Biomimetic monolithic material with affine selectivity similar to that of protein A and preparation method and application thereof

A technology of monolithic materials and antibodies, applied in the direction of immunoglobulin, chemical instruments and methods, carrier binding/immobilizing peptides, etc., can solve the problems of high price, poor selectivity, and high price of protein A or protein G, and achieve simple preparation process , Mild elution conditions and fast elution speed

Inactive Publication Date: 2014-04-30
NANJING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These types of ligands have their own advantages and disadvantages: the advantage of biospecific ligands is high selectivity, but their disadvantages are difficult elution, poor stability and high price; The shortcoming of the first ligand, but poor selectivity is its most obvious shortcoming; biospecific ligand mimics solve the shortcomings of the first two ligands well, but the selectivity is not as high as the biospecific ligand, and in stable In terms of sex and cost, it is not as advantageous as pseudobiospecific ligands
The chemical bonding method involves relatively cumbersome chemical reactions, and the binding of the antibody to the material is strong, but it will cause blocking and occupation of the antigen binding site, reducing the affinity and specificity of the antibody
The biospecific ligand binding method specifically binds the Fc end of the antibody through protein A or protein G, and the antigen binding region is not affected in any way, maintaining the original affinity and specificity of the antibody; however, protein A or protein G High price and poor stability

Method used

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  • Biomimetic monolithic material with affine selectivity similar to that of protein A and preparation method and application thereof
  • Biomimetic monolithic material with affine selectivity similar to that of protein A and preparation method and application thereof
  • Biomimetic monolithic material with affine selectivity similar to that of protein A and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1: Preparation of bulk protein A-like affinity-selective biomimetic monolithic material

[0026] (1) Take a suitable size centrifuge tube, dissolve 69mg of p-mercaptophenylboronic acid and 61mg of N,N'-bis(2-aminoethyl)oxalamide in 1.1g of PEG-200 solution, vortex, Mix evenly by ultrasonication, then add 240 mg of tris(2,3-epoxypropyl) isocyanate, vortex, ultrasonicate until uniform, seal, and put it in a constant temperature water bath at 80°C for 12 hours.

[0027] (2) Then take out the whole material, cut it into small pieces, put it into a Soxhlet extractor, add methanol at a temperature of 110°C, and extract for 24 hours to remove unreacted monomers and cross-linking agents.

[0028](3) Put the above materials in a vacuum drying oven and dry them at 100°C for 12 hours to obtain a protein A-like affinity-selective biomimetic monolithic material.

Embodiment 2

[0029] Embodiment 2: the preparation of the biomimetic monolithic capillary column of protein A affinity selectivity

[0030] Biomimetic monolithic materials with protein A-like affinity and selectivity were synthesized in capillaries with different inner diameters (such as 25 μm, 75 μm, 100 μm, 150 μm, and 250 μm):

[0031] (1) For the pretreatment of the capillary, first rinse the capillary empty column with 0.1M NaOH solution for 1 hour, then rinse the capillary with deionized water until the pH value of the effluent liquid is 7.0, then rinse the capillary with 0.1M HCl solution for 2 hours, and then use The capillary was rinsed with ionized water until the pH of the effluent liquid was 7.0, and then the capillary was rinsed with methanol solution for 30 minutes and dried with nitrogen. Inject a mixture of methanol and methacryloxypropyl-trimethoxysilane at a volume ratio of 1:1 into the capillary. React at a temperature of 20°C to 70°C for 5-24 hours. Then rinse with met...

Embodiment 3

[0036] Example 3: Preparation of biomimetic monolithic material with protein A-like affinity and selectivity in conventional chromatographic columns

[0037] Preparation of protein A-like affinity-selective biomimetic monolithic materials in conventional HPLC columns with different inner diameters:

[0038] (1) To clean the conventional HPLC column, first immerse the conventional HPLC column in deionized water, clean it ultrasonically for 1 hour, then replace it with methanol for ultrasonic cleaning for 1 hour, and dry it in an oven for 2 hours.

[0039] (2) Dissolve 115 mg of p-mercaptophenylboronic acid and 101 mg of N,N'-bis(2-aminoethyl)oxalamide in 1.845 g of PEG-200 solution, and then add 400 mg of tris(2 , 3-epoxypropyl) isocyanate, vortex, and sonicate to obtain a uniformly mixed solution.

[0040] (3) Carefully and slowly add the mixed solution prepared above into the cleaned HPLC conventional column with a pipette gun.

[0041] (4) Seal both ends of the chromatogr...

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PUM

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Abstract

The invention discloses a biomimetic monolithic material with the affine selectivity similar to that of a protein A, and a preparation method thereof, wherein the material is capable of purifying, separating and immobilizing antibodies. N, N'-di-(2-aminoethyl) oxamide and 4-mercapto-phenylboronic acid are used as common functional monomers, tri-(2, 3- epoxy group propyl group) isocyanate is used as a monomer, polyethylene glycol is used as a porogenic agent, and reaction is carried out in a mode of in-situ ring-opening polymerization to prepare the biomimetic monolithic material with the affine selectivity similar to that of the protein A. The material can be combined with antibodies specifically, and can be applied to the fields of antibody purification, separation, immobilization and the like. The material has the advantages of high selectivity, low cost, good stability, easiness in elution, reusability and the like, and the immunity affinity and the selectivity of the antibodies are not affected.

Description

technical field [0001] The invention relates to a protein A-like affinity-selective biomimetic integral material, as well as the purification, separation and immobilization of antibodies. Background technique [0002] Antibodies are an important class of biomolecules that play an important role in the diagnosis, treatment and biological research of diseases. Antibodies, especially monoclonal antibodies, have a market of tens of billions of dollars every year due to their important role in disease treatment (see G. Walsh, Nat. Biotechnol. 2010, 28, 917-924). The use of antibodies, especially in vivo applications, has strict requirements on purity, so effective purification methods are very important. Affinity method is currently the main method for antibody purification, including affinity chromatography and non-chromatographic affinity techniques. Affinity chromatography is the mainstream technology for antibody purification due to its advantages of automation and easy sca...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C08G59/66C08G59/56C08G59/32C08J9/28C07K16/00C07K1/22C07K17/08
Inventor 刘震刘云春路越
Owner NANJING UNIV
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