Method for preparing elspar modilfied by carbowax

An asparaginase and polyethylene glycol technology, which is applied in the directions of medical preparations, hydrolase, and pharmaceutical formulations containing active ingredients, can solve problems such as loss of therapeutic effect, biological injury, and impact on treatment, and achieve prolonged biological The effect of active half-life, reducing dosage and improving product yield

Inactive Publication Date: 2004-05-26
JIANGSU HENGRUI MEDICINE CO LTD
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  • Abstract
  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

Such parenterally administered biomacromolecular drug products usually have the following problems: (1) biomacromolecular drugs are often sensitizing, and once the organism produces a sensitization reaction, it will cause serious damage to the organism and cause damage to the organism. affect the treatment
(2) Due to the production of antibodies, biomacromolecular drugs combine with antibodies in vivo and rapidly lose their therapeutic effect
However, some technical difficulties also arise during the application of this technology, for example: (1) the modification of polyethylene glycol to biomacromolecules is a non-selective chemical re

Method used

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  • Method for preparing elspar modilfied by carbowax
  • Method for preparing elspar modilfied by carbowax

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1 Preparation of PEG-Asparaginase (PEG-Asparaginase)

[0057] First reaction

[0058] 1. Take 152ml of 0.1M pH 8.5 sodium phosphate buffer in a water bath and preheat it to 25°C, take 0.75 g of asparaginase, and slowly add it to the sodium phosphate buffer to fully dissolve it. Take 3.75 grams of activated polyethylene glycol (SS-PEG5000) with a molecular weight of 5000 and quickly add it to the above solution. Stir appropriately to completely dissolve the activated polyethylene glycol (SS-PEG5000) within 30 seconds, while avoiding a large number of bubbles. . At this time, the dosage ratio of asparaginase to polyethylene glycol is 1:5 (w / w). The reaction is carried out at 25°C for 30 minutes. After the reaction solution is concentrated and washed, it can be further purified or subjected to the second step reaction.

[0059] 2. The concentrated washing of polyethylene glycol-asparaginase reaction solution utilizes a hollow fiber separation column. First, use a peris...

experiment example 1

[0062] Experimental example 1 Analysis method Protein concentration analysis

[0063] Method 1: The Bradford-Lowry method was used to determine the protein concentration. The method is based on different concentrations of protein combined with the protein dye Coomassie Brilliant Blue. The data is also different. First, draw a standard curve based on the amount of dye bound to standard proteins of different concentrations known, and infer the protein sample concentration by measuring the amount of dye bound to the protein.

[0064] Method 2: Take the test solution for enzyme activity determination, and measure the absorbance at 280±1nm and 260±1nm wavelength according to the spectrophotometric method (Chinese Pharmacopoeia 1995 Edition Two Appendix), and calculate the content per ml by the following formula The number of milligrams of protein.

[0065] Protein content (mg / ml) = 1.55×D 280nm -0.76×D 260nm

experiment example 2

[0066] Experimental example 2 Protein purity analysis

[0067]Biological macromolecules, such as proteins, enzymes, peptides, amino acids, etc., have different moving directions and mobilities under the same electric field due to their different charged properties. Highly charged and low molecular weight, the electrophoresis speed is fast, so by SDS-polyacrylamide gel electrophoresis, different molecular weights, different types of proteins and other biological macromolecules can be separated. While determining whether the contaminated protein exists, it can also determine the purity of the unknown protein and the size of the protein molecule.

[0068] Asparaginase-polyethylene glycol activity determination

[0069] Use the Mashburn Wriston method. This method determines the rate of hydrolysis of the substrate by Asparaginase by measuring the concentration of amino acids in the product, and the amount of ammonia produced is determined by the color reaction between Nessler reagen...

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Abstract

A process for preparing the polyethanediol modified asparaginase includes reaction between asparaaginas and linear polyethanodiol whose linear chain length is 1000-5000 in ratio of 1:5-30, and reaction between linear polyethanediol whose linear chain length is 5000-3000 and biologic macro-molecular compound in ratio of 1:10.

Description

Technical field [0001] The invention relates to a new method for preparing polyethylene glycol-biological macromolecule medicine, in particular to a method for preparing polyethylene glycol modified asparaginase. Background technique [0002] Generally speaking, many biomacromolecule medicinal products obtained by biotechnology such as gene recombinant expression or natural extraction and purification enter the organism through parenteral administration to exert their pharmacological and medicinal effects. Such biomacromolecule drug products for parenteral administration usually have the following problems: (1) Biomacromolecule drugs are often allergenic. Once the organism has a sensitization reaction, it will cause serious harm to the organism. Affect the progress of treatment. (2) Due to the production of antibodies, biomacromolecule drugs bind to antibodies in the body and quickly lose their therapeutic effects. (3) The biological half-life of the biological macromolecule drug...

Claims

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Application Information

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IPC IPC(8): A61K38/50A61P31/12A61P35/00C12N9/80
Inventor 孟宪台彭滢孙飘扬周云曙
Owner JIANGSU HENGRUI MEDICINE CO LTD
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