CN108640840B

CN108640840B - Multi-column continuous chromatography method and equipment for purifying eicosapentaenoic acid ethyl ester

Info

Publication number: CN108640840B
Application number: CN201711329373.3A
Authority: CN
Inventors: 王季凤
Original assignee: Suzhou Microwants Biological Technology Co ltd
Current assignee: Suzhou Microwants Biological Technology Co ltd
Priority date: 2017-12-13
Filing date: 2017-12-13
Publication date: 2020-06-05
Anticipated expiration: 2037-12-13
Also published as: CN108640840A

Abstract

The invention provides a purified Eicosapentaenoic acid ethyl ester (Eicosapentaenoic acid, EPA-EE)The chromatographic method and the equipment are characterized in that: the apparatus is a multi-column continuous chromatography system divided into two major zones: a coarse separation zone and a fine separation zone; the crude separation zone consisted of five subregions: the fine separation zone consists of two subregions: each sub-zone consists of 1 tonThe branch chromatographic columns are connected in series; the chromatographic method on the equipment is as follows: sampling samples containing EPA-EE in a pulse mode in a region I; removing the precursor impurities in zone II (eluting EPA-EE to just penetrate zone II); eluting EPA-EE in the IV area until the EPA-EE enters the end column of the IV area completely; performing fine separation in the VI region (eluting EPA-EE to the VI region just penetrated); EPA-EE and EPA-EE containing a small amount of post impurities are eluted and collected in a VII region in a segmented way; post-impurities are removed in zones III and V. The method is realized by intelligent control such as valve switching, pump starting and stopping or/and flow rate regulation and the like.

Description

Multi-column continuous chromatography method and equipment for purifying eicosapentaenoic acid ethyl ester

Technical Field

The invention relates to a chromatographic method and equipment for purifying Eicosapentaenoic acid ethyl ester (EPA-EE), in particular to a multi-column continuous chromatographic method and equipment.

Background

EPA is a precursor of a plurality of important biological molecules, has a plurality of biological functions of regulating platelet aggregation, inflammation, immunity and the like, and has great treatment prospect for diseases such as nerve center, nervous diseases, cardiovascular diseases, immunological diseases and inflammatory diseases, including rheumatoid arthritis, inflammatory bowel diseases, Alzheimer diseases, Parkinson diseases, metabolic syndrome, tumors and the like. EPA-EE is approved by the FDA of the United states to be marketed as a hypolipidemic drug in 2013.

EPA is mainly derived from fish oil. Besides EPA, fish oil also contains various fatty acids such as docosahexaenoic acid, docosapentaenoic acid, stearidonic acid, linoleic acid, oleic acid, stearic acid, palmitic acid and the like, and the non-EPA fatty acids interfere with the treatment effect of EPA-EE due to similar molecular structures and similar physicochemical properties. Therefore, the demand for high-purity EPA-EE is increasing day by day.

The core of the technology for preparing EPA-EE is batch and cost. The challenge in making EPA-EE is: 1. the structure and the physical and chemical properties of the coexisting fatty acid are similar to those of EPA-EE; 2. EPA-EE has poor stability and is easily isomerized, oxidized or/and degraded in the preparation process.

Currently, the main methods disclosed for the preparation of EPA-EE, urea adduct crystallization (U.S. Pat. No. 6664405), metal salt precipitation (U.S. Pat. No. 6846942) and molecular distillation (U.S. Pat. No. 8889895), do not provide the high purity EPA-EE required by the market.

The 2014 Orochem Technologies company disclosed a three-pass SMB process for the preparation of high-purity EPA-EE (U.S. Pat. No. 9163198) in which the first chromatography was normal phase SMB and the second and third chromatography were reverse phase SMB. In 2016 BASF corporation, disclosed a three-time reverse phase SMB process for the production of high purity EPA-EE (U.S. Pat. No. 9695382).

