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Method for preparing antibody-maytansine alkaloid medicine conjugate

A drug conjugate, maytansine technology, applied in the biological field, can solve the problems of reduced plasma clearance and toxicity, increased cost, etc., to achieve the effect of improving efficiency and reducing cost

Active Publication Date: 2013-08-21
QILU PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] CN101087611A reported that the conjugate prepared by linking antibody and maytansine alkaloid with non-cleavable linker and the conjugate prepared by linking antibody and maytansine alkaloid with cleavable linker had the same anti-in vitro and in vivo Tumor activity, but the former showed a significant reduction in plasma clearance and toxicity
Moreover, in order to obtain a suitable coupling ratio, that is, the molar ratio of the drug to the antibody in the conjugate, it is generally necessary to add a 6-10 times excess drug during the reaction. For example, in the preparation of trastuzumab-SMCC-DM1, it is necessary to invest 7-9 times excess drug, generally between 7.5-8.5 times, which increases the cost

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Example 1. Preparation of trastuzumab-SMCC-DM1 conjugate, verifying the effect of adding a surfactant on the coupling efficiency.

[0062] The preparation method of trastuzumab-SMCC-DM1 conjugate, the steps are as follows:

[0063] (1) Antibody replacement buffer

[0064] Using desalting chromatography (Sephadex TM G25) to replace the trastuzumab monoantigen solution into the reaction buffer A1 (0.1M sodium phosphate, pH7.5), and concentrate the antibody concentration to 20mg / ml to prepare the trastuzumab antibody A1;

[0065] Using desalting chromatography (Sephadex TM G25) to replace the trastuzumab monoantigen solution into the reaction buffer A2 (0.1M sodium phosphate, pH7.5, 0.05% v / v Tween 20), and concentrate the antibody concentration to 20mg / ml to prepare trastuzumab Antibody A2;

[0066] (2) Preparation of SMCC-DM1 mother liquor

[0067] Weigh SMCC-DM1 and fully dissolve it with dimethylacetamide (DMA) to prepare 10mg / ml SMCC-DM1 mother liquor;

[0068...

Embodiment 2

[0081] Example 2, adding anion exchange chromatography in the coupling process can reduce the content of conjugated product aggregates and endotoxin content.

[0082] The preparation of trastuzumab-SMCC-DM1 conjugates, the steps are as follows:

[0083] (1) Antibody replacement buffer: first use desalting chromatography (Sephadex TM G25) to replace the trastuzumab antibody stock solution into the reaction buffer (0.1M sodium phosphate, pH7.5, 0.05%v / v Tween 20), and concentrate the antibody concentration to 20mg / ml;

[0084] (2) Preparation of SMCC-DM1 mother solution: Weigh SMCC-DM1 with a molar ratio of 5.5 times that of trastuzumab in step (1), and fully dissolve it with dimethylacetamide (DMA) to prepare 10 mg / ml of SMCC-DM1 Mother liquor;

[0085] (3) Coupling reaction:

[0086] Add the SMCC-DM1 mother solution prepared in step (2) to the trastuzumab prepared in step (1) to start the coupling reaction, the reaction temperature is 25°C, and the reaction time is 1.5h;

...

Embodiment 3

[0096] Embodiment 3, preparation of Pani-SMCC-DM1 conjugates, the steps are as follows:

[0097] (1) Antibody replacement buffer

[0098] First, use ultrafiltration concentration (pellicon system) to replace the Panitum monoantigen solution into the reaction buffer (20mM potassium phosphate buffer, 0.02% v / v Tween 20, pH 6.0), and concentrate the antibody concentration to 30mg / ml.

[0099] (2) Preparation of SMCC-DM1 mother liquor

[0100] Weigh SMCC-DM1 that is 5.0 times the molar ratio of panitumumab in step (1), and fully dissolve it with dimethylacetamide (DMA) to prepare a 10 mg / ml SMCC-DM1 mother solution.

[0101] (3) Coupling reaction

[0102] Add the SMCC-DM1 mother solution prepared in step (2) to the panitumumab prepared in step (1) to start the coupling reaction, the reaction temperature is 25°C, and the reaction time is 2.0h;

[0103] (4) Anion exchange chromatography

[0104] The above reaction solution was loaded on Capto Adhere anion exchange chromatograp...

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Abstract

The invention relates to a method for preparing antibody-maytansine alkaloid medicine conjugate. The method comprises the following steps of replacing the antibody into a reaction buffer solution; dissolving a dual-function bridging agent-maytansine alkaloid with an organic solvent so as to prepare the mother liquor of maytansine alkaloid medicine; mixing the replaced antibody with the mother liquor of maytansine alkaloid medicine for coupled reaction for 1-4 hours at 20-30 DEG C; and carrying out anion exchange chromatography on the reaction liquid, conducting Sephadex TMG25 desalination chromatographic column purification on collected flow liquid, and collecting a first peak sample as the prepared antibody-maytansine alkaloid medicine conjugate. The prepared antibody-maytansine alkaloid medicine conjugate is proper in coupling rate, high in purity and low in endotoxin content.

Description

technical field [0001] The invention relates to a preparation method of an antibody-maytansine alkaloid drug conjugate, belonging to the field of biotechnology. Background technique [0002] The treatment of cancer has made remarkable progress with the development of targeted therapy drugs. In recent years, researchers have developed many antibody drugs by using cell receptors and antigens selectively expressed on the surface of cancer cells, and these antibodies can specifically bind to tumor cells. On this basis, cytotoxic molecules, such as bacterial and plant toxins, radionuclides, and certain chemotherapeutic drugs, are chemically linked to antibodies to obtain antibody-drug conjugates. After the cell binds, the drug is released or the cytotoxic activity of the drug is activated to kill the tumor cells. The specificity of antibody-drug conjugates can reduce the toxicity to normal cells, thus improving the tolerability of the drug in patients. [0003] Maytansine was ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C07K1/107C07K1/18C07K1/16
Inventor 孙丽霞张宗辉周凯松艾现伟杨亚南王庆民张新强
Owner QILU PHARMA CO LTD
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