CN110272461B - Method for purifying beta-thymidine from fermentation liquor - Google Patents

Method for purifying beta-thymidine from fermentation liquor Download PDF

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CN110272461B
CN110272461B CN201910593511.1A CN201910593511A CN110272461B CN 110272461 B CN110272461 B CN 110272461B CN 201910593511 A CN201910593511 A CN 201910593511A CN 110272461 B CN110272461 B CN 110272461B
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membrane
thymidine
beta
ultrafiltration
nanofiltration
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CN110272461A (en
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张国银
何永进
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Chifeng Broad Biological Technology Co ltd
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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07HSUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00Processes for the preparation of sugar derivatives
    • C07H1/06Separation; Purification
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07HSUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/06Pyrimidine radicals
    • C07H19/073Pyrimidine radicals with 2-deoxyribosyl as the saccharide radical
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02PCLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00Technologies relating to chemical industry
    • Y02P20/10Process efficiency

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Abstract

The invention discloses a method for purifying beta-thymidine from fermentation liquor, which comprises the steps of filtering the fermentation liquor containing the beta-thymidine by using a microfiltration membrane with the pore diameter of 50-500 nm, filtering by using an ultrafiltration membrane with the molecular weight cutoff of 3500-20000 Da, collecting ultrafiltration permeate, filtering the ultrafiltration permeate by using a nanofiltration membrane with the molecular weight cutoff of 150-300 Da, and collecting nanofiltration concentrate; decolorizing the nanofiltration concentrated solution by active carbon, evaporating and concentrating in vacuum, and cooling and crystallizing to obtain wet beta-thymidine. The method does not use ion exchange resin and secondary crystallization, has simple process route, reduces the discharge of production wastewater, and has low production cost and high total yield.