Although the methods provided in U.S. Pat. Nos. 9163198 and 9695382 can produce EPA-EE in a content of 97% or more, the cost of high-purity EPA-EE provided by the triple SMB method is not widely accepted because chromatography is the technique with the lowest productivity, the highest cost, the high quality requirement of operators, and the delicate, time-consuming and labor-consuming operation among all separation and purification techniques.

In conclusion, the pharmaceutical market urgently needs a preparation method of high-purity EPA-EE with large batch and low cost.

Disclosure of Invention

In view of the above problems, the present invention aims to provide a method and equipment for preparing high-purity EPA-EE with large batch and low cost.

To achieve the above object, the present invention employs the following chromatographic equipment: a continuous chromatographic system is constructed by a plurality of short chromatographic columns, 1 multi-flow-path synchronous switching valve, 7 constant flow pumps, 2 ultraviolet detectors and 1 segmented collector, and the system is divided into two large areas: a coarse separation zone and a fine separation zone. The crude separation zone consisted of five subregions: the loading zone, the pre-impurity removal zone, the post-impurity removal 1 zone, the EPA-EE elution zone and the post-impurity removal 2 zone are sequentially defined as I, II, III, IV and V zones. The fine separation zone consists of two subregions: a fine separation zone and an EPA-EE elution zone, defined as the VI and VII zones in turn. Subregions are composed of 1 to n (n ═ 1, 2, 3, 4, 5, 6, or 7) chromatographic columns connected in series. The chromatographic method on the equipment is as follows: sampling EPA-EE sample in pulse in the I area; after one sample introduction, the valve is switched, the tail column of the I area is carried to be the first column of the II area, and the front impurities are removed in the II area (EPA-EE is eluted to the just penetrated area II); after valve switching, the end column carry of the II area is the first column of the IV area, and EPA-EE is eluted in the IV area until the EPA-EE just completely enters the end column of the IV area; post-impurity removal in zones III and V; after valve switching, the end column carry of the IV region is the first column of the VI region, and the sample is finely separated in the VI region (EPA-EE is eluted to the VI region just penetrated); through valve switching, the final column carry in the VI area is the first column in the VII area, and EPA-EE containing a small amount of post-impurity are collected in the VII area by elution and segmentation; the method is realized by intelligent control such as valve switching, pump starting and stopping or/and flow rate regulation and the like.

In the method and the equipment provided by the invention, chromatographic columns with the same geometric size are adopted: the diameter of the chromatographic column is 10-2000 mm, and the size of the diameter of the chromatographic column directly determines the height of the production energy; the length of the chromatographic column is 50-300 mm, and the shortest column length can accommodate all target compounds in one sample loading amount.

In the method and the equipment provided by the invention, the chromatographic columns can be filled with C₁₈Reversed phase silica gel with the particle size of 5-40 μm.

In the method and the device provided by the invention, the frequency of pulse sample introduction is equal to the switching frequency of the valve, and the sample introduction is carried out once and switched once or switched once and fed once.

In the method and the device provided by the invention, the raw material to be separated contains 30-90% (weight percentage) of EPA-EE.

In the method and the device provided by the invention, the sample loading concentration is 50-90% (weight percentage) of methanol solution containing the raw material to be separated.

In the method and the equipment provided by the invention, the mobile phase for eluting each zone is 95-100% methanol aqueous solution, and the flow velocity of the mobile phase is 3-5 cm/min.

In the method and the device provided by the invention, the switching of the valve is automatically controlled by the detection value of the area VI detector.

In the method and the equipment provided by the invention, the sampling quantity of the area I is automatically controlled by starting and stopping the constant flow pump of the area I at fixed time; the start-stop of the constant flow pump in the area II is reversely controlled by the detection value of the detector in the area II to automatically control the elution end point of the area I; automatically controlling the elution end point of the III, IV, V or VII area by starting and stopping the constant flow pump of the III, IV, V or VII area at fixed time; the EPA-EE and post-impurity-containing EPA-EE collection is automatically controlled by the timing switching of the VII region collector; the time for controlling the start and stop of the constant flow pumps in each area and the switching of the collectors is obtained by experiments.

