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Method for separating immune globulin IgG from human serum

A technology of immunoglobulin and human serum, which is applied in the field of separation and purification of biologically active substances, can solve the problems of high pH and salt concentration of feed liquid, low yield and purity, and influence of protein activity, etc., so as to maintain biological activity , High separation selectivity, high specificity guaranteed

Active Publication Date: 2018-05-22
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Chinese patent CN 1563091 A discloses a method for extracting immunoglobulin with a low-temperature organic solvent, with a purity of 65%, but this method has some limitations, the yield and purity are not high, and the protein activity will also be affected
U.S. Patent US 3,664,994 discloses the process of using DEAE dextran medium to separate IgG from horse serum; have higher requirements
Protein A affinity chromatography can obtain high-purity IgG, but the medium is expensive and the separation cost is high. Therefore, it is necessary to develop a new method for cost-effective IgG separation

Method used

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  • Method for separating immune globulin IgG from human serum
  • Method for separating immune globulin IgG from human serum
  • Method for separating immune globulin IgG from human serum

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Embodiment 1: Preparation of combined ligand

[0040] With the help of computer molecular simulation, analyze and evaluate the key residues of protein A and antibody Fc binding site, screen and design the combinatorial ligand of tripeptide-heterocyclic small molecule, whose sequence is phenylalanine-tyramine Acid-glutamine-5-aminobenzimidazole.

[0041] Combined ligand, the structural formula is as follows:

[0042]

[0043] The combinatorial ligand can be synthesized by the chemical synthesis method in the prior art, and the combinatorial ligand in this example is entrusted to China Peptide Biochemical Co., Ltd. to prepare it.

[0044] Carry out high-performance liquid chromatography and mass spectrometry characterization for the combined ligand in embodiment 1, as respectively figure 1 and 2 shown.

Embodiment 2

[0045] Example 2: Preparation of Combined Biomimetic Chromatography Medium

[0046] Take 3.0 g of drained agarose gel, add 3.0 g of 20% (v / v) dimethyl sulfoxide, 1.5 g of allyl bromide and 0.6 g of sodium hydroxide, activate it on a shaker at 150 rpm at 30 ° C for 24 hours, and pump Filter and wash with deionized water to obtain an activated chromatography matrix.

[0047] Mix the activated chromatographic matrix, 6.0g 50% (v / v) acetone and 0.9g N-bromosuccinimide for bromoalcoholization, react on a shaking table at 150rpm at 30°C for 3h, filter with suction, and use deionized Washing with water yields a bromoalcoholated substrate.

[0048] Mix 1.5g dimethyl sulfoxide and 3.0g 1M sodium carbonate buffer, add 0.3g phenylalanine-tyrosine-glutamine-5-aminobenzimidazolidine, fully dissolve, then add bromoalcohol The chromatographic matrix was reacted on a shaking table at 150 rpm at 30°C for 12 hours, and was repeatedly washed with deionized water, 0.1M HCl, and 0.1M NaOH to obt...

Embodiment 3

[0052] Example 3: Preparation of Combined Biomimetic Chromatography Medium

[0053]Take 3.0 g of drained agarose gel, add 1.5 g of 20% (v / v) dimethyl sulfoxide, 0.3 g of allyl bromide and 0.3 g of sodium hydroxide, activate it on a shaker at 150 rpm at 30 ° C for 24 hours, and pump Filter and wash with deionized water to obtain an activated chromatography matrix.

[0054] Mix the activated chromatographic matrix, 3.0g 50% (v / v) acetone and 0.3g N-bromosuccinimide for bromoalcoholization, react on a shaking table at 150rpm at 30°C for 1h, filter with suction, and use deionized Washing with water yields a bromoalcoholated substrate.

[0055] Mix 1.5g dimethyl sulfoxide and 3.0g 1M sodium carbonate buffer, add 0.3g phenylalanine-tyrosine-glutamine-5-aminobenzimidazolidine, fully dissolve, then add bromoalcohol The chromatographic matrix was reacted on a shaking table at 150 rpm at 30°C for 8 hours, and was repeatedly washed with deionized water, 0.1M HCl, and 0.1M NaOH to obtai...

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Abstract

The invention relates to a method for separating immune globulin IgG from human serum. The method comprises the following steps: 1) diluting the human serum by a buffer solution, adjusting a pH to 6.0to 8.0 and filtering by a filter membrane to obtain a human serum sample; 2) feeding the human serum sample into a chromatographic column, washing by a balancing buffer solution, then eluting by a eluting buffer solution and collecting eluent to obtain the immune globulin IgG solution, the chromatographic column is filled with a combined type bionic chromatography medium, the combined type bionicchromatography medium is prepared from a chromatography matrix and combined type ligand, the chromatography matrix is a hydrophilic porous microsphere with hydroxyl, and a sequence of the combined type ligand is phenylalanine-tyrosine-glutamine-5-aminobenzimidazole; 3) performing utlrafiltration and concentration on the immune globulin IgG solution to obtain the immune globulin IgG. The method can improve an efficiency and a purity for separating the immune globulin IgG from blood.

Description

technical field [0001] The invention belongs to the field of separation and purification of biologically active substances, and in particular relates to a method for separating immunoglobulin IgG from human serum. Background technique [0002] Immunoglobulin is a special glycoprotein produced by plasma cells differentiated from B lymphocytes in the host's immune response to antigen stimulation. According to the structure and biological properties, mammalian immunoglobulins can be divided into IgA, IgD, IgE, IgG and IgM, among which the most abundant is IgG, which is widely used in disease diagnosis and treatment. [0003] Blood is an important source of immunoglobulins. In the 1940s, Cohn developed the low-temperature ethanol separation process and realized the large-scale separation of plasma proteins. This method has been improved many times and has become the most widely used plasma protein separation method in the blood product industry. According to the differences in...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/00C07K16/06C07K1/36C07K1/34C07K1/16
CPCC07K16/065C07K2317/10
Inventor 张其磊邹徐俊卢慧丽林东强姚善泾
Owner ZHEJIANG UNIV
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