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Method for preparing affinity chromatography medium in reaction kettle

A chromatographic medium and reactor technology, applied in the field of biopharmaceuticals, can solve problems such as waste, increased operational complexity, time, and time-consuming, and achieve the effect of not wasting materials and shortening transfer and processing time

Active Publication Date: 2010-08-11
SHANGHAI NAT ENG RES CENT OF ANTIBODY MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

When the above method is prepared on a small scale, it needs to be repeatedly operated in multiple Erlenmeyer flasks and Buchner funnels, which increases the complexity and time of the operation on the one hand, and also easily causes the loss and waste of various reaction components on the other hand. Affinity chromatography medium is only available in hundreds of grams
If it is prepared on a larger scale, the time-consuming, labor-intensive, and material-consuming defects of the above method are more obvious

Method used

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  • Method for preparing affinity chromatography medium in reaction kettle
  • Method for preparing affinity chromatography medium in reaction kettle
  • Method for preparing affinity chromatography medium in reaction kettle

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: Preparation of affinity chromatography medium using a reactor

[0030] Recombinant protein A: rPA50, Repligen Company. Gel: polymerized agarose gel Spharose FF, GE company. Activator: N-hydroxysuccinimide. Mixer: W-600 type, Jiangyin Longchang Pharmaceutical Machinery Co., Ltd.

[0031] The affinity chromatography medium is formed by coupling recombinant protein A with activated gel, wherein the reaction processes of gel activation and activated gel coupled with recombinant protein A are all carried out in the reactor of the utility model. The pretreatment before the activation reaction, the posttreatment after the activation reaction, the pretreatment before the coupling reaction and the posttreatment after the coupling reaction are all carried out in the reactor of the utility model.

[0032] Activation: Add Sepharose FF into the reaction kettle, use kettle lid 4, 5 (such as figure 2 shown), rinse the gel with distilled water. Then change kettle cover...

experiment example

[0035] Experimental Example: Comparison of Dynamic Capacity (Human Immunoglobulin)

[0036] Self-made recombinant protein A affinity chromatography medium and rProteinA Sepharose Fast Flow column of GE Company were packed, using GE Company C10 / 10 type chromatography column, packed to 2mL; the chromatography system was AKTAPurifier of GE Company.

[0037] Balance solution (20mmol / LPB+250mmol / LNaCl pH7.0), equilibrate the chromatography column at a flow rate of 1.5mL / min.

[0038]Adjust human immunoglobulin (Gamma Rais, Shanghai Raas Blood Products Co., Ltd.) to 2 mg / mL, first bypass the ultraviolet detector of the chromatography system, read the OD280 of the sample and record it as A, and pass the sample through OD= At A / 10, the amount of immunoglobulin that has passed through the column is defined as the dynamic capacity.

[0039] The sample passes through the column at a flow rate of 0.6-1.5mL / min, and the OD280 is detected. When OD280=A / 10, stop loading the sample, calculat...

Embodiment 2

[0044] Example 2: Purification of recombinant anti-Her2 humanized monoclonal antibody using self-made recombinant protein A affinity chromatography medium

[0045] The self-made recombinant protein A affinity chromatography medium was loaded into the column, and the C10 / 10 type chromatography column of GE Company was used to pack to 2 mL; the chromatography system was AKTA Purifier of GE Company.

[0046] Balance solution (20mmol / L PB+250mmol / LNaCl pH7.0), equilibrate the chromatography column at a flow rate of 1.5mL / min.

[0047] Recombinant anti-Her2 humanized monoclonal antibody (Herceptin TM ) The serum-free culture supernatant was passed through the column at a flow rate of 1.5 mL / min.

[0048] The balance solution (20mmol / L PB+250mmol / LNaCl, pH7.0) passed through the column at a flow rate of 1.5mL / min. The eluate (20mmol / l citrate, pH3.0) passed through the column at a flow rate of 1.5mL / min.

[0049] Collect the elution peak with UV absorption at λ280.

[0050] After...

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Abstract

The invention belongs to the filed of biological pharmacy, more particularly, the invention discloses a method for preparing an affinity chromatography medium in a reaction kettle. The method is characterized in that gel activating reaction, coupling reaction of activated gel and recombinant protein A, pretreatment before reaction and after-treatment after reaction of reactant are all carried out in the same reaction kettle. The method reduces the inconvenience of using more containers in the process that reactant needs transferring or processing before or after reaction, and simultaneously also shortens the transferring and processing time to have the effects of saving time, labor and material. The invention further discloses the use of affinity chromatography medium purified antibody protein prepared by using the method.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals, and more specifically, the invention discloses a method for preparing an affinity chromatography medium in a reaction kettle. Background technique [0002] At present, most of the reactions in the field of biopharmaceuticals are solid-liquid or liquid-liquid reactions, and the final products are mostly solid-liquid mixtures. During the production process, the reactions are often carried out in the reactor. After the reaction is completed, the solid-liquid mixture is transferred to another Workup in one vessel or separate by centrifugation. Sometimes it is necessary to place the reactant in a container for pretreatment before the reaction, and then transfer the pretreated reactant to the reactor for reaction during the reaction. Many solid-liquid or liquid-liquid reactions require repeated pretreatment-reaction and / or reaction-posttreatment processes to achieve the desired reaction results. For...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): B01J19/00B01J20/30
Inventor 侯盛寇庚陶静谈珉张大鹏
Owner SHANGHAI NAT ENG RES CENT OF ANTIBODY MEDICINE
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