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Method for separating purified cytidine diphosphate choline by hydrophobic chromatography

A technology for separation and purification of citicoline, applied in the field of biological separation, can solve the problems of high product loss rate, lengthy production process, and low separation efficiency, achieve short loss, increase product yield, and reduce separation process Effect

Active Publication Date: 2010-12-08
NANJING UNIV OF TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] Although these methods have achieved the separation of CDP-choline, they all need to use two resin or activated carbon columns, resulting in lengthy production process and high product loss rate, which has become the bottleneck of its development.
Qiu Weiran et al. (CN101096380A.) proposed for the first time the use of ion-exchange resin combined chromatography to separate CDP-choline and reclaim unreacted cytidylic acid at the same time. This method can reclaim a small amount of unreacted cytidine in the cell transformation solution acid, but the yield of CDP-choline in the purification process is only 80.9%, which also fails to solve the problems of low separation efficiency and low product yield

Method used

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  • Method for separating purified cytidine diphosphate choline by hydrophobic chromatography

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Effect test

Embodiment 1

[0028] According to the whole-cell catalytic synthesis method above, 15 L of CDP-choline conversion liquid was prepared, wherein the concentration of CDP-choline was 9.47 g / L.

[0029] The treatment process is as follows:

[0030] (1) Adjust the pH of the whole-cell catalytic conversion solution to 2.0 with hydrochloric acid to inactivate the enzyme, and remove solid impurities after centrifugation at 8000 rpm for 20 minutes. The centrifuged supernatant was processed by ultrafiltration, and the ultrafiltration permeate was collected; the ultrafiltration membrane was a polysulfone membrane, the molecular weight cut-off of the ultrafiltration membrane was 5000Dolton, and the working pressure of the ultrafiltration membrane was 1MPa.

[0031](2) Sodium chloride was added to the ultrafiltration permeate to prepare a column liquid containing 2.5 mol / L sodium chloride and 5 g / L CDP-choline, and the pH was adjusted to 4.0 with hydrochloric acid. The sulfonic acid-based polystyrene r...

Embodiment 2

[0034] According to the above whole-cell catalytic synthesis method, 15 L of CDP-choline conversion liquid was prepared, wherein the concentration of CDP-choline was 10.75 g / L.

[0035] The treatment process is as follows:

[0036] (1) Adjust the pH of the whole-cell catalytic conversion solution to 3.0 with hydrochloric acid to inactivate the enzyme, and remove solid impurities after centrifugation at 10,000 rpm for 20 minutes. The centrifuged supernatant was processed by ultrafiltration, and the ultrafiltration permeate was collected; the ultrafiltration membrane was a polysulfone membrane, the molecular weight cut-off of the ultrafiltration membrane was 3000Dolton, and the working pressure of the ultrafiltration membrane was 1.5MPa.

[0037] (2) Ammonium sulfate is added to the permeate of the ultrafiltration to prepare a column solution containing 0.01mol / L ammonium sulfate and 5g / L CDP-choline, and the pH is adjusted to 2.0 with hydrochloric acid, and the phosphoric acid-...

Embodiment 3

[0040] According to the whole-cell catalytic synthesis method above, 15 L of CDP-choline conversion solution was prepared, wherein the concentration of CDP-choline was 12.8 g / L.

[0041] The treatment process is as follows:

[0042] (1) Adjust the pH of the whole-cell catalytic conversion solution to 2.0 with hydrochloric acid to inactivate the enzyme, and remove solid impurities after centrifugation at 8000 rpm for 20 minutes. The centrifuged supernatant was processed by ultrafiltration, and the ultrafiltration permeate was collected; the ultrafiltration membrane was a polysulfone membrane, the molecular weight cut-off of the ultrafiltration membrane was 8000Dolton, and the working pressure of the ultrafiltration membrane was 0.5MPa.

[0043] (2) Potassium dihydrogen phosphate was added to the ultrafiltration permeate to prepare a column solution containing 1 mol / L potassium dihydrogen phosphate and 10 g / LCDP-choline, and the pH was adjusted to 4.0 with hydrochloric acid. Th...

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Abstract

The invention discloses a method for separating purified cytidine diphosphate choline by hydrophobic chromatography. The method comprises the following steps of: (1), performing enzyme inactivation, centrifugation and ultra-filtration of a cytidine diphosphate choline conversion liquid; (2), after adjusting the pH of the pre-processed cytidine diphosphate choline conversion liquid to between 2.0 and 14.0, adding an inorganic salt to prepare column-loading liquid which contains 0.01 to 5mol / L of inorganic salt and 0.1 to 40g / L of cytidine diphosphate choline with the cytidine diphosphate choline conversion liquid, absorbing the column-loading liquid with a hydrophobic chromatography resin and eluting with pure water of which the pH value is 2.0 to 14.0; and (3), nano-filtering, desalinating and concentrating the eluate, and obtaining the cytidine diphosphate choline through crystallization. In the method, the separation process is simple; through regeneration, the resin after elution can be repeatedly used; the separation cost is low; the product is easily crystallized; and the CDP-choline product with high purity and high yield can be obtained.

Description

technical field [0001] The invention belongs to the technical field of biological separation, and in particular relates to a method for separating and purifying citicoline by hydrophobic chromatography. Background technique [0002] Citicoline (CDP-choline) (I) is a nucleoside derivative and a precursor of phosphatidylcholine, which can increase the levels of norepinephrine and dopamine in the central nervous system. It is widely used clinically to treat various cerebrovascular diseases, senile dementia, depression, and also has certain curative effect on Parkinson's syndrome, neurological deafness and tinnitus, tardive dyskinesia, cerebellar and spinocerebellar ataxia . [0003] [0004] In the whole-cell catalytic conversion solution of CDP-choline, there are some compounds and intermediate metabolites with similar physical and chemical properties such as cytidine monophosphate, cytidine diphosphate and cytidine triphosphate, etc., as well as unconverted substrates suc...

Claims

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Application Information

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IPC IPC(8): C07H19/10C07H1/06
Inventor 应汉杰沈素芹周锡群苑巍金乃纯熊健柏建新张磊
Owner NANJING UNIV OF TECH
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