WO2021036386A1

WO2021036386A1 - Simulated moving bed continuous chromatography system and application thereof, and method for purifying coenzyme q10

Info

Publication number: WO2021036386A1
Application number: PCT/CN2020/094047
Authority: WO
Inventors: 胡泽君; 徐鲁明; 廖炜程; 王炳荣; 窦婵玉; 甄明; 王丽
Original assignee: 内蒙古金达威药业有限公司; 厦门金达威集团股份有限公司
Priority date: 2019-08-23
Filing date: 2020-06-03
Publication date: 2021-03-04
Also published as: CN110465114B; CN110465114A

Abstract

A simulated moving bed continuous chromatography system and an application thereof, and a method for purifying coenzyme Q10. The simulated moving bed continuous chromatography system comprises at least four chromatographic columns sequentially communicated end to end. A feed liquid inlet, an eluent inlet 1#, an eluent inlet 2# and an eluent inlet 3# are sequentially arranged in the direction of chromatographic column arrangement. The simulated moving bed continuous chromatography system is divided into a feed zone, an elution zone, a desorption zone and a regeneration zone by means of the four material inlets, and by switching a porous distribution valve, the chromatographic columns are sequentially and cyclically switched to the feed zone, the elution zone, the desorption zone and the regeneration zone.

Description

Simulated moving bed continuous chromatography system and application thereof and method for purifying coenzyme Q10

Technical field

The invention belongs to the field of purification of coenzyme Q10, and specifically relates to a simulated moving bed continuous chromatography system and application thereof, and a method for purifying coenzyme Q10 by using simulated moving bed continuous chromatography.

Background technique

Coenzyme Q10 (Coenzyme Q10, abbreviated as CoQ10), also known as ubiquinone, is a vitamin-like substance that is widely present in animals, plants and microorganisms. Coenzyme Q10 is a spontaneously synthesized cell metabolism activator and antioxidant. It can act on certain enzymes to cause changes in their three-dimensional structure, thereby affecting their physiological activities. Past studies and clinical trials have proved that Coenzyme Q10 has the effect of increasing the body’s immunity, preventing cardiovascular and cerebrovascular sclerosis, and is helpful for improving hypertension, congestive heart failure, neurological diseases and tumor treatment. Currently, as a precious natural product, Coenzyme Q10 is widely used in the production of biochemical drugs, health foods and cosmetics.

Coenzyme Q10 production methods mainly include chemical synthesis, animal and plant cell culture methods, and microbial fermentation methods. Among them, the microbial fermentation method has the advantages of high process stability, easy large-scale production, simple operation, high product biological activity, easy absorption, etc., and is currently a research hotspot in the production of coenzyme Q10. The fermentation broth prepared by the microbial fermentation method is centrifuged, filtered, lyophilized, and pulverized to obtain bacterial residue. The crude Coenzyme Q10 extract is obtained by extraction, and the high-purity Coenzyme Q10 product is obtained by further purification treatment. The existing extraction methods usually firstly use solvent extraction, saponification, and supercritical fluid extraction for crude purification, and then combine silica gel column chromatography, recrystallization and other techniques to further purify the crude product of Coenzyme Q10. However, the crude extract of Coenzyme Q10 mainly contains Coenzyme Q homologues with different numbers of isopentene units on the side chain, which is difficult to separate.

Simulated moving bed chromatography is currently the most promising preparative chromatography technology for industrialization. The existing simulated moving bed chromatography usually only includes four material inlets and outlets: feed liquid inlet, eluent inlet, extract liquid outlet, and raffinate outlet. These four material inlets and outlets divide all chromatographic columns into four with different flow rates. Each area has different functions. It uses the timing switching of four inlet and outlet materials to simulate the countercurrent movement of the eluent and the stationary phase, so as to realize the continuity of the inlet and outlet. The mixed solution containing the strong adsorption component and the eluent is continuously collected at the extraction liquid outlet, and the mixed solution containing the weak adsorption component and the eluent is continuously collected at the raffinate outlet. On the one hand, this operation allows continuous sample injection, so the production capacity is high; on the other hand, because the eluent is recycled, the solvent consumption is low, which can reduce the cost of large-scale preparation.

For example, CN108017530A discloses a method for continuously separating coenzyme Q10 from bacterial residues, which includes: (1) dissolving the crude coenzyme Q10 extract in a non-polar organic solvent to prepare a feed liquid; (2) dissolving the feed liquid And the eluent are continuously passed into the simulated moving bed chromatography system, and the raffinate is continuously collected from the raffinate port of the simulated moving bed chromatography system; (3) the raffinate obtained in step (2) is concentrated under reduced pressure and re-dissolved, and then After crystallization, filtration, and drying, a high-quality coenzyme Q10 with a purity of more than 98% is obtained. CN108084007A discloses a method for separating coenzyme Q10 and coenzyme Q11 by simulated moving bed chromatography, which includes: (1) dissolving a mixture of coenzyme Q10 and coenzyme Q11 in an organic solvent to form a feed liquid; (2) combining the feed liquid with The eluent is continuously passed into the simulated moving bed chromatography system, and the Q11-rich extract is continuously collected from the extraction port of the simulated moving bed chromatography system, and the raffinate rich in Coenzyme Q10 is continuously collected from the raffinate port; (3) Extraction The remaining liquid is post-processed to obtain coenzyme Q10 monomer; the extract is post-processed to obtain coenzyme Q11 monomer. The above methods all use the traditional simulated moving bed method to purify Coenzyme Q10. These traditional simulated moving beds only include one eluent inlet, and the eluent introduced from the inlet flows through the entire simulated moving bed, that is, the entire simulated moving bed. The eluent used in the bed is fixed, and when the traditional simulated moving bed is in operation, the outlet flow rate, inlet flow rate, switching time, system temperature, material concentration, eluent polarity, number of zones, number of columns in each zone and The flow rate and packing composition are fixed. This simulated moving bed is mainly suitable for the separation of two-component Coenzyme Q10 crude extract. However, the components of the crude extract of Coenzyme Q10 to be separated and purified in industry are more complicated. In addition to Coenzyme Q10, it usually contains a variety of impurities with different properties. When a traditional simulated moving bed is used to separate and purify it, Some impurities cannot be desorbed cleanly with a single gradient polar eluent in the column, and they are easy to tail, which affects the efficiency of the column. The yield of Coenzyme Q10 obtained cannot reach more than 98%, and it will also reduce the life of the packing. Different batches of sample solution It is difficult to achieve a fixed material concentration, difficult to unify the impurity content, easy volatility of the eluent, and deviation of polarity, which leads to the unfixed switching time, the process is difficult to operate stably, and it is not suitable for large-scale promotion and application.

