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Method for removing endotoxin from influenza vaccine formulation

A flu vaccine and endotoxin technology, applied in the preparation method of peptides, chemical instruments and methods, antiviral agents, etc., can solve the problems of low flow rate, low processing capacity, and difficult to scale up, and achieve good specificity and activity recovery rate High effect with high removal rate

Inactive Publication Date: 2005-06-01
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But the flow rate is low, the resistance is high, the processing capacity is small, and it is not easy to scale up
[0009] Since there are many types of recombinant proteins, the steps of separation and purification (methods and steps) are different, and the requirements for the content of endotoxin in them are also different in the production process, so the method and effect of removing endotoxin cannot be completely universal

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1. Bonding of polyethyleneimine to particles:

[0023] Dissolve 0.3g of polyethyleneimine with different molecular weights (600, 60000) in 12ml of potassium phosphate buffer solution with pH = 9 and a concentration of 1mol / L, and add 2g of N,N'-methylene-methoxy(methacrylic acid) Amide) copolymer particles of glycidyl methacrylate-propenyl glycidyl ether and methacrylamide; stand at room temperature for 72-96 hours, and remove the liquid by suction filtration; the particles are buffered with 50ml of 0.1mol / LPH=9 potassium phosphate The solution was washed three times and set aside. The nitrogen content of the prepared affinity media was determined by Kjeldahl method, and they were 7.0% and 6.8%, respectively.

Embodiment 2

[0024] Example 2. Bonding of polyethyleneimine to cellulose membrane:

[0025] Take 50 pieces of cellulose membranes with a diameter of 47 mm, rinse them with a large amount of water, put them in a 200ml beaker, add 40ml of epichlorohydrin, 120ml of 1M NaOH solution and 0.19g of NaBH 4 , mix evenly, and stand overnight at room temperature for activation. The next day, take the activated membrane, wash it with a large amount of water until it is neutral, drain it, and then add 125ml of 1% polyethyleneimine solution (molecular weights are 600, 1800, 10000 respectively), and place it overnight at a constant temperature of 30°C (more than 16 hours). . The next day, the activated membrane was taken, washed with a large amount of water until neutral, drained, and then added with 100ml of 0.25% sodium borohydride solution (prepared with 0.1M pH8.5 phosphate buffer) for reduction reaction at room temperature for 4 hours. Take the reduced membrane, wash it with plenty of water until ...

Embodiment 3

[0026] Embodiment 3. Preparation of charged microporous affinity membrane:

[0027] Cellulose is formulated with cellulose diacetate of 7% by weight, 43.8% of dimethylformamide, 22.7% of acetone, and 26.5% of glycerin, and is prepared into a film-making solution, and 50% polyethyleneimine (molecular weight is 60000) aqueous solution, To prepare a polymer solution, a 7% solution of polyisocyanate (a multimer of diphenylmethyl diisocyanate) (DMF and acetone are divided into half and half as solvent), according to the weight ratio of polyisocyanate and 50% polyvinyl imine 0.23: 1, after crosslinking, add 50% polyvinylimine and glycerin to prepare film-making solution, its final weight percent is cellulose diacetate 7%, dimethylformamide 43.8%, acetone 22.8%, 50% 1.6% aqueous solution of polyvinylimine, 0.25% polyisocyanate, 24.5% glycerol, scrape the film solution on a glass plate with a thickness of 400 μm, and deposit it in air at 50°C with a relative humidity higher than 95% f...

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PUM

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Abstract

The present invention is method of eliminating endotoxin from influenza vaccine. Affinity medium is prepared into affinity column or affinity membrane separator, which is used in processing influenza vaccine to eliminate endotoxin through static adsorption or filtration. The present invention features that the affinity medium has polyethylene imine as affinity ligand and nitrogen content of 0.75-7.0 %. The present invention has high endotoxin eliminating rate, high specificity and high active influenza vaccine recovering rate, and is suitable for production.

Description

Technical field: [0001] The invention relates to the production process of recombinant protein medicine, and particularly provides a method for removing endotoxin from influenza vaccine preparations. Background technique: [0002] With the increasing maturity of genetic engineering technology, more and more recombinant protein drugs have been developed and utilized. Since the production process involves Gram-negative Escherichia coli and the separation and purification process is complicated, it will cause endotoxin contamination. [0003] Affinity technology is an effective means of separating and purifying biomacromolecules, which mainly utilizes the principle of affinity between ligands and biomacromolecules. The ligand is covalently bonded to the porous medium to obtain an affinity medium, which can be used to selectively adsorb endotoxin and achieve the goal of removing endotoxin from the recombinant protein solution. Existing affinity ligands and affinity media inclu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/145A61P31/16C07K1/36C12N7/02
Inventor 李京华邵英光陈兆安丛润滋王俊德
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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