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A kind of separation and purification method of antibiotic nvp018 intermediate

A technology for separation and purification of intermediates, applied in the field of separation and purification of intermediates of antibiotic NVP018, can solve the problems of high cost, large amount of use, no reports, etc., and achieve the effects of high cost, large amount of solvent use, and cost saving

Active Publication Date: 2019-03-15
SUZHOU BIOSYNTHETICA CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, the separation and purification of HS457 is known to use silica gel column chromatography, and the elution solvent is ethyl acetate-methanol. In this method, the yield of HS457 with a purity of more than 85% is very low; acetone and cyclohexane are used for elution. Solvent, the amount of solvent used is large, the cost is high, the total recovery rate can reach 87%, and the yield of components with a purity of more than 80% is only 29.0%. At present, there is no industrialized separation and purification report
[0007] And about the separation and purification method of HS496, there is no report at present

Method used

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  • A kind of separation and purification method of antibiotic nvp018 intermediate
  • A kind of separation and purification method of antibiotic nvp018 intermediate
  • A kind of separation and purification method of antibiotic nvp018 intermediate

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Embodiment 1 Streptomyces bacterial classification is cultivated

[0048] Strain: Streptomyces sp.BIOT-4746 (purchased from Isomerase Therapeutics Ltd)

[0049] A. Primary seed cultivation

[0050] Put the bacteria stored in the glycerol tube into the sterilized shaker flask culture medium, place it on a shaker, culture at 27°C for 36-48 hours at 220r / min, when the pH reaches 5.2-5.4 and PMV≥18%, you can get first-class seed liquid;

[0051] Among them, shake flask medium components: glycerin 40g / L, soybean peptone (A3) 10g / L, malt extract 21g / L, pH7.0, sterilized at 121±2°C for 30min;

[0052] B. Secondary seed cultivation

[0053] Put 400-800ml of primary seed liquid into sterilized 1000L secondary seed medium for cultivation. The cultivation temperature is 27°C, the dissolved oxygen is greater than 30%, and the cultivation is 36-50h. When the pH reaches 5.2-5.4, PMV≥20 %, to obtain the secondary seed liquid;

[0054] Secondary seed medium is the same as the shake ...

Embodiment 2

[0060] First Purification of Macroporous Adsorbent Resins

[0061] After the secondary seed solution was inoculated into the fermentation medium and fermented for 24 hours, 10t of pretreated substrate FS45 and 1600kg of non-polar macroporous adsorption resin were added, cultivated for 110-120 hours, and the pretreated and sterilized non-polar At this time, the content of HS457 can reach 175-185mg / L. Continue to cultivate until the amount of product no longer increases, which means the end of fermentation.

[0062] Among them, the substrate FS45 was dissolved in DMSO one day in advance, and its final concentration was controlled at 2mM / L after adding to the fermenter; the non-polar macroporous adsorption resin was soaked in methanol overnight in advance, washed with water to remove methanol, and sterilized at 121°C for 30 minutes Add to the fermenter. In addition, the selected non-polar macroporous adsorption resins include D101, LXT-081, and Aberlite XAD18 resins, and of cour...

Embodiment 3

[0066] Purification by HS457 silica gel column chromatography

[0067] Get 4.5kg ethyl acetate concentrated solution and carry out pretreatment, add cyclohexane of equal volume and stir for 30 minutes, static layering, remove upper strata cyclohexane phase, measure and take the lower floor material liquid phase and add dichloromethane to dissolve, and quality is 18kg, The measured content of HS457 is 17.11g / kg, waiting to be loaded on the column.

[0068] Column loading and sample loading: Wet column loading and wet loading; weigh 34kg of silica gel and soak in dichloromethane overnight, the silica gel is 100-200 mesh, stir the silica gel after soaking overnight into a homogenate, and slowly add it to the glass chromatography In the column, 30cm×2m chromatography column, the excess dichloromethane eluent flows out, and 1-2cm of anhydrous sodium sulfate is added to the upper layer, and the total height of the column bed is about 1.5-1.6m; then take 18kg of samples and add them ...

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Abstract

The invention discloses a separation and purification method of an antibiotic NVP018 intermediate. A fermentation culture medium is inoculated with streptomycete for fermentation culture for 24 h, a substrate FS45 is added, nonpolar macroporous adsorption resin is added in batches, then, the antibiotic NVP018 intermediate is desorbed from the resin and concentrated, and separation and purification are performed with a silica gel column chromatography method, wherein an adopted eluent is dichloromethane-methanol, and gradient elution is performed. By means of the separation and purification method, HS457 with the purity being 92% or higher can be obtained through separation, the yield with the purity being 85% or higher can reach 70%, and the overall recovery rate of HS457 can reach 93%; meanwhile, HS457 can be separated and purified with the method, HS496 with the highest purity being 95% can be obtained, the yield with the purity being 85% or higher can reach 70%, and the overall recovery rate can reach 95%; by means of the method, solid powdery HS496 with the yield being 50% can be obtained.

Description

technical field [0001] The invention is applicable to the fields of biopharmaceutical and biochemical industry, and in particular relates to a separation and purification method of an antibiotic NVP018 intermediate. Background technique [0002] Immunosuppressants are a class of drugs with immunosuppressive effects, which are mainly used clinically for rejection of organ transplantation and autoimmune response diseases. Currently clinically used are cyclosporin A (cyclosporin A, CoA), adrenocortical hormone and so on. These drugs lack selectivity and specificity, and long-term use will reduce the body's resistance and induce infection or increase the probability of tumor occurrence. Therefore, the development of new immunosuppressants has good clinical prospects. [0003] NVP018 (Formula 1) is a new type of macrolide antibiotic produced by Streptomyces, which is used for the treatment of hepatitis B and hepatitis C virus and is currently in clinical trials. Compared with ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P21/02C07K5/062C07K1/16C12N1/20C12R1/465
CPCC07K5/06052C12N1/20C12P21/02
Inventor 王欣彤胡志浩蔡昌银刘斌
Owner SUZHOU BIOSYNTHETICA CO LTD