Summary of the invention

The purpose of the present invention is to overcome the traditional simulated moving bed that is suitable for the separation of two-component Coenzyme Q10 crude extract. When the multi-component Coenzyme Q10 crude extract is separated, the yield of Coenzyme Q10 obtained is low, and the impurities cannot be desorbed cleanly. To reduce the shortcomings of packing life, provide a new simulated moving bed continuous chromatography system and its application, and a method for purifying coenzyme Q10 by using simulated moving bed continuous chromatography. The simulated moving bed continuous chromatography system is adopted. Both the two-component Coenzyme Q10 crude extract and the multi-component Coenzyme Q10 crude extract can achieve good separation and purification, and have good universality. Not only the purity and yield of the obtained Coenzyme Q10 are very high, but also the impurities can be It is easily desorbed from the chromatographic column and the packing has a long life.

Specifically, the present invention provides a simulated moving bed continuous chromatography system, wherein the simulated moving bed continuous chromatography system includes at least four chromatographic columns connected end to end in sequence along the direction in which the chromatographic columns are arranged. It is provided with a feed liquid inlet, 1# eluent inlet, 2# eluent inlet and 3# eluent inlet. These four material inlets divide the simulated moving bed continuous chromatography system into a feeding zone, In the elution zone, desorption zone and regeneration zone, the positions of the feed liquid inlet, 1# eluent inlet, 2# eluent inlet and 3# eluent inlet are switched by a porous distribution valve so that each chromatographic column Switch to the feed zone, elution zone, desorption zone and regeneration zone sequentially and cyclically.

Further, the total number of the chromatographic columns is 4 to 32, and the feed zone, the elution zone, the desorption zone and the regeneration zone each independently include 1 to 8 chromatographic columns.

Further, the filler packed in the chromatographic column is selected from at least one of polar macroporous adsorption resin, ion exchange resin, alumina and silica gel.

Further, in the simulated moving bed continuous chromatographic chromatography system, the material liquid and the filler flow in countercurrent.

The invention also provides the application of the simulated moving bed continuous chromatography system in the purification of coenzyme Q10.

In addition, the present invention also provides a method for purifying Coenzyme Q10 by using simulated moving bed continuous chromatography, wherein the method includes the following steps:

(1) Dissolve the crude Coenzyme Q10 extract in a non-polar organic solvent to prepare a feed solution;

(2) Take the feed liquid, 1# eluent, 2# eluent and 3# eluent from the feed liquid inlet and 1# eluent inlet of the above-mentioned simulated moving bed continuous chromatography system respectively. , 2# eluent inlet and 3# eluent inlet are continuously introduced into the feed zone, elution zone, desorption zone and regeneration zone; the chromatographic column in the feed zone is switched to the elution zone and adopts 1 #The eluent performs elution, while the remaining chromatographic columns in the feed zone that have not completed the loading and adsorption continue to feed; the chromatographic column in the elution zone collects the eluent rich in Coenzyme Q10 and then switches to the desorption zone And use 2# eluent for desorption, while the remaining chromatographic columns in the elution zone that have not completed the elution continue to elute; after the chromatographic column in the desorption zone completes desorption, switch to the regeneration zone and use 3# eluent for Regeneration, but the remaining chromatographic columns in the desorption zone that have not been desorbed continue to desorb; the chromatographic column in the regeneration zone is switched to the feed zone after regeneration is completed and the feed liquid is introduced for sample loading and adsorption, while the chromatographic column in the regeneration zone is not completed The rest of the regenerated chromatographic columns continue to be regenerated, so as to recycle;

(3) Recrystallize the coenzyme Q10-rich eluate collected from the elution zone to obtain coenzyme Q10.

Further, the non-polar organic solvent is selected from at least one of n-hexane, cyclohexane, n-heptane, n-octane and petroleum ether or 3# eluent.

Further, the concentration of coenzyme Q10 solids in the feed liquid is 50-400 mg/mL.

Further, the 1# eluent, 2# eluent and 3# eluent each independently contain component A and/or component B, and the component A is selected from petroleum ether, ethyl ether, isopropyl At least one of ether, diisopropyl ether, ethyl butyl ether, n-hexane, n-heptane, n-octane, cyclopentane, methylcyclopentane, cyclohexane and methylcyclohexane, so The component B is selected from acetone, methyl ethyl ketone, methyl formate, ethyl formate, propyl formate, ethyl acetate, methyl acetate, tetrahydrofuran, dimethyl sulfoxide, N,N-dimethylformamide and carbon At least one of monohydric alcohols having 1 to 4 atoms.

Further, the solvent polarity index of the #1 eluent is 0.2-4, containing component A and optional component B, and the volume percentage of component A is greater than 80%, preferably greater than 90%.