Description

Method for purifying beta-thymidine from fermentation liquor
Technical Field
The invention belongs to the technical field of biological pharmacy, and particularly relates to a method for purifying beta-thymidine from fermentation liquor.
Background
Beta-thymidine, also known as thymidine, is a compound formed by connecting N-1 of thymine and C-1 of deoxyribose through a beta glycosidic bond, is one of four nucleosides forming deoxyribonucleic acid (DNA), and is also a key starting material of zidovudine as an anti-AIDS drug.
With the increasing demand of anti-AIDS drugs worldwide, how to produce beta-thymidine with high efficiency, low cost and large scale is a very urgent problem. Because the conventional synthesis method for producing beta-thymidine has complicated process and uses a large amount of organic solvent, the safety and the manufacturing cost cannot meet the requirements of the growing market. The method for separating and purifying beta-thymidine from fermentation liquor developed by Shanghai Chuanno pharmaceutical company Limited CN107383135A is the method which has the highest extraction yield and the best product chemical purity and content and is published so far, and is implemented in mass production.
In the prior art, the method for purifying beta-thymidine from fermentation liquor by the company is as follows: carrying out microfiltration, ultrafiltration, cation exchange resin, anion exchange resin, reverse osmosis concentration, evaporation concentration, cooling and crystallization on the fermentation liquor, and separating the crystallization liquor to obtain a solid; and after the solid is dissolved, decoloring by using active carbon, evaporating and concentrating, cooling and crystallizing, separating a crystallization liquid to obtain a wet product, and drying the wet product to obtain a finished product. The method uses ion exchange resin and secondary crystallization processes, a large amount of wastewater is generated in the production process, great pressure is brought to environment-friendly treatment, the process route is complex, and the total yield is low.
Disclosure of Invention
The invention aims to provide a method for purifying beta-thymidine from fermentation liquor, which is simple and convenient to operate, low in pollution and high in yield.
The technical scheme of the invention is realized as follows: a method for purifying beta-thymidine from a fermentation broth, comprising: it comprises the following steps:
s1, microfiltration: filtering the fermentation liquor containing the beta-thymidine content by using a microfiltration membrane with the pore diameter of 50-500 nm, concentrating the volume of the fermentation liquor to 1/3-2/3 under the conditions that the temperature is 40-90 ℃ and the pressure is 0.1-0.4 MPa, adding water which is 3-5 times of the volume of the concentrated fermentation liquor for washing, and collecting microfiltration permeate;
s2, ultrafiltration: filtering the microfiltration permeate by using an ultrafiltration membrane with the molecular weight cutoff of 3500-20000 Da, concentrating the microfiltration permeate to 1/8-1/20 in volume under the conditions that the temperature is 30-45 ℃ and the pressure is 0.5-1.5 MPa, adding water of 1/10-1/3 in volume of the original fermentation liquid for washing, and collecting the ultrafiltration permeate;
s3, nanofiltration: filtering the ultrafiltration permeate by using a nanofiltration membrane with the molecular weight cutoff of 150-300 Da, concentrating the ultrafiltration permeate to 1/5-1/12 of the original volume under the conditions that the temperature is 30-45 ℃ and the pressure is 0.5-2.5 MPa, and collecting nanofiltration concentrate;
s4, decoloring: decolorizing the nanofiltration concentrated solution by using activated carbon, wherein the adding proportion of the activated carbon is 5-20% (w/w) of the nanofiltration concentrated solution, decolorizing for 1.0-2.5 hours at the temperature of 50-90 ℃, and collecting the decolorized concentrated solution;
s5, concentrating: carrying out vacuum evaporation concentration on the decolorized concentrated solution, concentrating the decolorized concentrated solution under the conditions that the vacuum degree is-0.07 to-0.09 Mpa and the temperature is 40 to 60 ℃ until the concentration of beta-thymidine is 150 to 300g/L, and collecting the evaporation concentrated solution;
s6, crystallization: and cooling the evaporation concentrated solution to 0-10 ℃, crystallizing, and filtering to obtain a wet product of the beta-thymidine.
Further, in step S1, the microfiltration membrane is an inorganic ceramic membrane or a polyvinylidene fluoride membrane.
Further, in step S2, the ultrafiltration membrane is a roll-type membrane, and the material is polyethersulfone or polyacrylonitrile.
Further, in step S3, the nanofiltration membrane is a roll membrane, and the material is polyamide or polysulfone.
Further, in step S4, the activated carbon is one or more of NoritSX, NoritCA, and 767 activated carbon, and needle-type activated carbon is preferably used.
Compared with the prior art, the invention has the advantages that:
(1) the invention uses nanofiltration technology to desalt, which greatly reduces the amount of waste water generated by the desalination compared with cation and anion resin, and greatly improves the automation degree of operation.
(2) The nanofiltration technology of the invention greatly enhances the removal effect of soluble fermented small molecular impurities.
(3) The invention uses active carbon to remove impurities before concentration, realizes one-time crystallization to obtain a high-quality finished product, simplifies operation and improves product yield.
(4) The model and material of the selected microfiltration membrane, ultrafiltration membrane and nanofiltration membrane are scientific and reasonable, and the temperature, pressure, material consumption and concentration ratio of each process step are all the most suitable parameters obtained through a large number of scientific experiments.
(5) The process method has the advantages of simple operation, low pollution and high yield, and is suitable for industrial production and application.
