections. 96 hours after the last injection, 0.5 ml of blood was collected from the ophthalmic venous plexus through the capillaries, and the serum was separated by centrifugation at 3000 rpm for 15 minutes. The IgG antibody titer against the unmodified canine uric acid oxidase analog protein was detected by conventional ELISA method. The results showed that the IgG antibod...

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Abstract

The invention discloses a polyethylene glycol (PEG) dog-source urate oxidase analogue, wherein the dog-source urate oxidase analogue is natural dog-source urate oxidase and chimeric protein and mutant protein containing part of dog-source and human-source urate oxidase amino acid sequences, the average molecular weight of polyethylene glycol is about 1kDa-40kDa, and each urate oxidase monomer is coupled with 2-15 polyethylene glycol molecules in average. According to the method for preparing mammal-source urate oxidase subjected to covalent modification by the polyethylene glycol, the enzyme activity retention rate is higher than 85.0%, and the purification is higher than 95.0%. According to the method, the dog-source urate oxidase analogue subjected to covalent modification by the polyethylene glycol and drug compositions thereof can be used for preventing and / or treating acute hyperuricemia caused by hematologic tumor chemotherapy and hyperuricemia and chronic gout caused by metabolic disorders.

Description

technical field [0001] The invention belongs to the fields of biotechnology and pharmacy. More specifically, the present invention relates to a pegylated canine urate oxidase analogue with high retention rate of enzyme activity, low immunogenicity, long half-life and repeated administration, and its preparation method and application. The present invention is an extension of the invention patent named "Humanized Urate Oxidase" (application number: 200910191240.3). Background technique [0002] The statement in the Background section is not an overall acceptance of the prior art, but instead reflects the inventor's subjective evaluation and interpretation of the technical situation at the time of making this invention. These explanations may include the inventor's own hitherto undisclosed insights into the prior art. And these insights themselves are not part of the existing technology. [0003] Hyperuricemia is a disease caused by excessive purine metabolism. Purine in t...

Claims

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Application Information

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IPC IPC(8): C12N9/06A61K38/44A61K47/48A61P19/02A61P19/06A61P13/12
CPCC12N9/0048A61K38/00C12Y107/03003A61K47/48215C12N9/96A61K38/44A61K47/60A61P13/12A61P19/02A61P19/06
Inventor 张淳范开马雪丰杨黎胡春兰罗华梅翔
Owner SHANDONG NEWTIME PHARMA
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