Further, the solvent polarity index of the 2# eluent is ≥4, containing component B and optional component A, and the volume percentage of component B>20%, preferably>60%, most preferably 100% .

Further, the solvent polarity index of the 3# eluent is less than or equal to 0.2, containing component A and optional component B, and the volume percentage of component A is greater than 90%, preferably greater than 95%, and most preferably 100% .

Further, the operating parameters of the simulated moving bed continuous chromatography system are controlled as follows: the elution temperature is 0-60°C, the feed liquid flow rate is 1 to 1000 L/h, and the eluent flow rate is 1 to 1000 L/h, The switching time is 0.5～2h.

Further, the method of recrystallization is to concentrate the coenzyme Q10-rich eluent and re-dissolve it with an organic solvent, and then sequentially perform crystallization, filtration and drying to obtain coenzyme Q10.

Further, the method of recrystallization is to concentrate the coenzyme Q10-enriched eluate and re-dissolve it in an organic solvent at 40-75°C, and then stir to cool down and crystallize, and the stirring speed is controlled at 15-20r/min. , The cooling rate is controlled at 5-15℃/h, and the final temperature is controlled at 0-25℃. After the cooling and crystallization is completed, it is centrifuged and filtered and dried to obtain Coenzyme Q10.

Further, the volume-to-mass ratio of the organic solvent to the concentrate is (2-15) L: 1 kg.

Further, the organic solvent is selected from the group consisting of acetone, methyl ethyl ketone, methanol, ethanol, n-propanol, isopropanol, methyl formate, ethyl formate, propyl formate, ethyl acetate, methyl acetate, petroleum ether, diethyl ether , At least one of isopropyl ether, diisopropyl ether, ethyl butyl ether, n-hexane, n-heptane and n-octane.

The beneficial effects of the present invention are:

Although the traditional simulated moving bed chromatography technology uses continuous chromatography technology, it can improve the utilization rate of the stationary phase and reduce the consumption of the stationary phase. In theory, it can realize the continuous production of coenzyme Q10, make the production process fully automated, and reduce labor intensity and production costs. , But during operation, the outlet flow rate, inlet flow rate, switching time, system temperature, material concentration, eluent polarity, number of zones, number of columns in each zone and flow rate, and packing composition are fixed, which is mainly suitable for two-component When using this process for industrial purification of coenzyme Q10, there is a complex composition of the feed solution. Some impurities cannot be desorbed in the column with a single gradient polar eluent, and they are easy to tail, which affects the efficiency of the column. The purity of the obtained coenzyme Q10 The sum yield is low, the packing life is short, the material concentration of different batches of feed liquid is difficult to achieve, the impurity content is difficult to be unified, the eluent is volatile, and the polarity is biased, which makes the switching time unable to be fixed, and the process is difficult to operate stably Such problems are not suitable for large-scale promotion and application. On the basis of the existing purification method of coenzyme Q10, the present invention improves the traditional simulated moving bed chromatography technology, provides a simulated moving bed continuous chromatography chromatography system and uses the simulated moving bed continuous chromatography chromatography system to purify the coenzyme Q10 method. The simulated moving bed continuous chromatography system consists of multiple chromatographic columns and a porous distribution valve. The entire simulated moving bed chromatography system includes a feed liquid inlet and multiple eluent inlets, which pass through the porous distribution valve. The switching of the port makes the role of different chromatographic columns switch, so that the chromatographic columns complete the adsorption in a process cycle and use the same or different eluents for all the processes of elution, desorption and regeneration, so that the material concentration can be adjusted. The polarity of the eluent is variable, so that the impurities can be desorbed cleanly, which not only ensures the stability of the column efficiency, improves the life of the packing, reduces the amount of solvent, and the purity and yield of the purified coenzyme Q10 can also be greatly improved. improve. In the simulated moving bed continuous chromatography system provided by the present invention, all the process steps of chromatographic separation are carried out at the same time, and each zone works continuously and is independent of each other. Continuous production is realized, the production process is fully automated, and labor intensity and production cost are reduced. . In addition, the purification process provided by the present invention is simple, has higher adaptability and stability, and is suitable for industrialized large-scale promotion and application.

Description of the drawings

Fig. 1 is a diagram showing the usage state of each zone at the _{start time T 0} of the simulated moving bed continuous chromatography system of embodiment 1;

Fig. 2 is a diagram showing the usage state of each zone of the simulated moving bed continuous chromatography system of Fig. 1 after 1/4 cycle (that is, when T ₀ +1/4T), the valve of the porous distribution valve is switched once.

detailed description

As shown in Figures 1 and 2, the simulated moving bed continuous chromatography system provided by the present invention includes at least four chromatographic columns connected end to end in sequence, and feed liquid inlets and 1# are sequentially arranged along the direction in which the chromatographic columns are arranged. The eluent inlet, the 2# eluent inlet and the 3# eluent inlet, these four material inlets divide the simulated moving bed continuous chromatography system into a feed zone, an elution zone, a desorption zone and a regeneration zone , The positions of the feed liquid inlet, 1# eluent inlet, 2# eluent inlet and 3# eluent inlet are switched by a porous distribution valve so that each chromatographic column is switched to the feed zone sequentially and cyclically , Elution zone, desorption zone and regeneration zone. Wherein, the feed liquid inlet, the 1# eluent inlet, the 2# eluent inlet and the 3# eluent inlet are respectively located at the upper part of different chromatographic columns. The area from the feed liquid inlet to the 1# eluent inlet is the feed zone, and the area from the 1# eluent inlet to the 2# eluent inlet is the elution zone, from the 2# eluent inlet The area from the inlet to the 3# eluent inlet is the desorption zone, and the area from the 3# eluent inlet to the feed liquid inlet is the regeneration zone. The total number of the chromatographic columns is at least 4, preferably 4 to 32. The number of chromatographic columns contained in the feed zone, elution zone, desorption zone and regeneration zone may be the same or different, and preferably each independently includes 1-8 chromatographic columns. The isocratic operation mode can be adopted between the zones, or the gradient operation mode can be adopted. When in use, pre-set the operating parameters of each zone, such as the flow rate, switching time, switching times, and column temperature, continuously pump the feed liquid and eluent, and after the system reaches a steady state, continuously collect the coenzyme Q10-rich in the elution zone The eluent, the eluent depleted in coenzyme Q10 is connected to the unfinished chromatographic column to enter the next elution zone, and the components rich in impurities are continuously collected in the desorption zone, and the components depleted in impurities enter the next elution zone. A desorption chromatographic column, thereby saving solvent consumption.