Due to the adoption of the technical scheme, the invention has the following advantages: 1. the EPA-EE raw material containing a large amount of foreimpurities and postimpurities is removed from a large amount of foreimpurities and postimpurities in a crude separation zone and then is finely separated by a fine separation zone, thereby greatly reducing the chromatographic load and the total peak width of the fine separation zone, enhancing the chromatographic separation capacity, improving the sample introduction frequency and improving the productivity; 2. the chromatographic processes of the coarse separation area and the fine separation area are simultaneously carried out in different subregions, so that the time required by the chromatographic process is further reduced, and the productivity is improved.

The invention provides a multi-column continuous chromatographic method and equipment with higher sampling frequency and stronger separation capability for preparing high-purity EPA-EE, can further improve the productivity and reduce the cost, and can meet the urgent demand of the medical market on the high-purity EPA-EE.

Drawings

FIG. 1 is a schematic diagram of the structure and chromatographic process of the method and its equipment provided by the present invention;

FIG. 2 is a chromatogram for preparation of example two of the present invention;

FIG. 3 is a diagram showing the analysis of the components of a preparative chromatogram of example two of the present invention;

FIG. 4 is a preparative chromatogram of example three of the present invention; (ii) a

FIG. 5 is a diagram showing the analysis of the components of a preparative chromatogram of example III of the present invention;

FIG. 6 is a monitoring diagram of a zone II detector in accordance with an embodiment of the present invention;

FIG. 7 is a diagram of the monitoring of an IV zone detector in accordance with an embodiment of the present invention;

FIG. 8 is a schematic diagram of another structure of the method and its equipment and its chromatographic process;

Detailed Description

The invention is described in detail below with reference to the figures and examples.

Example one

As shown in FIG. 1, the present example provides an equipment system comprising 1 12 columns, 7 flow path rotary valves, 12 columns (20mm ID. times.100 mm, C)₁₈Reverse phase silica gel, 10 μm, column efficiency not less than 4000), 7 constant flowThe pump (1-50 ml/min), 2 ultraviolet detectors and 1 segmented collector are constructed, and the system is divided into two large areas: a coarse separation zone and a fine separation zone. The crude separation zone consisted of five subregions: the sample loading zone (column No. 1), the pre-impurity removal zone (columns No. 2 and 4), the post-impurity removal zone 1 (column No. 3), the sample elution zone (columns No. 5 and 7), and the post-impurity removal zone 2 (column No. 6) are defined as zones I, II, III, IV, and V (dotted line frame) in this order. The fine separation zone consists of two subregions: the fine separation zones (

columns

8, 9, 10 and 11) and the sample elution zone (column 12), in turn, are defined as zones VI and VII. 7 pumps independently convey multi-component sample liquid F and 6 mobile phases S₂～S₇. The outlet end of the No. 4 column in the zone II is connected with an ultraviolet detector, and the signal intensity detected by the detector controls the mobile phase S₂And starting and stopping the liquid supply pump. And the outlet end of the 11 # column in the VI area is connected with an ultraviolet detector, and the signal intensity detected by the detector controls the switching of the rotary valve. The constant flow pumps in the I, III, IV, V or VII areas are controlled by timing start and stop. And the outlet end of the column VII with the number 12 is connected with a sectional collector, and the collector is controlled by timing switching.

