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Antibody affinity purification material and its application

An affinity and antibody technology, applied in the field of biological separation engineering and biomedicine, can solve the problems of intolerance to strong acid and alkali, high price, poor durability and biological safety, etc., and achieve strong alkali resistance, no shedding, biological good safety effect

Inactive Publication Date: 2018-04-20
SHANGHAI HYCHARM
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The results of literature search on the separation and purification technology of existing drug antibodies show that the current production and purification of monoclonal antibody drugs is mainly based on Staphylococcus aureus protein A immunoaffinity chromatography technology, but the price of protein A / G affinity chromatography column Expensive, not resistant to strong acids and alkalis, coupled ligands are easy to fall off, harsh column storage conditions, poor durability and biological safety and many other factors; international patent NOVEL AFFINITY LIGANDS AND THEIR USE (WO1997010887) and Chinese patent Human gamma globulin affinity ligand and its use (CN99119936.7) respectively disclosed small molecule ligand affinity purification materials capable of binding antibodies, but the evaluation data of purified monoclonal antibody drugs showed that affinity with protein A / G Compared with the medium, the binding capacity and specificity of these affinity media are not high enough, and they are not widely used in the production of antibody drugs. Therefore, it is necessary to develop antibodies with better binding capacity and specificity.

Method used

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  • Antibody affinity purification material and its application
  • Antibody affinity purification material and its application
  • Antibody affinity purification material and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Sepharose 6B is activated with trichlorotriazazine (for operation, refer to "Introduction to Affinity Chromatography" (written by C.R.Lowe; translated by Liu Yuxiu. Science Press, first edition in May 1983), take 5 parts of 200ml each, estimate The number of moles of triazoxide, respectively weighed 5-fold molar excess amino compound (R1) 4-(2-aminoethyl) benzenesulfonamide, N-(3-aminopropyl) imidazole, 4-amino-1-naphthalene Phenol, 2-amino-4,6-dihydroxypyrimidine, dibenzylamine, dissolved in 300ml of DMF, respectively added to the triazoxide activation medium, mixed and stirred at 50 ° C for 24h, saturated NaHCO during the reaction 3 The pH of the solution was maintained at 7-8. After the reaction was completed, the medium was washed with 10 times the volume of DMF (N'N dimethylformamide) and water.

[0019] Then weigh the 5-fold molar excess amino compound (R2) L-serine, N-(3-aminopropyl) imidazole, 3,5-diaminobenzoic acid, γ-aminobutyric acid and dibenzylamine and di...

Embodiment 2

[0021] Physical and chemical property verification of affinity purification materials:

[0022] Affinity purification material C1-2 was packed into a column (160mm×10mm), and the antibody solution was used to continuously inject samples until the UV absorption signal remained unchanged. The original antibody concentration was 1mg / ml, and the sample flow rate was 2ml / min;

[0023] Antibodies were dissolved in 10 mMPBS (pH 7.0, 150 mM NaCl) and equilibrated for 10 column volumes until UV280 stabilized near baseline.

[0024] Record the loading volume of the sample under different flow-through ratios when the flow rate of the sample in the pipeline is 150cm / h, until the protein concentration at the outlet of the column reaches more than 10% of the inlet concentration, stop the injection, and wash the affinity purification material with the loading buffer When the UV absorption value of the column drops to the baseline, the column is washed with Gly-HCl (100mM, pH3), and the total...

Embodiment 3

[0035] Affinity purification material column C1-2 purification of monoclonal antibody drugs cultured in CHO cells:

[0036] Prepare 10 mM HAc buffer as a balance solution, adjust pH 5.0 with 1N NaOH, prepare 100 mM Gly-HCl buffers with pH 4.5, pH 4.0 and pH 3.0.

[0037] Take a 1ml prepacked column of affinity purification material and connect it to AKTA, equilibrate to the baseline level with 10Mm PBS (150Mm NaCl, pH7.4) at a flow rate of 1ml / min, and prepare for loading.

[0038] CHO cell culture supernatant (4000rpm, centrifuged for 5in and discarded pellet), centrifuged at 3000g for 10min, took the supernatant and diluted it 10 times before loading the sample, washed with 10mM buffer solution at pH5.0, the flow rate was 1ml / min, To the baseline level, use 100mM Gly-HCl of pH 4.5, pH 4.0 and pH 3.0 to elute and collect the eluted products;

[0039] SDS-PAGE (12% SDS-PAGE gel) was used to detect the eluted sample, and the BCA kit was used to measure the protein concentratio...

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Abstract

The invention belongs to the fields of bioseparation engineering and biomedicines, relates to an affinity separation material for purifying an antibody, and aims at providing an affinity separation material which has stable physicochemical property and good biosecurity and is capable of purifying antibodies of various sources. The affinity separation material is composed of an activated basic matrix, a spacer arm with more than one active site, and a functional chemical group. The affinity separation material is not only capable of purifying antibodies in a laboratory, but also suitable for large-scale purification of antibodies in industry; besides, the affinity separation material is high in specificity in antibody purification, good in biosecurity, low in price and easy to preserve, and can be used repeatedly.

Description

technical field [0001] The invention belongs to the field of bioseparation engineering and biomedicine, and specifically relates to a novel affinity purification material for purifying antibody substances and its application in the separation and purification of antibody substances. Background technique [0002] Antibodies, also known as immunoglobulin (Ig), are a group of proteins secreted by plasma cells that have antigen-binding ability, mainly exist in blood and interstitial fluid, and have extensive clinical disease treatment and scientific research value. Monoclonal antibody drug is based on cell engineering and genetic engineering technology, using the wireless reproduction of myeloma cells and the antigen secretion ability of plasma cells to establish a clinical disease treatment with high production uniformity, strong specific needles, and targeting specific antigenic epitopes. Use drugs. It has broad application prospects in the diagnosis and treatment of various ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): B01J20/22C07K16/00C07K1/22
Inventor 李荣秀潘金亭程超任敬钢马国荣马贵军
Owner SHANGHAI HYCHARM
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