The position of the feed liquid inlet, 1# eluent inlet, 2# eluent inlet and 3# eluent inlet can be adjusted at the same time when the porous distribution valve is switched once, and one, two or three of them can also be selectively adjusted. The location of each entrance depends on the situation. For example, when one or more chromatographic columns located in any area have completed the corresponding work, but the chromatographic columns in other areas have not completed the corresponding work, you can switch the valve once so that the chromatographic column that has completed the work enters the next area first; When one or more chromatographic columns located in any two areas have completed the corresponding work, but the chromatographic columns in the remaining two areas have not completed the corresponding work, the valve can be switched once so that the chromatographic column that has completed the work enters the next area first ; When one or more chromatographic columns located in any three areas have completed the corresponding work, and the chromatographic column in the remaining area has not completed the corresponding work, you can switch the valve once so that the chromatographic column that has completed the work enters the next area first ; When one or more chromatographic columns in the four regions have completed the corresponding work, the valve can be switched once so that the completed chromatographic column enters the next region at the same time.

When working, the feed liquid is continuously introduced into the feed zone from the feed liquid inlet. When the feed (loading and adsorption) is completed, the valve of the porous distribution valve is switched once, and the 1# eluent inlet is switched to the just loaded and adsorbed When the elution is completed, the valve of the porous distribution valve is switched once, and the 2# eluent inlet is switched to the chromatographic column that has just been eluted to enter the desorption zone; when the desorption is completed Afterwards, the valve of the porous distribution valve is switched once, and the 3# eluent inlet is switched to the chromatographic column that has just been desorbed so that it enters the regeneration zone; when the regeneration is completed, the valve of the porous distribution valve is switched once, and the feed liquid inlet is switched to The chromatographic column that has just been regenerated allows it to enter the feed zone, so as to continue to circulate. For example, FIG. 1 and FIG. 2 are diagrams of the use state of the simulated moving bed continuous chromatography system of Example 1. Among them, Figure 1 is a state diagram of each zone at the beginning of a cycle (T ₀ ). It can be seen from Figure 1 that the simulated moving bed continuous chromatography system includes a total of 6 chromatographic columns connected end to end (named 1 in turn). No. Column, No. 2 Column, No. 3 Column, No. 4 Column, No. 5 Column and No. 6 Column), from this moment to the next valve switch, the feed liquid inlet, 1# eluent inlet, 2# wash The deagent inlet and the 3# eluent inlet are respectively located at the upper part of No. 1, No. 3, No. 4, and No. 6 columns. At this time, No. 1 and No. 2 columns are in the feed zone, and No. 3 is in the wash area. Dezone, No. 4 column and No. 5 column are located in the desorption zone, and No. 6 column is located in the regeneration zone. The feed liquid, 1# eluent, 2# eluent and 3# eluent are respectively from the feed liquid inlet, 1 # Eluent inlet, 2# eluent inlet and 3# eluent inlet are continuously introduced to load, elute, desorb and regenerate the chromatographic columns in different regions respectively. Figure 2 is a diagram of the usage status of each zone after the valve of the porous distribution valve is switched once after 1/4 cycle (that is, when T ₀ +1/4T). It can be seen from Figure 2 that after the valve is switched, the feed liquid inlet Switch to the upper part of No. 6 column to load the regenerated No. 6 column, switch 1# eluent inlet to the No. 2 column that has been loaded so that No. 2 column enters the elution zone, and 2# eluent inlet Switch to the eluted No. 3 column to make the No. 3 column enter the desorption zone, and switch the 3# eluent inlet to the desorbed No. 5 column to make the No. 5 column enter the regeneration zone.

In the present invention, the fillers packed in the chromatographic column can be selected from at least one of polar macroporous adsorption resins, ion exchange resins, alumina and silica gels. These fillers are rich in polar groups such as hydroxyl groups and can be combined with The carbon groups in the coenzyme Q homologs form hydrogen bonds, and the small structural differences between the homologs can be recognized according to the difference in the strength of the hydrogen bond.

In the present invention, in the simulated moving bed continuous chromatographic chromatography system, the feed liquid and the filler flow in a reverse direction, that is, the flow direction of the feed liquid is opposite to the switching direction of the material inlet. The feed area is the sample loading area, and the elution area uses 1# eluent for elution, and at the same time collects the eluent rich in coenzyme Q10; the desorption area uses 2# eluent for desorption and washing column; the regeneration area uses 3# The eluent performs column regeneration.

The method for purifying coenzyme Q10 by using simulated moving bed continuous chromatographic chromatography provided by the present invention includes the following steps:

In the present invention, the crude extract of Coenzyme Q10 is extracted from the bacterial powder obtained by microbial fermentation. For details, please refer to the method described in the patent application with publication number CN101314782A, CN101619330A or CN105886562A to cultivate the bacterial species, filter the fermentation broth, Bacterial powder is obtained after drying and crushing; the extraction method for extracting Coenzyme Q10 crude extract from the bacterial powder can be percolation extraction, organic solvent extraction, alcohol-alkali saponification or supercritical fluid extraction. For details, please refer to the publication number Extract for the method described in the patent application of CN106146278A, CN101381747A, CN102391092A or CN104694613A.

