Polyethylene glycol modified L-Asparaginasum and modification method thereof

A technology of asparaginase and polyethylene glycol, applied in the direction of hydrolase, etc., can solve the problems of inhomogeneous modification products, difficulty in separation of modified mixtures, prolongation of half-life of biological activity, etc.

Inactive Publication Date: 2009-11-25
BEIJING SL PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are also some shortcomings in the modification of polyethylene glycol in the past: 1. Due to the non-selectivity of the modification of the first-generation polyethylene glycol modifier (such as polyethylene glycol succinimide ester), the modified prod

Method used

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  • Polyethylene glycol modified L-Asparaginasum and modification method thereof
  • Polyethylene glycol modified L-Asparaginasum and modification method thereof
  • Polyethylene glycol modified L-Asparaginasum and modification method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] The preparation of embodiment 1 polyethylene glycol modified asparaginase

[0037] Take 100ml of asparaginase stock solution (buffer system is pH7.0, 20mMPB buffer, protein concentration is 5mg / ml), and another 100mg of mPEG-ALD (20kd), add it to the asparaginase stock solution, stir at room temperature to make it Dissolve rapidly, and stir slowly at 10°C for 8 hours. Then add 100mg of mPEG-ALD (20kd), stir slowly at 10°C for 8 hours, and purify the modification by gel exclusion chromatography and ion exchange chromatography.

Embodiment 2

[0038] Embodiment 2 SDS-PADE electrophoresis detection

[0039] Electrophoresis instrument: DYY-6C

[0040] Stacking Gel: 5%

[0041] Separating gel: 12%

[0042] Loading volume: 25μl

[0043] Voltage setting: 80V, 30min; 180V, 60min

[0044] Test results: After analysis by the gel imaging system, the total modification efficiency of polyethylene glycol is 76.25%, and the modification efficiency of the main band is 63.41%. The molecular weight of the main band is 81400 Daltons, while the theoretical molecular weight should be 75500 Daltons. This is because when polyethylene glycol migrates in polyacrylamide gel, polyethylene glycol looks like a long and flexible tail, which slows down the overall migration speed.

Embodiment 3

[0045] Example 3 Enzyme Activity Determination

[0046] Utilizes the Mashburn Wriston method. The method determines the hydrolysis rate of the substrate by asparaginase (Asparaginase) by measuring the concentration of amino acids in the product, and the amount of ammonia produced is determined by the color reaction between the Nessler reagent and the protein.

[0047]

[0048] A t : sample value

[0049] A 0 : negative control value

[0050] df: dilution factor

[0051] A s : positive control value

[0052] C pr : protein sample concentration

[0053]

[0054] Asparaginase modified by two polyethylene glycols has an enzyme activity retention rate of 41.14%, and the enzyme activity has not decreased due to the connection of the second polyethylene glycol.

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Abstract

The invention provides polyethylene glycol modified L-Asparaginasum shown as formula (1) and a modification method thereof. The method uses the modification reaction property of polyethylene glycol molecules to modify the L-Asparaginasum under the condition of proper pH value of a reaction system. The polyethylene glycol modified L-Asparaginasum is characterized in that two polyethylene glycol molecules are covalently bound in a reaction product; and the substance has lower glomerular filtration rate and lower immunogenicity compared with a single polyethylene glycol molecule modifier so as to provide better antitumor effect.

Description

technical field [0001] The invention relates to a novel polyethylene glycol-modified asparaginase and a modification method thereof. Background technique [0002] Asparaginase is an amidohydrolase that specifically catalyzes asparagine to generate aspartic acid and ammonia. Some tumor cells lack asparagine synthase, so they cannot synthesize asparagine necessary for growth by themselves, and their growth depends on asparagine synthesized by normal host cells. Asparaginase can hydrolyze asparagine in serum to aspartic acid and ammonia. The use of asparaginase can cause a sharp loss of asparagine in the blood. Tumor cells can neither rely on the host to obtain enough asparagine nor synthesize it by themselves, resulting in the lack of amino acids in the process of protein synthesis, inhibited proliferation, and a large number of tumor cells. Destroyed and unable to grow and survive. [0003] Asparaginase is clinically applicable to the treatment of acute lymphocytic leukemi...

Claims

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Application Information

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IPC IPC(8): C12N9/82
Inventor 徐明波连治国王俊玲许可张鹏杨仲璠吴彦卓
Owner BEIJING SL PHARMA
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