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Chromatographic medium and preparation method thereof

A chromatographic medium and matrix technology, applied in the field of chromatographic medium and its preparation, can solve the problems that the purification efficiency has not been significantly improved, cannot remove protein A well, and has limited adsorption performance, so as to achieve continuous separation and adsorption characteristics, The effect of low catalyst and ligand usage and high ligand coupling efficiency

Inactive Publication Date: 2019-03-19
杭州纽龙生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

These MMC media mainly bind to antibodies through hydrophobic interactions. The ligand content is not high, and the adsorption performance is limited. Generally, traditional ion exchange chromatography is needed to separate antibodies, and the purification efficiency has not been significantly improved.
Additionally, these MMC media do not do a good job of removing Protein A that is shed from antibodies during purification
In addition, conventional antibody purification methods require protein A to be purified and then purified by ion exchange chromatography and hydrophobic interaction chromatography in sequence, requiring three steps to achieve purification
This conventional method is time-consuming and has many steps, resulting in relatively high purification costs

Method used

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  • Chromatographic medium and preparation method thereof
  • Chromatographic medium and preparation method thereof
  • Chromatographic medium and preparation method thereof

Examples

Experimental program
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Effect test

preparation Embodiment 1

[0039] Prepare chromatography media by the following steps:

[0040] Step 1), wash 10g of agarose gel microspheres with deionized water, drain, add 0.015g of sodium borohydride and 0.5g of anhydrous sodium sulfate, then add 1.5mL of 50% sodium hydroxide solution, 50 °C for 1 hour. Then add 3.5mL of allyl glycidyl ether, activate at 50°C for 8 hours, filter with suction, wash with 50ml of deionized water, 50ml of absolute ethanol, 50ml of 0.2M acetic acid and 100ml of deionized water to obtain activated chromatography matrix;

[0041] Step 2), mix 10g of activated chromatography matrix, 0.1g sodium acetate and 1.5ml deionized water to obtain a mixed solution, add bromine water dropwise while stirring the mixed solution at room temperature until the mixed solution turns light yellow and The color does not change within 1 minute, then add sodium formate dropwise until it becomes colorless, and finally filter and wash to obtain a brominated chromatographic medium;

[0042] Step...

preparation Embodiment 2-7

[0044] Preparation Example 2-7 Prepare the chromatographic medium according to the method of Example 1, the difference is that the amount of reactant and the reaction time in Example 2-7 are somewhat different from those in Example 1, and the prepared chromatographic medium Ligand densities are different. See Table 1 below for details.

[0045]

Embodiment 1

[0047] Use the chromatographic medium Adhere NUPharose FF prepared in Preparation Example 6 to measure the saturation adsorption capacity Q of bovine IgG at 25°C and at different pH m , and the adsorption isotherm was determined, the results are shown in figure 1 middle.

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Abstract

The invention discloses a chromatographic medium and a preparation method thereof. The chromatographic medium comprises a chromatographic substrate and a functional ligand; the chromatographic substrate is agarose gel microspheres; the functional ligand is coupled N-benzyl-N-methylethanolamine activated via allyl glycidyl ether. The preparation method includes the steps of 1) activating the chromatographic substrate with allyl glycidyl ether to obtain an activated chromatographic substrate; 2) brominating the activated chromatographic substrate of step 1) via bromine water to form a brominatedchromatographic substrate; 3) subjecting the brominated chromatographic substrate of step 2) to reaction with the functional ligand N-benzyl-N-methylethanolamine to form the chromatographic medium. The preparation process of the chromatographic medium is simple; antibody adsorbing capacity is high; exfoliated protein A can be well removed by changing pH at the premise of achieving antibody adsorbing and desorbing, the protein A can be used to capture and purify the medium, and two-step purification of an antibody is achieved.

Description

technical field [0001] The invention belongs to the field of protein chromatography separation in the field of biochemical industry, and in particular relates to a chromatography medium and a preparation method thereof. Background technique [0002] With the rapid development of modern biotechnology, new requirements have been put forward for biological separation methods, and more and more biological products require efficient biological downstream technology for separation and purification. Among them, chromatography technology is currently the most effective biological separation and purification technology, and chromatography medium is the key and core of chromatography technology. [0003] Much attention has been paid to the separation and purification of antibodies. Antibodies have the characteristics of strong targeting, high specificity, and low toxicity and side effects. They are widely used in the treatment of major diseases such as tumors and autoimmune diseases,...

Claims

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Application Information

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IPC IPC(8): B01J20/28B01J20/286B01D15/36B01D15/34C07K1/16
CPCB01D15/34B01D15/36B01J20/28016B01J20/286C07K1/165
Inventor 严军钱永常
Owner 杭州纽龙生物科技有限公司
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