The chromatographic process parameters on the system of the present embodiment are: the raw material to be separated contains 70 percent of EPA-EE; the sample injection concentration is 500 mg/ml; 6 mobile phases S₂～S₇Same, 95% methanol water solution; the flow rate of a constant flow pump in the area I is 5ml/min, and the sample injection time is 1 min; the flow rates of the constant flow pumps in the II to VII regions are the same and are all 15 ml/min; the detection wavelength is 210 nm; the pump stop peak value of the II area is 940mV (the running time of the constant flow pump is about 9.2 min); IV area rotary valve switching peak value V870mV (constant flow pump running time 16.3 min); the running time of the constant flow pumps in the areas III and V is 12 min; the running time of the constant flow pump in the IV area is 5.4 min; the operation time of the VII-region constant flow pump is 15min, the switching time of the segmented collector is 4min, and the EPA-EE collecting time is 0-4 min. 9.2+5.4+12+12+16.3+15

The chromatographic method on the system of the embodiment is as follows: sampling 500mg/ml of sample F in a pulse mode in a region I, wherein the sampling frequency is equal to the switching frequency of a valve, and the sampling is carried out once and the switching is carried out once; after one sample introduction, the column No. 1 in the area I is carried to the column No. 2 in the area II by switching the valve, and the mobile phase S is used in the area II₂Eluting, and automatically stopping the pump when the detection value of the II area reaches 940mV (figure 6); switched by a valve, zone II No. 4The column carries the column No. 5 in the IV region from the mobile phase S₄Automatically stopping the pump after elution is carried out for 5.4 min; after valve switching, the No. 7 column in IV region is carried to No. 8 column in VI region by mobile phase S₆Eluting, and automatically switching a rotary valve when the VI area detection value reaches 870mV (figure 7); when the rotary valve is switched, all the constant flow pumps in the areas I to VII stop; after valve switching, the 11 # column in VI region is carried to the 12 # column in VII region, where the mobile phase S is introduced₇Eluting, collecting EPA-EE in 0-4 min, and automatically switching the section collector in 4 min. In zones III and V, respectively, from the mobile phase S₃And mobile phase S₅The impurities were removed by elution for 12min each. The above process is performed automatically and circularly, and the time process is detailed in table 1. The results are shown in Table 2.

TABLE 1 time course of the system

Example two process condition confirmation experiment of example one

The equipment system provided by this example consists of 2 chromatography columns (20mm ID. times.100 mm, C)₁₈Reverse phase silica gel, 10 μm, column efficiency not less than 4000), 2 constant flow pumps (1-50 ml/min), 1 ultraviolet detector and 1 segmented collector: 2 chromatographic columns are used in series, 2 pumps are used in parallel, 1 is used for sample injection, and the other is used for elution. The chromatographic process parameters on the system of the embodiment are as follows: the raw material to be separated contains 70 percent of EPA-EE; the sample injection concentration is 500 mg/ml; the mobile phase was 95% aqueous methanol.

The chromatographic method of the system of the embodiment comprises the following steps: injecting a sample of 500mg/ml at a flow rate of 5ml/min for 1 min; the sample was completely eluted with 95% aqueous methanol at a flow rate of 15ml/min while the collector collected all chromatographic peaks in sections and analyzed for the composition of the eluate in each section. The results are shown in FIGS. 2 and 3. According to the results of this example, the peak value of pump stop in zone II in example I was 940mV, and the running time of constant flow pump in zone IV was 5.4 min.

Example three example one Process Condition confirmation experiment

The equipment system provided by this example consists of 6 chromatography columns (20mm ID. times.100 mm, C)₁₈Reverse phase silica gel, 10 μm, column efficiency not less than 4000), 2 constant flow pumps (1-50 ml/min), 1 ultraviolet detector and 1 segmented collector: 2 and 4 chromatographic columns are respectively connected in series, 2 pumps are used in parallel, 1 is used for sample injection, and the other is used for elution. The chromatographic process parameters on the system of the embodiment are as follows: the raw material to be separated contains 70 percent of EPA-EE; the sample injection concentration is 500 mg/ml; the mobile phase was 95% aqueous methanol.