Detailed Description
In order to make the technical solutions clear, the following describes embodiments of the present invention in further detail by specific examples.
Example 1 (prior art): microfiltering the fermentation liquor containing beta-thymidine with 50nm ceramic membrane, concentrating the fermentation liquor to 1/3 at 85 deg.C, adding 4 times volume of concentrated fermentation liquor, top washing, and collecting microfiltered permeate; cooling the microfiltration permeate to 40 ℃, performing ultrafiltration by using a 10000Da roll type ultrafiltration membrane, concentrating the volume to 1/8, adding 1/3 volume of the original fermentation liquor, washing with water, and collecting the ultrafiltration permeate; respectively allowing the ultrafiltration permeate to flow through D001 and D213 resins, adding water with the volume 5 times that of the resins to wash the resins, and collecting the resin permeate; performing reverse osmosis concentration on the resin permeate at 2.0MPa and the temperature of 40 ℃ to 1/15, performing vacuum concentration at the temperature of 50 ℃ to 200g/L of beta-thymidine, and cooling to 10 ℃ for crystallization; the crystals were suction filtered to give 34.8g of a wet product having a content of beta-thymidine (dry basis) of 95.5%.
Example 2 (prior art): the wet product of example 1 was added to water, 15% NoritSX activated carbon was added, the mixture was maintained at 75 ℃ for 2.0 hours, the activated carbon was removed by filtration, the mixture was concentrated at 45 ℃ in vacuo to 300g/L, then cooled to 10 ℃ for crystallization, filtered to obtain a wet product, and dried at 60 ℃ in vacuo to obtain 32.5g of dry β -thymidine product, with a total yield of 80.1%, a white appearance, an HPLC purity of 99.75% and an external standard content of 99.71%.
Example 3 (prior art): adding the wet product obtained in the example 1 into water, adding 767% of 20% of activated carbon, keeping the mixture at 90 ℃ for 1.5 hours, filtering to remove the activated carbon, carrying out vacuum concentration at 45 ℃ to 200g/L, cooling to 4 ℃ for crystallization, carrying out suction filtration to obtain a wet product, and carrying out vacuum drying at 50 ℃ to obtain 32.2g of a dry finished product of the beta-thymidine, wherein the total yield is 79.4%, the appearance is white, the HPLC purity is 99.80%, and the external standard content is 99.85%.
Example 4: microfiltering the fermentation liquor containing beta-thymidine by using a 100nm ceramic membrane, concentrating the fermentation liquor to 1/2 at the temperature of 65 ℃ and the pressure of 0.2MPa, adding water with the volume 4 times of that of the concentrated fermentation liquor for top washing, and collecting the microfiltered permeate; cooling the microfiltration permeate to 30 ℃, performing ultrafiltration by using a 5000Da roll type ultrafiltration membrane, concentrating the microfiltration permeate to 1/10 in volume under the condition that the pressure is 1MPa, adding water in the original fermentation liquor 1/5 volume for washing, and collecting the ultrafiltration permeate; nano-filtering the ultrafiltration permeate by using a 200Da roll type nano-filtration membrane under the conditions of 2.0MPa and 35 ℃, concentrating to 1/12 of the original volume, adding 15 percent Norit CA activated carbon, keeping the temperature at 75 ℃ for 2.0 hours, concentrating in vacuum at 55 ℃ to 300g/L of beta-thymidine, and cooling to 10 ℃ for crystallization; the crystallized solution was suction filtered to obtain 33.2g of wet product with a total yield of 81.8%, white appearance, HPLC purity of 99.77%, external standard content of 99.80%.
Example 5: microfiltering the fermentation liquor containing beta-thymidine with 200nm ceramic membrane, concentrating the fermentation liquor to 1/3 at 75 deg.C and 0.3MPa, adding water 3 times the volume of the concentrated fermentation liquor, top-washing, and collecting microfiltered permeate; cooling the microfiltration permeate to 30 ℃, performing ultrafiltration by using a 3500Da roll-type ultrafiltration membrane, concentrating the microfiltration permeate to 1/10 in volume under the condition that the pressure is 1.2MPa, adding water in volume of 1/3 of the original fermentation liquor for washing, and collecting the ultrafiltration permeate; nano-filtering the ultrafiltration permeate by using a 250Da roll type nano-filtration membrane under the conditions of 1.5MPa and 40 ℃, concentrating to 1/10 of the original volume, adding 20 percent of Norit SX activated carbon, keeping the temperature at 65 ℃ for 1.5 hours, concentrating in vacuum at 45 ℃ to 200g/L of beta-thymidine, and cooling to 4 ℃ for crystallization; 32.8g of wet product is obtained by suction filtration of the crystallization liquid, the total yield is 80.8%, the appearance is white, the HPLC purity is 99.77%, and the external standard content is 99.70%.
Example 6: microfiltering the fermentation liquor containing beta-thymidine with 50nm ceramic membrane, concentrating the fermentation liquor to 1/3 at 75 deg.C and 0.4MPa, adding water 3 times the volume of the concentrated fermentation liquor, top-washing, and collecting microfiltered permeate; cooling the microfiltration permeate to 35 ℃, performing ultrafiltration by using a 5000Da roll-type ultrafiltration membrane, concentrating the microfiltration permeate to 1/10 in volume under the condition that the pressure is 0.8MPa, adding water in the volume of 1/3 of the original fermentation liquor for washing, and collecting the ultrafiltration permeate; carrying out nanofiltration on the ultrafiltration permeate by using a 180Da roll type nanofiltration membrane under the conditions of 2.0MPa and 40 ℃, carrying out nanofiltration concentration to 1/10 of the original volume, then adding 10 percent of Norit SX active carbon and 10 percent of 767 active carbon, keeping the mixture at 75 ℃ for 1.5 hours, carrying out vacuum concentration at 45 ℃ to 270g/L beta-thymidine, and cooling to 4 ℃ for crystallization; the crystalline liquid was filtered with suction to obtain 33.5g of wet product, total yield 82.6%, white appearance, HPLC purity 99.72%, external standard content 99.80%.