In the present invention, specific examples of the non-polar organic solvent include but are not limited to: at least one of n-hexane, cyclohexane, n-heptane, n-octane and petroleum ether or 3# eluent. The concentration of coenzyme Q10 solids in the feed liquid is preferably 50-400 mg/mL. If the feed concentration is too low, the production capacity will be reduced and the process economy will be reduced; if the feed concentration is too high, the complete separation zone will be significantly reduced , The difficulty of designing operating conditions increases, and the difficulty of separation increases. The feed volume of the feed liquid is equivalent to 10%-50% of the mass of the single column packing.

According to a preferred embodiment of the present invention, in the elution zone, after collection of the eluate rich in coenzyme Q10, the eluate depleted in coenzyme Q10 is connected to the chromatographic column that has not completed the loading and adsorption; and/or In the desorption zone, after the collection of the components rich in impurities, the components poor in impurities enter the next desorption chromatographic column, which can increase the yield of coenzyme Q10 and save the amount of solvent. In the present invention, the purity of coenzyme Q10 in the eluate rich in coenzyme Q10 is relatively high, for example, it may be more than 85%, preferably more than 90%, and more preferably more than 95%; the poor content of coenzyme Q10 The purity of coenzyme Q10 in the eluate is relatively low, for example, it may be 15% or less, preferably 10% or less, more preferably 5% or less; the impurity content of the component rich in impurities is relatively high, for example, It can be 60% or more; the impurity content in the impurity-poor component is relatively low, for example, it can be 40% or less.

In the present invention, the 1# eluent, 2# eluent and 3# eluent may each independently contain component A and/or component B, and the component A is selected from petroleum ether and diethyl ether. At least one of, isopropyl ether, diisopropyl ether, ethyl butyl ether, n-hexane, n-heptane, n-octane, cyclopentane, methylcyclopentane, cyclohexane and methylcyclohexane The component B is selected from the group consisting of acetone, methyl ethyl ketone, methyl formate, ethyl formate, propyl formate, ethyl acetate, methyl acetate, tetrahydrofuran, dimethyl sulfoxide, N,N-dimethylformate At least one of an amide and a monohydric alcohol having 1 to 4 carbon atoms. According to a preferred embodiment of the present invention, the solvent polarity index of the #1 eluent is 0.2-4, containing component A and optional component B, and the volume percentage of component A is greater than 80%, preferably ＞90%; the solvent polarity index of the 2# eluent is ≥4, containing component B and optional component A, the volume percentage of component B is more than 20%, preferably more than 60%, most preferably 100 %; The solvent polarity index of the 3# eluent is less than or equal to 0.2, containing component A and optional component B, and the volume percentage of component A is greater than 90%, preferably greater than 95%, and most preferably 100%. When the crude Coenzyme Q10 extract to be purified has two components, the 1# eluent, 2# eluent and 3# eluent may be the same; when the crude Coenzyme Q10 extract to be purified is multi-component (That is, when it contains various impurities, especially the properties of these impurities are quite different), the 1# eluent, 2# eluent and 3# eluent are usually different, and the polarity is 3# elution Eluent <1# eluent <2# eluent, by adjusting the polarity of the eluent, the coenzyme Q10 can be separated smoothly and the impurities can be desorbed from the chromatographic column, which improves the purity and yield of coenzyme Q10 and ensures The column efficiency is stable and the service life of the packing is improved. In addition, the types of 1# eluent, 2# eluent and 3# eluent used at different times may be the same or different.

In the present invention, the operating parameters of the simulated moving bed continuous chromatography system are controlled as follows: the elution temperature is preferably 0-60°C, the feed liquid flow rate is preferably 1 to 1000 L/h, and the eluent flow rate is preferably 1. ~1000L/h, and the switching time is preferably 0.5-2h. Wherein, the switching time refers to the time it takes for the porous valve to switch from a certain state to the next state.

According to a specific embodiment of the present invention, the method of recrystallization may include concentrating the eluent rich in Coenzyme Q10 and re-dissolving it with an organic solvent, and then sequentially performing crystallization, filtration and drying to obtain Coenzyme Q10. . More specifically, the eluent rich in coenzyme Q10 is concentrated and re-dissolved in an organic solvent at 40-75°C, and then stirred for cooling and crystallization, the stirring speed is controlled at 15-20r/min, and the cooling rate is controlled at 5 ～15℃/h, the final temperature is controlled at 0～25℃, after the cooling and crystallization is completed, centrifugal filtration, drying, obtain coenzyme Q10. Wherein, the volume-to-mass ratio of the organic solvent to the concentrate may be (2-15) L: 1 kg. Specific examples of the organic solvent include, but are not limited to: acetone, methyl ethyl ketone, methanol, ethanol, n-propanol, isopropanol, methyl formate, ethyl formate, propyl formate, ethyl acetate, methyl acetate, petroleum At least one of ether, diethyl ether, isopropyl ether, diisopropyl ether, ethyl butyl ether, n-hexane, n-heptane, and n-octane.

The following describes the embodiments of the present invention in detail. The examples of the embodiments are intended to explain the present invention, but should not be construed as limiting the present invention. Where specific techniques or conditions are not indicated in the examples, the procedures shall be carried out in accordance with the techniques or conditions described in the literature in the field or in accordance with the product specification. The reagents or instruments used without the manufacturer's indication are all conventional products that can be purchased on the market.