The chromatographic method of the system of the embodiment comprises the following steps: sampling 2 series-connected chromatographic columns with a sample of 500mg/ml at a flow rate of 5ml/min for 1 min; eluting with 95% methanol water solution at 15ml/min flow rate to peak value of 940mV, stopping the pump; removing the column No. 1, and respectively connecting the column No. 2 with the other 4 chromatographic columns in series; the sample was further completely eluted with 95% aqueous methanol at a flow rate of 15ml/min while the collector collected all chromatographic peaks in sections and analyzed for the composition of the eluate in each section. The results are shown in FIGS. 4 and 5. Based on the results of this example, the peak 870mV for the VI region of example one was set as the rotary valve switch command and the EPA-EE collection time for region VII was set as 4 min.

Example four Single column chromatography comparative experiments

The equipment system provided by this example consists of 6 chromatography columns (20mm ID. times.100 mm, C)₁₈Reverse phase silica gel, 10 μm, column efficiency not less than 4000), 2 constant flow pumps (1-50 ml/min), 1 ultraviolet detector and 1 segmented collector: the 6 chromatographic columns are respectively connected in series, 2 pumps are used in parallel, 1 is used for sample injection, and the other is used for elution. The chromatographic process parameters on the system of the embodiment are as follows: the raw material to be separated contains 70 percent of EPA-EE; the sample injection concentration is 500 mg/ml; the mobile phase was 95% aqueous methanol.

The chromatographic method of the system of the embodiment comprises the following steps: injecting a sample of 500mg/ml at a flow rate of 5ml/min for 1 min; the sample was completely eluted with 95% aqueous methanol at a flow rate of 15ml/min while the collector collected all chromatographic peaks in sections and analyzed for the composition of the eluate in each section. The results are shown in Table 2.

TABLE 2 comparison of Multi-column continuous chromatography with Single column chromatography

EXAMPLE five

As shown in FIG. 8, the present example provides an equipment system comprising 1 column, 10 columns, 7 flow path rotary valves, 11 columns (wherein 10 columns are 200mm ID. times.100 mm, C)₁₈Reverse phase silica gel with the diameter of 10 mu m and the column efficiency of more than or equal to 4000; another chromatographic column is 200mmID × 200mm, C₁₈Reversed phase silica gel, 10 μm, column efficiency more than or equal to 8000), 7 constant flow pumps (1-50 ml/min), 2 ultraviolet detectors and 1 segmented collector, wherein the system is divided into two major areas: a coarse separation zone and a fine separation zone. The crude separation zone consisted of five subregions: the sample loading zone (column No. 1), the pre-impurity removal zone (columns No. 2 and 4), the post-impurity removal zone 1 (column No. 3), the sample elution zone (columns No. 5 and 7), and the post-impurity removal zone 2 (column No. 6) are defined as zones I, II, III, IV, and V (dotted line frame) in this order. The fine separation zone consists of two subregions: the fine separation zones (

columns

8, 9 and 10) and the sample elution zone (column 11), in turn, are defined as zones VI and VII. 7 pumps independently convey multi-component sample liquid F and 6 mobile phases S₂～S₇. The outlet end of the No. 4 column in the zone II is connected with an ultraviolet detector, and the signal intensity detected by the detector controls the mobile phase S₂And starting and stopping the liquid supply pump. And the outlet end of the 11 # column in the VI area is connected with an ultraviolet detector, and the signal intensity detected by the detector controls the switching of the rotary valve. The constant flow pumps in the I, III, IV, V or VII areas are controlled by timing start and stop. And the outlet end of the column VII with the number 12 is connected with a sectional collector, and the collector is controlled by timing switching.

The chromatographic method of the system of the embodiment is the same as that of the first embodiment.

In this embodiment, 2 100mm long columns in the first embodiment are combined into 1 200mm long column, and the column is arranged outside the rotary valve, so that the diameter of rotation can be reduced, and the equipment cost can be reduced.

The present invention is illustrated only by the above examples, and the construction of the apparatus and the implementation of the chromatographic process provided by the present invention may vary. On the basis of the technical scheme of the invention, the improvement or equivalent transformation of the equipment construction and implementation method according to the principle of the invention is not excluded from the protection scope of the invention.

