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Pegylated recombinant Candida utilis urate oxidase lyophilized injection

A technology for producing Candida utilis and PEGylation, which is applied in the directions of oxidoreductase, lyophilized delivery, and enzymes, and can solve problems such as activity decline and loss

Active Publication Date: 2019-02-26
SHENYANG SUNSHINE PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] Due to the complexity of the structure of PEGylated recombinant Candida utilis uric acid oxidase, the three stages of freeze-drying (low temperature effect, freezing effect and dehydration effect) may lead to a decrease or loss of activity

Method used

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  • Pegylated recombinant Candida utilis urate oxidase lyophilized injection
  • Pegylated recombinant Candida utilis urate oxidase lyophilized injection
  • Pegylated recombinant Candida utilis urate oxidase lyophilized injection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Effects of Polyethylene Glycol Modification Products with Different Molecular Weights on Enzyme Activity

[0040] The polyethylene glycol linking group was selected as succinimidyl succinate, abbreviated as mSS-PEG. Choose mSS-PEG with molecular weights of 5K, 20K, and 30K to modify the purified recombinant urate oxidase protein. The pH of the modification reaction is controlled at 7.5-8.0. The buffer system is 100mM phosphate buffer. The recombinant urate oxidase protein and mSS -The reaction molar ratio of PEG is 1:30, and the reaction is allowed to stand at room temperature for 120 minutes. After the reaction was finished, an ultrafiltration centrifuge tube with a molecular weight cut-off of 30KD was used to perform ultrafiltration and centrifugation for 10 times to obtain purified PEGylated recombinant urate oxidases of different molecular weights ( figure 1 ). The enzymatic activities of products before modification and after modification with different molecular...

Embodiment 2

[0045] Immunogenicity experiment of polyethylene glycol modified products with different molecular weights

[0046] 70 SPF mice. They were randomly divided into 3 groups, 20 rats in each group, and blank serum was taken from the other 10 rats as blank control. By subcutaneous injection, three groups of mice were given recombinant Candida urate oxidase, recombinant Candida urate oxidase-PEG-5K and recombinant Candida urate oxidase-PEG-20K respectively, with a dosage of 1.54 mg / kg, administered twice a week for 5 consecutive weeks. Before the administration, blood was collected from 10 blank mice as a blank control; blood was collected from each mouse at the 6th and 8th weeks, and the serum anti-uric acid oxidase antibody concentration was detected by ELISA method. The volume of each blood collection is not less than 250ul. The experimental results are shown in Table 2.

[0047] Table 2 ELIAS method to detect serum anti-uric acid oxidase antibody titer

[0048]

[0049]...

Embodiment 3

[0051] The isoelectric point of the pegylated recombinant urate oxidase is detected, and a suitable pH value range of the formulation is selected.

[0052] Capillary isoelectric focusing is an ideal method to obtain the actual isoelectric point of protein, so capillary isoelectric focusing is used to detect the isoelectric point of PEGylated recombinant urate oxidase. Since recombinant urate oxidase undergoes PEGylation, the interference of polyethylene glycol on isoelectric detection is very serious, so only the isoelectric point of recombinant urease can be detected, and the test results are shown in figure 2 . pass figure 2 The results showed that the isoelectric point of recombinant urate oxidase is about 7.3, because the binding site of recombinant urate oxidase and polyethylene glycol is mainly lysine, which is a basic amino acid, so recombinant urate oxidase and polyethylene glycol After ethylene glycol is combined, the isoelectric point shifts toward acidity. There...

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Abstract

The invention provides a PEGylated recombinant Candida utilis urate oxidase lyophilized injection for treating various diseases caused by elevated blood uric acid in the human body, which is characterized in that it consists of the following components: the active ingredient is poly Glycolated recombinant Candida utilis uric acid oxidase, a lyoprotectant for maintaining protein activity and stability during freezing or lyophilization, a bulking agent, an acid-base regulator and a buffer for maintaining a certain pH value. It is characterized by maintaining the complete spatial configuration and biological activity of PEGylated recombinant Candida utilis urate oxidase after freeze-drying, which is not only beneficial to the preservation of the drug, but also conducive to the redissolution and reconstitution of the drug in a short time. sex.

Description

technical field [0001] The invention relates to a new freeze-dried preparation of biological products, a preparation method and application in related therapeutic fields. Specifically, the invention relates to the administration of PEGylated recombinant products for various diseases caused by elevated blood uric acid in the human body. Candida prion urate oxidase lyophilized injection. Background technique [0002] Uric acid is the end product of purine metabolism in birds, reptiles and primates including humans, because in these animals there is a lack of uric acid that uses molecular oxygen as the receptor to catalyze the further decomposition of uric acid into allantoin, carbon dioxide and hydrogen peroxide oxidase. Uric acid is excreted by the kidneys as the end product of purine metabolism in the human body. When uric acid production exceeds renal metabolism, such as excessive intake of purine-rich foods, increased purine metabolism caused by leukemia and lymphoma and ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K47/60A61K38/44A61K9/19A61P19/06
CPCA61K9/0019A61K9/19A61K38/44C12Y107/03003
Inventor 娄竞袁杰徐秀玲靳征
Owner SHENYANG SUNSHINE PHARMA
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