PEGylated asparaginase and applications thereof

A technology of asparaginase and PEGylation, which is applied in the field of protein drugs, can solve the problems of easy shedding, reduced activity, and low yield, and achieve the effects of reducing immunogenicity, prolonging half-life, and firm binding

Pending Publication Date: 2016-07-27
ZONHON BIOPHARMA INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, Oncaspar has poor stability in vitro and in vivo, is easy to fall off, and has relatively large side effects
However, the PEG-modified L-asparaginase currently on the market in China is only "pegaspargase" of Hengrui Company, which is a generic drug of Oncaspar, and there are also problems such as PEG is easy to degrade and fall off, has poor uniformity, and seriously reduces activity.
[0010] At the same time, there are currently some papers or patents related to PEG-specific modification of L-asparaginase, but these modifiers are more expensive, and the modification process is more complicated, the yield is low, and the entire manufacturing cost is high, so it is not suitable for the industry. application

Method used

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  • PEGylated asparaginase and applications thereof
  • PEGylated asparaginase and applications thereof
  • PEGylated asparaginase and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Example 1: Preparation and analysis of PEG conjugates of asparaginase

preparation example 1

[0064] Preparation example 1, pegylated asparaginase of the present invention is prepared, purified and identified by the following methods:

[0065] Step 1: Buffer Exchange

[0066] The freeze-dried powder of asparaginase was dissolved in 20 mM Tris-Hcl (pH 8.0) buffer solution to prepare a solution with a protein concentration of 5 mg / ml. Then the sample was loaded through the sample pump of the AKTA chromatography system, and the sample was sucked into the Q ion exchange column (purchased from GE Company, HiTrapQHP5mL). After loading the sample, equilibrate the chromatographic column with equilibration buffer A. After equilibrating for 5 column volumes, perform one-step elution with elution buffer B, and collect the elution peaks.

[0067] (A solution: 20mM phosphate buffer (pH8.0), B solution: 20mM phosphate buffer + 0.2M sodium chloride (pH7.5))

[0068] Step 2: Modification reaction and purification of modified products

[0069]Using M-SPA-5000 (purchased from Beijing...

Embodiment 2

[0081] Example 2: Comparison of PEGylated ASP modification uniformity

[0082] We compared the modification uniformity of PEGylated ASP by SDS-PAGE electrophoresis. Protein stacking gel is 5%, separating gel is 8%. The stacking gel buffer is 0.5MTris-HCl buffer (pH6.8); the separating gel buffer is 1.5mol / LTris-HCl buffer (pH8.8). Take 10ug protein sample, mix it with sample buffer in equal volume, boil at 100°C for 5min, load the sample and run, and stain with Coomassie Brilliant Blue R250 (purchased from Sinopharm Group) after electrophoresis.

[0083] Depend on figure 2 It can be seen that compared with similar products on the market - Pegaspargase (purchased from Jiangsu Hengrui Medicine Co., Ltd.) and Oncaspar (purchased from SigmaTau Pharmaceutical Company), the electrophoresis of our modified product SPA5K-ASP and Oncaspar The band is relatively narrow, which shows that the modification uniformity of SPA5K-ASP and the original drug is slightly better than pegasparga...

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Abstract

The invention discloses a PEGylated asparaginase as well as applications thereof to medicament preparation and clinic treatment. One molecule of asparaginase in the asparaginase modified by PEG is coupled to 13-45 molecules of PEG, and the PEG is straight-chain PEG whose average molecular weight is 2-20KDa. The PEGylated asparaginase is modified by PEG. The PEGylated asparaginase has the advantages of reduced immunogenicity, obviously prolonged half life, etc.; PEG and asparaginase are coupled more stable. Compared with branded drugs and generic drugs of commercially available PEGylated asparaginase, the PEGylated asparaginase has the advantages of more stable structure, firm combination of PEG, uneasy falling, higher homogeneity, and higher activity.

Description

technical field [0001] The present invention relates to polyethylene glycol-modified protein medicine, especially pegylated asparaginase and its application in medicine preparation and clinical treatment. Background technique [0002] Asparaginase (ASP), also known as L-asparaginase, L-asparaginase or L-asparaginase, is an enzyme that catalyzes the hydrolysis of asparagine into aspartic acid. Asparaginase is effective in the treatment of acute lymphoblastic leukemia (ALL) in children or adults. In recent years, drugs containing L-asparaginase have been used in combination with chemotherapy to treat NK / T cell lymphoma, and achieved good therapeutic effects. NK / T-cell lymphoma is a special type of non-Hodgkin's lymphoma, which is more common in Asia and Latin America, and the incidence rate in my country is relatively high. According to the tumor site, NK / T-cell lymphoma can be divided into nasal NK / T-cell lymphoma and non-nasal NK / T-cell lymphoma. In addition, L-asparagina...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/82A61K38/50A61K47/48A61K47/10A61P35/02A61P35/00
Inventor 马永王俊吴鼎龙闻兴元王和徐春林陈一飞王耀方
Owner ZONHON BIOPHARMA INST
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