Claims

1. A multi-column continuous chromatographic method for purifying eicosapentaenoic acid ethyl ester is characterized in that: the equipment used for the method is a multi-column continuous chromatography system consisting of 1 12 columns, 7 flow path rotary valves, 12 chromatographic columns, and parameters of 20mm ID × 100mm, C₁₈10 microns of reverse phase silica gel, more than or equal to 4000 column efficiency, 7 constant flow pumps, 1-50 ml/min of parameters, 2 ultraviolet detectors and 1 segmented collector; the system is divided into two major zones: a coarse separation zone and a fine separation zone; the column No. 1 of the sample loading area, the columns No. 2 and 4 of the pre-impurity removing area, the column No. 3 of the post-impurity removing area 1, the columns No. 5 and 7 of the sample elution area and the column No. 6 of the post-impurity removing area 2 are sequentially defined as I, II, III, IV and V areas; the fine separation zone consists of two subregions: columns 8, 9, 10 and 11 of the fine separation zone and column 12 of the sample elution zone, which are sequentially defined as zones VI and VII;

the 7-constant flow pump independently conveys multi-component sample liquid F and 6 mobile phases S₂～S₇The outlet end of the No. 4 column in the zone II is connected with an ultraviolet detector, and the signal intensity detected by the detector controls the mobile phase S₂The method comprises the following steps that a liquid supply pump is started and stopped, the outlet end of a No. 11 column in a VI area is connected with an ultraviolet detector, the switching of a rotary valve is controlled by the signal intensity detected by the detector, a constant flow pump in an I, III, IV, V or VII area is controlled by timing start and stop, the outlet end of a No. 12 column in the VII area is connected with a segmented collector, and the collector is controlled by timing switching;

the chromatographic process parameters are as follows: the raw material to be separated contains 70 percent of EPA-EE; the sample injection concentration is 500 mg/ml; 6 mobile phases S₂～S₇Same, 95% methanol water solution; the flow rate of a constant flow pump in the area I is 5ml/min, and the sample injection time is 1 min; the flow rates of the constant flow pumps in the II to VII regions are the same and are all 15 ml/min; the detection wavelength is 210 nm; the pump stop peak value of the II area is 940mV, and the running time of the constant flow pump is about 9.2 min; the peak value V870mV is switched by the rotary valve in the IV area, and the running time of the constant flow pump is 16.3 min; the running time of the constant flow pumps in the areas III and V is 12 min; the running time of the constant flow pump in the IV area is 5.4 min; VII zone constant flow pump operationThe time is 15min, the switching time of a segmented collector is 4min, and the EPA-EE collecting time is 0-4 min;

the chromatographic method comprises the following steps: sampling 500mg/ml of sample F in a pulse mode in a region I, wherein the sampling frequency is equal to the switching frequency of a valve, and the sampling is carried out once and the switching is carried out once; after one sample introduction, the column No. 1 in the area I is carried to the column No. 2 in the area II by switching the valve, and the mobile phase S is used in the area II₂Eluting, and automatically stopping the pump when the detection value of the II area reaches 940 mV; after valve switching, the No. 4 column in zone II is carried to No. 5 column in zone IV, and the mobile phase S is used in zone IV₄Automatically stopping the pump after elution is carried out for 5.4 min; after valve switching, the No. 7 column in IV region is carried to No. 8 column in VI region by mobile phase S₆Eluting, and automatically switching the rotary valve when the detection value of the VI area reaches 870 mV; when the rotary valve is switched, all the constant flow pumps in the areas I to VII stop; after valve switching, the 11 # column in VI region is carried to the 12 # column in VII region, where the mobile phase S is introduced₇Eluting, collecting EPA-EE in 0-4 min, automatically switching segment collectors in 4min, and respectively separating III and V by mobile phase S₃And mobile phase S₅After 12min of each elution, impurities are removed, and the processes are automatically and circularly carried out.