Claims (3)

1. A method for purifying beta-thymidine from a fermentation broth, comprising: it comprises the following steps:
s1, microfiltration: filtering a fermentation liquor containing beta-thymidine by using a microfiltration membrane with the aperture of 50-500 nm, concentrating the volume of the fermentation liquor to 1/3-2/3 under the conditions that the temperature is 40-90 ℃ and the pressure is 0.1-0.4 MPa, adding water with the volume 3-5 times of the volume of the concentrated fermentation liquor for washing, and collecting microfiltration permeate, wherein the microfiltration membrane is an inorganic ceramic membrane or a polyvinylidene fluoride membrane;
s2, ultrafiltration: filtering the microfiltration permeate by using an ultrafiltration membrane with the molecular weight cutoff of 3500-20000 Da, concentrating the microfiltration permeate to 1/8-1/20 at the temperature of 30-45 ℃ and under the pressure of 0.5-1.5 MPa, adding water with the volume of 1/10-1/3 of the original fermentation liquid for washing, and collecting the ultrafiltration permeate, wherein the ultrafiltration membrane is a roll-type membrane and is made of polyether sulfone or polyacrylonitrile;
s3, nanofiltration: filtering the ultrafiltration permeate by using a nanofiltration membrane with the molecular weight cutoff of 150-300 Da, concentrating the ultrafiltration permeate to 1/5-1/12 of the original volume under the conditions that the temperature is 30-45 ℃ and the pressure is 0.5-2.5 MPa, and collecting nanofiltration concentrate, wherein the nanofiltration membrane is a roll membrane and is made of polyamide or polysulfone;
s4, decoloring: decolorizing the nanofiltration concentrated solution by using activated carbon, wherein the adding proportion of the activated carbon is 5-20 percent of that of the nanofiltration concentrated solution, and the addition proportion of the activated carbon is w/w, decolorizing for 1.0-2.5 hours at the temperature of 50-90 ℃, and collecting the decolorized concentrated solution;
s5, concentrating: carrying out vacuum evaporation concentration on the decolorized concentrated solution, concentrating the decolorized concentrated solution under the conditions that the vacuum degree is-0.07 to-0.09 Mpa and the temperature is 40 to 60 ℃ until the concentration of beta-thymidine is 150 to 300g/L, and collecting the evaporation concentrated solution;
s6, crystallization: and cooling the evaporation concentrated solution to 0-10 ℃, crystallizing, and filtering to obtain a wet product of the beta-thymidine.
2. The method of purifying β -thymidine from a fermentation broth of claim 1, wherein: in step S4, the activated carbon is one or more of NoritSX, NoritCA, and 767 activated carbon.
3. The method of claim 2, wherein the beta thymidine is purified from a fermentation broth, comprising: the active carbon is needle-shaped active carbon.
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CN111171097B (en) * 2019-12-29 2023-04-07 赤峰蒙广生物科技有限公司 Separation and purification method for producing adenosine by fermentation
CN114032265A (en) * 2021-12-03 2022-02-11 江西诚志生物工程有限公司 Process for producing thymidine by fermentation method
CN114853823A (en) * 2022-05-26 2022-08-05 宁夏华吉生物有限公司 Method for extracting thymidine
CN116496336A (en) * 2023-05-06 2023-07-28 浙江昊清生物科技有限公司 Method for extracting thymidine from fermentation broth

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