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Method for preparing cyclosporine A

A technology of cyclosporin and fermented liquid, which is applied in the field of biomedicine, can solve the problems of many types of organic solvents, poor pigment removal effect, complex process, etc., and achieve reduction of energy consumption and labor intensity, significant concentration and enrichment, and use less effect

Active Publication Date: 2012-10-10
鲁南新时代生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] Aiming at the disadvantages of cyclosporin A separation and purification in the prior art, such as complex process, cumbersome steps, many types of organic solvents and large dosage, poor pigment removal effect and low total yield, the inventors separated and purified cyclosporine A A large number of studies have been carried out on the technology. Aiming at the biochemical properties of cyclosporine A, the experimental conditions and process parameters have been explored, and the separation method combined with macroporous adsorption and silica gel column chromatography has achieved good technical results. The present invention adopts macroporous Adsorption resin chromatography technology, the organic solvent in the extract can be directly applied to the macroporous adsorption resin without replacement, and the amount of eluent is small. It is determined by experiments that only 1.5 to 2 times the column volume is needed to completely remove cyclosporine A. The elution has a very significant concentration and enrichment effect on cyclosporin A, and can remove most of the pigments, which solves the shortcomings of the original extraction process and improves the product yield; because the macroporous adsorption resin chromatography has already To remove most impurities, the present invention adopts non-gradient elution in silica gel column chromatography, and uses low-toxic organic solvents acetone and petroleum ether as mobile phases, which simplifies the chromatography process and reduces environmental pollution compared with gradient elution

Method used

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  • Method for preparing cyclosporine A
  • Method for preparing cyclosporine A
  • Method for preparing cyclosporine A

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] (1) Take 50L of cyclosporin A fermentation broth, add 5mol / l NaOH to adjust the pH to neutral, add 1 volume of acetone and stir and extract at 30°C for 8 hours, vacuum filter at -0.06Mpa to remove the bacteria, and collect the filtrate to be Upper column.

[0034] (2) Pack a column (Φ10cm×120cm) with 5kg D101 macroporous adsorption resin, wash and process the resin with pure water, 3% sodium hydroxide solution and 3% hydrochloric acid solution respectively, and extract about 10 times the column volume after treatment Put on the column; wash the column with about 2 column volumes of 3% hydrochloric acid and wash it with pure water to pH 6.5-7.5; elute with acetone, collect about 1.5 times the column volume eluate; add 0.5 times the volume of petroleum in the eluent The layers were separated by ether extraction, the lower aqueous phase was removed, and the upper liquid phase was concentrated to be applied to a silica gel column.

[0035] (3) Appropriately treat 200-300 m...

Embodiment 2

[0038] (1) Take 50L of cyclosporine A fermentation broth, add 5mol / l NaOH to adjust the pH to neutral, add 2 times the volume of methanol and stir and extract at 40°C for 5 hours, vacuum filter at -0.06Mpa to remove the bacteria, and collect the filtrate to be Upper column.

[0039] (2) Pack a column (Φ10cm×120cm) with 5kg H103 macroporous adsorption resin, wash and treat the resin with pure water, 3% sodium hydroxide solution and 3% hydrochloric acid solution respectively, and put about 8 times column volume extract on the Column, wash the column with 1.5 times the column volume of 3% hydrochloric acid and wash it with pure water to pH 6.5-7.5, elute with ethyl acetate, collect about 2 times the column volume eluate, add 0.5 times the volume of petroleum to the eluate The layers were separated by ether extraction, the lower aqueous phase was removed, and the upper liquid phase was concentrated to be applied to a silica gel column.

[0040] (3) This step is the same as step (...

Embodiment 3

[0043] (1) Take 50L of cyclosporine A fermentation broth, add 5mol / l NaOH to adjust the pH to neutral, add 1.5 times the volume of ethyl acetate, stir and extract at 35°C for 6 hours, remove the bacteria by vacuum filtration at -0.06Mpa, and collect The filtrate was loaded onto the column.

[0044] (2) Pack a column (Φ10cm×120cm) with 5kg X-5 macroporous adsorption resin, wash and treat the resin with pure water, 3% sodium hydroxide solution and 3% hydrochloric acid solution respectively, and extract about 7 times the column volume after treatment Liquid on the column; wash the column with 2 times the column volume of 3% hydrochloric acid and wash it with pure water to pH 6.5-7.5; elute with acetone, collect about 1.5 times the column volume eluate; add 0.5 times the volume of petroleum in the eluent The layers were separated by ether extraction, the lower aqueous phase was removed, and the upper liquid phase was concentrated to be applied to a silica gel column.

[0045] (3)...

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Abstract

The invention belongs to the field of biomedical technology and provides an industrialized method for preparing high-purity cyclosporine A. According to the method, the cyclosporine A is obtained through extraction by utilizing fermentation liquid, chromatography by utilizing a nonpolar macroporous absorption resin, silica gel column chromatography, crystallization and purification. In the invention, isoconcentration elution is adopted without complicated intermediary steps of dissolvent substitution processes; the process is easy to operate and is suitable for industrialized production; the purity of products is greater than 98.5; and the yield is above 65%.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a separation and purification process of cyclosporine A. Background technique [0002] Cyclosporin A (Ciclosporin A, CsA) is a cyclic ring containing 11 amino acids isolated from the culture of Tolypocladium inflatum CSP3, later named Beauveria bassiana. Polypeptide is a powerful immunosuppressant. In addition to cyclosporin A, there are more than 20 homologues such as B, C, and D. Cyclosporin A is used in the immunosuppressive treatment of kidney, liver and heart transplantation, and it can also be used to treat some autoimmune diseases, such as lupus erythematosus, rheumatoid arthritis, idiopathic thrombocytopenic purpura, ulcerative Various autoimmune diseases such as colitis, myasthenia gravis, chronic nephritis, chronic active hepatitis and nephrotic syndrome. [0003] [0004] The main impurities in cyclosporin A fermentation products are cyclosporin A homologues w...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K7/64C07K1/36A61P37/06
Inventor 赵志全董惠钧赵军杰
Owner 鲁南新时代生物技术有限公司
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