Example 1

The multi-component Coenzyme Q10 crude extract (among which, the impurities mainly contain Coenzyme Q9, reduced coenzyme Q9, 5-demethoxy coenzyme Q10, reduced coenzyme Q10, coenzyme Q11, and reduced coenzyme Q11) are dissolved in n-hexane In the feed solution, the solid concentration of coenzyme Q10 is 80 mg/mL, and the content of coenzyme Q10 is about 75.5%. As shown in Figure 1, the simulated moving bed continuous chromatography system is equipped with 6 chromatographic columns, including 2 in the feed zone, 1 in the elution zone, 2 in the desorption zone and 1 in the regeneration zone, all of which are 3.5cm in size ×50cm; the stationary phase is silica gel (particle size 45μm, pore size 10nm); the eluent is a mixture of cyclohexane and ethyl acetate, and the volume percentage of ethyl acetate in the #1 eluent is 8%, 2 #The volume percentage of ethyl acetate in the eluent is 35%, and the volume percentage of ethyl acetate in the 3# eluent is 1%; the operating temperature is 30°C; the operating parameters are optimized and determined as: eluent flow rate 4L/h , The feed liquid flow rate is 2L/h, and the switching time is 1h. A high-concentration eluate rich in Coenzyme Q10 is collected from the exit of the elution zone. Analysis showed that the purity of Coenzyme Q10 in the high-concentration eluate collected in one cycle was 98.3%. Concentrate the high-concentration eluate into a solid, add ethanol under stirring at 75°C until it is just completely dissolved, the solid-to-liquid mass-volume ratio is 1kg:10L, the stirring speed is 15r/min, the temperature is gradually reduced to 20°C, and the temperature is lowered. The rate is 5℃/h, after cooling and crystallization for 3h, filtering, put in a vacuum drying oven and drying at 30℃ for 24h to obtain Coenzyme Q10 product. The purity of the coenzyme Q10 product is 99.6% by liquid chromatography analysis, the recovery rate of the whole process is 99.3%, the impurities can be easily desorbed from the chromatographic column, and the life of the packing is 600h.

Example 2

The two-component Coenzyme Q10 crude extract (wherein the impurity is Coenzyme Q11) is dissolved in n-hexane to prepare a feed solution with a solid concentration of Coenzyme Q10 of 120 mg/mL, in which the content of Coenzyme Q10 is about 70.3%. The simulated moving bed is equipped with 12 chromatographic columns, of which 4 are in the feed zone, 2 are in the elution zone, 4 are in the desorption zone, and 2 are in the regeneration zone, all with a size of 3.5cm×50cm; the stationary phase is silica gel (particle size 25μm, pore size 20nm); the eluent is a mixture of petroleum ether and ethyl acetate, in which the volume percentage of ethyl acetate in the eluents 1#, 2#, and 3# are all 10%; the operating temperature is 30°C; The parameters are optimized and determined as: eluent flow rate 6L/h, feed liquid flow rate 4L/h, switching time 0.5h. A high-concentration eluate rich in Coenzyme Q10 is collected from the exit of the elution zone. The analysis showed that the purity of Coenzyme Q10 in the high-concentration eluate collected in one cycle was 97.5%. Concentrate the high-concentration eluate into a solid, add isopropanol under stirring at 30°C until it is just completely dissolved, the solid-liquid mass-volume ratio is 1kg:12L, the stirring speed is 15r/min, and the temperature is gradually reduced to 25°C , Cooling rate 8℃/h, cooling and crystallizing for 5h, filtering, putting it in a vacuum drying oven and drying at 30℃ for 24h to obtain Coenzyme Q10 product. The purity of the coenzyme Q10 product is 99.5% by liquid chromatography analysis, the recovery rate of the whole process is 99.5%, the impurities can be easily desorbed from the chromatographic column, and the life of the packing is 1000h.

Example 3

The multi-component Coenzyme Q10 crude extract (among which, the impurities mainly contain Coenzyme Q9, reduced coenzyme Q9, 5-demethoxy coenzyme Q10, reduced coenzyme Q10, coenzyme Q11, and reduced coenzyme Q11) are dissolved in n-hexane In the feed solution, the solid content of coenzyme Q10 is 200 mg/mL, and the content of coenzyme Q10 is about 65.3%. The simulated moving bed is equipped with 18 chromatographic columns, of which 5 are in the feed zone, 5 are in the elution zone, 4 are in the desorption zone, and 4 are in the regeneration zone, all of which are 3.5cm×50cm in size; the stationary phase is alumina (particles). Diameter 20μm, pore size 22nm); the eluent is a mixture of n-hexane and acetone, in which the volume percentage of acetone in 1# eluent is 3%, and the volume percentage of acetone in 2# eluent is 20%, 3# The eluent is pure n-hexane; the operating temperature is 32°C; the operating parameters are optimized and determined as follows: the eluent flow rate is 8L/h, the feed liquid flow rate is 5L/h, and the switching time is 0.8h. A high-concentration eluate rich in Coenzyme Q10 is collected from the exit of the elution zone. The analysis showed that the purity of Coenzyme Q10 in the high-concentration eluate collected in one cycle was 98.1%. Concentrate the high-concentration eluent into a solid, add methanol under stirring at 30°C until it is just completely dissolved, the solid-liquid mass-volume ratio is 1kg:8L, the stirring speed is 20r/min, the temperature is gradually reduced to 15°C, and the temperature is lowered. The rate is 10℃/h, after cooling and crystallizing for 5h, filtering, putting it in a vacuum drying oven and drying at 30℃ for 24h to obtain Coenzyme Q10 product. The purity of Coenzyme Q10 product is 99.6% by liquid chromatography analysis, the recovery rate of the whole process is 99.2%, the impurities can be easily desorbed from the chromatographic column, and the life of the packing is 580h.

