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Preparing method of high-purity polymyxin B

A polymyxin, high-purity technology, applied in the field of medicine, can solve the problems such as difficult to remove B3 and B1-1, and achieve the effect of high purity, high yield and simple process

Inactive Publication Date: 2013-06-05
HUBEI RUIHAO ANKE MEDICINE TECH DEV CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The purpose of the present invention is to overcome the technical problems that the existing technology is difficult to remove impurities such as B3 and B1-1, and to provide a method that can effectively remove various impurities and increase the general purity (B1+B2 chromatographic purity at 40-80.0%). Colistin B is purified to B1+B2 ≥ 99.5%, high yield and suitable for the preparation of high-purity polymyxin B method for large-scale industrial production

Method used

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  • Preparing method of high-purity polymyxin B
  • Preparing method of high-purity polymyxin B
  • Preparing method of high-purity polymyxin B

Examples

Experimental program
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Effect test

Embodiment 1

[0028] (1) Preparation of upper column liquid: 1 g of polymyxin B sulfate (the chromatographic purity of polymyxin B1+polymyxin B2 is 74.0%) is dissolved in 100 ml of water through fermentation and separation and extraction of raw material polymyxin B. Filter through a microporous 0.45 μm filter membrane to remove impurities to obtain a clear solution; sample the clear solution for HPLC detection, see chromatographic purity figure 1 , impurity B3 is 3.9%, impurity B1-1 is 8.4%;

[0029] (2) upper column separation: the mobile phase is methanol and sodium sulfate-phosphate buffer solution with a volume ratio of 15:85, and the sodium sulfate-phosphate buffer solution is an aqueous sodium sulfate solution of 20 mM adjusted to pH=2.1 with phosphoric acid; (1) The obtained clarified solution is injected into a preparative HPLC column with a flow rate of 1.0ml / min. The HPLC preparative column is Kromasil 100-3.5-C18 (250 × 4.6mm), and the particle diameter of filler C18 is 5 μm. Th...

Embodiment 2

[0032] (1) Preparation of the upper column liquid: the polymyxin B sulfate raw material is the same as in Example 1, 1.0 g of the sample is taken, dissolved in 25 ml of water, and filtered through a 0.45 μm filter membrane to obtain a clear solution;

[0033] (2) Upper column separation: the chromatographic column is Kromasil 100-5-C8 (250×4.6mm), the particle diameter of packing C8 is 5 μm, the buffer solution is 35mM sodium sulfate aqueous solution (phosphoric acid adjusts pH=2.8), and the mobile phase is volume ratio Acetonitrile and buffer solution of 23:77, the clarified solution obtained in step (1) was injected, the flow rate was 1.2ml / min, the working temperature was 33°C, the pressure was 9.2MPa, the UV detection wavelength used was 215nm, and the target component B1 was collected .

[0034] The target component was concentrated by rotary evaporation at -0.1MPa and 50°C, acetonitrile and part of water were evaporated, and the concentrated solution was lyophilized, and...

Embodiment 3

[0036] Kromasil 100-3.5-C4 was used to replace Kromasil 100-5-C8 in Example 1, and the others were the same as in Example 1 to obtain polymyxin B2 with a purity of more than 99.5%.

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Abstract

The invention discloses a preparing method of high-purity polymyxin B. The preparing method comprises the steps of preparation of column-loading solution; injecting the column-loading solution into a high performance liquid chromatography preparative column, wherein sodium sulfate - phosphate buffer / organic solvent is used as mobile phase, collecting target components, removing impurities, especially B1 - 1 and B3, and determining collected solution by means of high performance liquid chromatography (HPLC), wherein chromatographic purity of the target components B1 or B2 or mixture of the B1 and the B2 is more than 99.5%; and purification, wherein vacuum concentration is carried out on the collected target components so that the organic solvent is removed, organic solvent desalination concentration is carried out on balance phase, concentrated solution is spray-dried or freeze-dried to produce the polymyxin B with purity of more than 99.5%. The preparing method of the high-purity polymyxin B has the advantages of being high in purity, high in yield, simple in process, and capable of injecting sample solution continuously. Quality of products is far better than the medical standard regulated by the European Pharmacopoeia (EP) and the United States Pharmacopeia (USP). One-time investment of equipment is slightly high, while operation cost is not high, and therefore the preparing method of the high-purity polymyxin B is suitable for industrial production.

Description

technical field [0001] The invention belongs to the technical field of medicine, and in particular relates to a preparation method of high-purity polymyxin B. Background technique [0002] Polymyxin B is a basic polypeptide antibiotic composed of various amino acids and fatty acids produced by Bacillus polymyxa. Polymyxin B is a multicomponent product (see formula I), including polymyxin B1, B2, B3, B1-1, wherein, B 1 and B 2 Is the active ingredient, B3 and B1-1 as the main impurities. Clinical use of its sulfate, polymyxin B sulfate has antibacterial effect on almost all Gram-negative bacteria, because it is highly effective against Pseudomonas aeruginosa, it used to be the drug of choice against Pseudomonas aeruginosa, and is currently effective against superbugs However, the impurities B3 and B1-1 have relatively large toxic and side effects, which have seriously affected the clinical use of polymyxin B. At present, it is mainly used externally, and it is only used lo...

Claims

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Application Information

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IPC IPC(8): C07K7/62C07K1/16
Inventor 李小兵赵平
Owner HUBEI RUIHAO ANKE MEDICINE TECH DEV CO LTD
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