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Chromatography medium as well as preparation method and application thereof

A chromatographic medium and matrix technology, applied in the field of chromatographic medium and its preparation, can solve the problems of limited processing capacity, no effective improvement of adsorption capacity, and advantages of difficult antibodies, and achieves high dynamic binding capacity and non-specific medium. The effect of small anisotropic adsorption and significant adsorption capacity

Active Publication Date: 2017-10-13
DALIAN UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But in general, the existing mixed-mode chromatography media are mainly used for the separation of antibody aggregates, and have not been able to replace protein A for the fine separation of antibodies. The main reason is that the processing capacity is limited and the adsorption capacity has not been effectively utilized. Improvement, it is difficult to show advantages in the process of antibody separation and purification on an industrial scale

Method used

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  • Chromatography medium as well as preparation method and application thereof
  • Chromatography medium as well as preparation method and application thereof
  • Chromatography medium as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1 Using 3,3'-diaminodipropylamine as a spacer to couple 5-carboxybenzotriazole

[0058] ⑴Activation

[0059] Take cross-linked agarose gel (GE Healthcare, Sepharose TM CL-6B) 10g, rinse with deionized water several times, then wash with appropriate amount of acetone for 3-5 times, drain and add to 20-40mL acetone solution containing 0.1-1.0g of N,N'-carbonyldiimidazole in a shaker at 25-37°C, 150-200rpm for 2-5 hours, after activation, wash with acetone, and filter with suction to obtain activated cross-linked agarose gel;

[0060] ⑵Grafted spacer arm

[0061] Add the activated cross-linked agarose gel and 1~3g of 3,3'-diaminodipropylamine into 20~40mL acetone solution, and react in a shaker at 30~37℃, 150~200rpm for 5~8 After the reaction, wash with acetone to remove unreacted 3,3'-diaminodipropylamine, and drain to obtain a cross-linked agarose with 3,3'-diaminodipropylamine grafted at the end as a spacer arm gel;

[0062] ⑶Coupling functional ligand

[...

Embodiment 2

[0064] Embodiment 2 utilizes 1,6-hexamethylenediamine as a spacer coupling ligand

[0065] ⑴Activation

[0066] Take cross-linked agarose gel (GE Healthcare, Sepharose TM CL-6B) 10g, rinsed with deionized water several times to remove 20% ethanol, then washed with an appropriate amount of acetone for 3 to 5 times, drained and added to the In 20-40mL acetone solution, activate in a shaker at 25-37°C, 150-200rpm for 2-5 hours, wash with acetone after activation to remove unreacted N,N'-carbonyldiimidazole, and obtain activated The cross-linked agarose gel;

[0067] ⑵Grafted spacer arm

[0068] Add the activated cross-linked agarose gel and 1-3g of 1,6-hexanediamine into a solution of 20-40mL acetone, react in a shaker at 30-37°C, 150-200rpm for 5-8 hours, and react After washing with acetone, unreacted 1,6-hexanediamine was removed, and dried to obtain a cross-linked agarose gel with 1,6-hexamethylenediamine grafted at the end as a spacer arm;

[0069] ⑶Coupling functional ...

Embodiment 3

[0071] Embodiment 3 utilizes 1,2-ethylenediamine as spacer arm coupling ligand

[0072] ⑴Activation

[0073] Take cross-linked agarose gel (GE Healthcare, Sepharose TM CL-6B) 10g, rinse with deionized water several times to remove 20% ethanol, then wash with appropriate amount of acetone for 3 to 5 times, drain and add to 20 In ~40mL acetone solution, activate in a shaker at 25-37°C, 150-200rpm for 2-5 hours, wash with acetone after activation to remove unreacted N,N'-carbonyldiimidazole, and obtain activated Cross-linked agarose gel;

[0074] ⑵Grafted spacer arm

[0075] Add the activated cross-linked agarose gel and 1-3g of 1,2-ethylenediamine into 20-40mL acetone solution, react in a shaker at 150-200rpm at 30-37°C for 5-8 hours, and use it after the reaction Wash with acetone to remove unreacted 1,2-ethylenediamine, and drain to obtain a cross-linked agarose gel with 1,2-ethylenediamine grafted on the end as a spacer arm;

[0076] ⑶Coupling functional ligand

[0077...

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PUM

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Abstract

The invention discloses a chromatography medium comprising a substrate coupled with spacer arms and a functional ligand. The functional ligand is a five-substituted benzotriazole, and the structure thereof is as shown in the description, wherein R is carboxyl, amino, sulfydryl or hydroxyl. The chromatography medium is prepared through a simple process and is low in cost, and compared with other types of antibody adsorption medium, the chromatography medium has the advantage of high dynamic adsorption capacity. Target protein is desorbed and recycled under weak acid condition by regulating the pH of a buffer solution. The novel chromatography medium is relatively high in stability when contacting with acid and alkaline solutions and organic solvents, and can suffer from high-temperature sterilization. The chromatography medium can be used for separating or purifying antibodies in a hydrophobic charge-induced chromatography mode, and can be used as an antibody selective absorbent for blood purification treatment of autoimmune diseases.

Description

technical field [0001] The invention relates to a chromatographic medium and a preparation method thereof, belonging to the technical field of protein chromatographic separation in the field of biochemical industry. Background technique [0002] Antibody drugs are currently the most important class of biotechnology drugs. Because of their strong specificity and high affinity, they have broad application prospects in the fields of medicine and research. For example, in autoimmune diseases, in vitro diagnosis and detection, etc. The scale of such drugs in the biopharmaceutical market has exceeded 20%, and is increasing year by year. With the continuous expansion of market demand and production scale, how to quickly separate and purify antibodies from complex feed solutions has become an unavoidable problem, which also puts forward higher and higher requirements for antibody separation technology. [0003] Usually, there are extremely high requirements on the purity of antibo...

Claims

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Application Information

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IPC IPC(8): B01J20/286B01D15/20C08B37/00B01J20/30
CPCB01D15/20B01J20/286B01J2220/80C08B37/0039
Inventor 任军贾凌云高明
Owner DALIAN UNIV OF TECH
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