Example 4

The two-component Coenzyme Q10 crude extract (wherein the impurity is Coenzyme Q11) was dissolved in n-hexane to prepare a feed solution with a solid concentration of Coenzyme Q10 of 300 mg/mL, in which the content of Coenzyme Q10 was about 77.2%. The simulated moving bed is equipped with 24 chromatographic columns, of which 6 are in the feed zone, 6 are in the elution zone, 6 are in the desorption zone, and 6 are in the regeneration zone, all of which are 3.5cm×50cm in size; the stationary phase is alumina (particles). The diameter is 50μm, the pore diameter is 25nm); the eluent is a mixture of petroleum ether and acetone, and the volume percentage of acetone in the eluents 1#, 2#, and 3# are all 5%; the operating temperature is 35°C; the operating parameters are optimized Determined as: eluent flow rate 10L/h, feed liquid flow rate 6L/h, switching time 0.6h. A high-concentration eluate rich in Coenzyme Q10 is collected from the exit of the elution zone. The analysis showed that the purity of Coenzyme Q10 in the high-concentration eluate collected in one cycle was 98.6%. Concentrate the high-concentration eluate into a solid, add n-propanol under stirring at 30°C until it is just completely dissolved, the solid-liquid mass-volume ratio is 1kg:15L, the stirring speed is 20r/min, and the temperature is gradually reduced to 10°C , Cooling rate of 15℃/h, cooling and crystallization for 5h, filtering, putting it in a vacuum drying cabinet and drying at 30℃ for 24h to obtain Coenzyme Q10 product. The purity of Coenzyme Q10 product was 99.7% by liquid chromatography analysis, and the recovery rate of the whole process was 99.4%. Impurities can be easily desorbed from the chromatographic column. The life of the packing is 1100h.

Comparative example 1 (using the traditional simulated moving bed chromatography technology disclosed in CN108017530A Example 1 to purify the multi-component Coenzyme Q10 crude extract)

The multi-component Coenzyme Q10 crude extract (among which, the impurities mainly contain Coenzyme Q9, reduced coenzyme Q9, 5-demethoxy coenzyme Q10, reduced coenzyme Q10, coenzyme Q11, and reduced coenzyme Q11) are dissolved in n-hexane In the feed solution, the solid concentration of coenzyme Q10 is 80 mg/mL, and the content of coenzyme Q10 is about 75.5%. The simulated moving bed device of CN108017530A is used, and the chromatographic technology in Example 1 of CN108017530A is used to process the above-mentioned material and liquid. Specifically: the simulated moving bed is equipped with 8 chromatographic columns, all of which are 1cm×25cm in size; the stationary phase is silica gel (Particle size 45μm, pore size 10nm); eluent is a mixture of n-hexane and ethyl acetate, where the volume percentage of ethyl acetate is 10%; operating temperature is 30°C; operating parameters are optimized and determined as: eluent flow rate 16mL /min, feed liquid flow rate 2mL/min, extraction liquid flow rate 9.5mL/min, raffinate flow rate 8.5mL/min, switching time 5min. After switching continuously for 32 times, the system reached equilibrium, and the raffinate rich in coenzyme Q10 was collected from the raffinate outlet. Analysis showed that the content of coenzyme Q10 in the raffinate was 95.3%. Concentrate the eluate into a solid, add ethanol under stirring at 75°C until it is just completely dissolved, the solid-liquid mass-volume ratio is 1kg:10L, the stirring speed is 15r/min, the temperature is gradually reduced to 20°C, and the cooling rate is 5°C /h, after cooling and crystallizing for 3h, filtering, putting it in a vacuum drying oven and drying at 30℃ for 24h to obtain Coenzyme Q10 product. The purity of the coenzyme Q10 product was 99.1% by liquid chromatography analysis, and the recovery rate of the whole process was 95.5%. Impurities could not be desorbed in the column, and tailing was easy to affect the column efficiency. The life of the packing was 120h.

Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art will not depart from the principle and purpose of the present invention. Under the circumstances, changes, modifications, substitutions and modifications can be made to the above-mentioned embodiments within the scope of the present invention.

Claims

A simulated moving bed continuous chromatographic chromatography system, characterized in that the simulated moving bed continuous chromatographic chromatography system comprises at least four chromatographic columns connected end to end in sequence, and feed liquids are sequentially arranged along the direction in which the chromatographic columns are arranged. Inlet, 1# eluent inlet, 2# eluent inlet and 3# eluent inlet, these four material inlets divide the simulated moving bed continuous chromatography system into a feed zone, an elution zone, and a desorption zone. Zone and regeneration zone, the positions of the feed liquid inlet, 1# eluent inlet, 2# eluent inlet and 3# eluent inlet are switched by a porous distribution valve so that each chromatographic column is switched in sequence and cyclically To the feed zone, elution zone, desorption zone and regeneration zone.
The simulated moving bed continuous chromatography system according to claim 1, wherein the total number of the chromatographic columns is 4 to 32, and the feed zone, the elution zone, the desorption zone and the regeneration zone are each Independently includes 1 to 8 chromatographic columns.
The simulated moving bed continuous chromatographic chromatography system according to claim 1 or 2, wherein the fillers packed in the chromatographic column are selected from at least polar macroporous adsorption resins, ion exchange resins, alumina and silica gels. One kind.
The simulated moving bed continuous chromatographic chromatography system according to claim 1 or 2, characterized in that, in the simulated moving bed continuous chromatographic chromatography system, the feed liquid and the filler flow in countercurrent.
The application of the simulated moving bed continuous chromatography system according to any one of claims 1 to 4 in the purification of coenzyme Q10.
A method for purifying coenzyme Q10 by using the simulated moving bed continuous chromatography system according to any one of claims 1 to 4, characterized in that the method comprises the following steps:

(1) Dissolve the crude Coenzyme Q10 extract in a non-polar organic solvent to prepare a feed solution;

(2) Separate the feed liquid, 1# eluent, 2# eluent and 3# eluent from the simulated moving bed continuous chromatography system according to any one of claims 1 to 4 Feed liquid inlet, 1# eluent inlet, 2# eluent inlet and 3# eluent inlet are continuously introduced into the feed zone, elution zone, desorption zone and regeneration zone; the chromatographic column in the feed zone is completed After the sample is adsorbed, switch to the elution zone and use 1# eluent for elution, while the remaining chromatographic columns in the feed zone that have not completed the sample adsorption adsorption continue to feed; the chromatographic column in the elution zone has collected the rich After the eluent of Coenzyme Q10 is switched to the desorption zone and desorbed with 2# eluent, the remaining chromatographic columns in the elution zone that have not completed the elution continue to elute; the chromatographic column in the desorption zone is switched after the desorption is completed Go to the regeneration zone and use 3# eluent to regenerate, while the remaining chromatographic columns in the desorption zone that have not completed desorption continue to desorb; the chromatographic columns in the regeneration zone are switched to the feed zone after regeneration is completed and the feed liquid is passed into it. The sample is loaded and adsorbed, and the remaining chromatographic columns in the regeneration zone that have not been regenerated continue to be regenerated, so as to cycle;

(3) Recrystallize the coenzyme Q10-rich eluate collected from the elution zone to obtain coenzyme Q10.
The method for purifying coenzyme Q10 using simulated moving bed continuous chromatography according to claim 6, wherein the non-polar organic solvent is selected from the group consisting of n-hexane, cyclohexane, n-heptane, n-octane and petroleum. At least one of the ether or 3# eluent; the concentration of the solid coenzyme Q10 in the feed liquid is 50-400 mg/mL.
The method for purifying coenzyme Q10 using simulated moving bed continuous chromatography according to claim 6 or 7, wherein the 1# eluent, 2# eluent and 3# eluent each independently contain Component A and/or component B, the component A is selected from petroleum ether, diethyl ether, isopropyl ether, diisopropyl ether, ethyl butyl ether, n-hexane, n-heptane, n-octane, cyclopentane At least one of alkane, methylcyclopentane, cyclohexane and methylcyclohexane, the component B is selected from acetone, methyl ethyl ketone, methyl formate, ethyl formate, propyl formate, ethyl acetate , At least one of methyl acetate, tetrahydrofuran, dimethyl sulfoxide, N,N-dimethylformamide, and monohydric alcohols with 1 to 4 carbon atoms.
The method for purifying coenzyme Q10 using simulated moving bed continuous chromatography according to claim 8, wherein the solvent polarity index of the #1 eluent is 0.2-4, and contains component A and optional The volume percentage of component B and component A>80%.
The method for purifying coenzyme Q10 by using simulated moving bed continuous chromatography according to claim 9, wherein the volume percentage of component A in the #1 eluent is greater than 90%.
The method for purifying coenzyme Q10 using simulated moving bed continuous chromatography according to claim 8, wherein the solvent polarity index of the 2# eluent is ≥4, and it contains component B and optional components A, the volume percentage of component B>20%.
The method for purifying Coenzyme Q10 by using simulated moving bed continuous chromatography according to claim 11, wherein the volume percentage of component B in the 2# eluent is greater than 60%.
The method for purifying coenzyme Q10 using simulated moving bed continuous chromatography according to claim 12, wherein the volume percentage of component B in the 2# eluent is 100%.
The method for purifying coenzyme Q10 using simulated moving bed continuous chromatography according to claim 8, wherein the solvent polarity index of the 3# eluent is ≤ 0.2, and it contains component A and optional components B, the volume percentage of component A>90%.
The method for purifying Coenzyme Q10 by using simulated moving bed continuous chromatography according to claim 14, wherein the volume percentage of component A in the 3# eluent is> 95%.
The method for purifying Coenzyme Q10 using simulated moving bed continuous chromatography according to claim 15, wherein the volume percentage of component A in the 3# eluent is 100%.
The method for purifying coenzyme Q10 using simulated moving bed continuous chromatography according to claim 6 or 7, wherein the operating parameters of the simulated moving bed continuous chromatography system are controlled as follows: the elution temperature is 0-60 ℃, the feed liquid flow rate is 1 to 1000 L/h, the eluent flow rate is 1 to 1000 L/h, and the switching time is 0.5 to 2 h.
The method for purifying Coenzyme Q10 using simulated moving bed continuous chromatography according to claim 6 or 7, wherein the method of recrystallization is to concentrate the eluent rich in Coenzyme Q10 and then use organic The solvent is re-dissolved, and then crystallization, filtration and drying are carried out in sequence to obtain Coenzyme Q10.
The method for purifying Coenzyme Q10 by using simulated moving bed continuous chromatography according to claim 18, wherein the method of recrystallization is to concentrate the eluent rich in Coenzyme Q10 and then use an organic solvent in it. Re-dissolve at 40～75℃, then stir and cool to crystallize. The stirring speed is controlled at 15～20r/min, the cooling rate is controlled at 5～15℃/h, and the final temperature is controlled at 0～25℃. After the cooling and crystallization are completed, centrifugal filtration , Dried to obtain Coenzyme Q10.
The method for purifying coenzyme Q10 using simulated moving bed continuous chromatography according to claim 18, wherein the volume-to-mass ratio of the organic solvent to the concentrate is (2-15) L: 1 kg; the organic solvent Selected from acetone, methyl ethyl ketone, methanol, ethanol, n-propanol, isopropanol, methyl formate, ethyl formate, propyl formate, ethyl acetate, methyl acetate, petroleum ether, diethyl ether, isopropyl ether, diisopropyl ether At least one of propyl ether, ethyl butyl ether, n-hexane, n-heptane, and n-octane.