Purification method of cocarboxylase tetrahydrate chloride

Abstract

The invention provides a purification method of cocarboxylase tetrahydrate chloride, and belongs to the technical field of purification. The purification method of the chlorinated cocarboxylase tetrahydrate comprises the following steps: loading a crude product of the chlorinated cocarboxylase tetrahydrate on an ion exchange chromatography column, sequentially carrying out first elution with water and second elution with a phosphate aqueous solution, and collecting an effluent corresponding to the chlorinated cocarboxylase tetrahydrate in the second elution process, removing water in the effluent to obtain a chlorinated cocarboxylase tetrahydrate pure product; a filler in the ion exchange chromatography column is an ion exchange type agarose medium. According to the method provided by the invention, the pure product of the cocarboxylase tetrahydrate chloride with the chromatographic purity of 99% or above can be obtained only through one-time purification, and the operation is simple and convenient. In addition, the method disclosed by the invention is stable in whole separation process, good in reproducibility and environment-friendly, and has an application value of further industrial production.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of purification, in particular to a purification method of chlorinated cocarboxylase tetrahydrate. BACKGROUND

[0002] Cocarboxylase tetrahydrate, also known as thiamine pyrophosphate tetrahydrate, is one of the ingredients of 12 kinds of injection complex vitamins, which was approved by FDA in 1999 for injection, and it is different from the combination of simple water-soluble vitamins and simple fat-soluble vitamins. It is a kind of complex vitamins with water-soluble and fat-soluble, which can provide comprehensive vitamins. Cocarboxylase tetrahydrate is a stable chemical form of thiamine pyrophosphate (TPP) in vitro, and thiamine pyrophosphate is converted from vitamin B1 in vivo, so it can generally play a catalytic role in biochemistry and participate in energy metabolism in cells, and can convert lactic acid and tartaric acid into energy. Cocarboxylase tetrahydrate is widely used in medicine, food science and biological engineering, such as treating lactic acidosis and alcoholism in medicine; serving as an additive ingredient in food science; and serving as a catalyst in the synthesis and conversion process of new drugs in biological engineering.

[0003] At present, the related process is usually to prepare cocarboxylase tetrahydrate crude product first, and then purify the cocarboxylase tetrahydrate crude product to obtain cocarboxylase tetrahydrate pure product. The main impurities in the cocarboxylase tetrahydrate crude product are thiamine triphosphate and thiamine monophosphate. In the related technology, D315 type weak basic anion exchange resin and HZD-2 type weak acid cation exchange resin are usually used to remove thiamine triphosphate and thiamine monophosphate, respectively, which needs multiple chromatography treatment, and the operation is complex. If only one step of chromatography treatment is performed, the product purity still needs to be improved. SUMMARY

[0004] The purpose of the present application is to provide a purification method of chlorinated cocarboxylase tetrahydrate, which can obtain high-purity product by only one purification step.

[0005] In order to achieve the above-mentioned purpose of the application, the present application provides the following technical solutions: The present application provides a purification method of chlorinated cocarboxylase tetrahydrate, comprising the following steps: The chlorinated cocarboxylase tetrahydrate crude product is loaded on an ion exchange chromatography column, and water is used for first elution and phosphate aqueous solution is used for second elution in sequence. The effluent corresponding to the chlorinated cocarboxylase tetrahydrate in the second elution process is collected, and the water in the effluent is removed to obtain the chlorinated cocarboxylase tetrahydrate pure product. The filler in the ion exchange chromatography column is ion exchange type agarose medium.

[0006] Preferably, the ion exchange chromatography column has an inner diameter of 10-2000 mm and an effective column length of 150-500 mm.

[0007] Preferably, the mass of the chlorinated tetrahydrate co-carboxylase crude product is 0.2-1% of the mass of the packing material in the ion exchange chromatography column.

[0008] Preferably, the loading of the chlorinated tetrahydrate co-carboxylase crude product into the ion exchange chromatography column comprises dissolving the chlorinated tetrahydrate co-carboxylase crude product in water, filtering the solution, and collecting the filtrate and loading it into the ion exchange chromatography column.

[0009] Preferably, the concentration of the solution obtained after dissolving the chlorinated tetrahydrate co-carboxylase crude product in water is 20-50 mg / mL; and the filter membrane used in the filtering is a 0.45 μm water-based microporous filter membrane.

[0010] Preferably, the flow rate of the water used in the first elution is 6-150 cm / h, and the volume of the water used in the first elution is 3-10 column volumes.

[0011] Preferably, the concentration of the aqueous phosphate solution is 10-100 mmol / L; the flow rate of the aqueous phosphate solution used in the second elution is 6-150 cm / h, and the volume of the aqueous phosphate solution used in the second elution is 5-15 column volumes.

[0012] Preferably, the first elution and the second elution comprise on-line monitoring of the UV response value of the effluent using a UV detector; and the detection wavelength of the UV detector is 232 nm.

[0013] Preferably, the collection of the effluent corresponding to the chlorinated tetrahydrate co-carboxylase in the second elution comprises starting the collection of the effluent when the UV response value in the second elution increases to 200-500 mAu, and ending the collection of the effluent when the UV response value decreases to 200-500 mAu.

[0014] Preferably, the removal of water from the effluent comprises freeze-drying.

[0015] The present application provides a method for purifying chlorinated tetrahydrate co-carboxylase, comprising the following steps: loading a chlorinated tetrahydrate co-carboxylase crude product into an ion exchange chromatography column, sequentially performing first elution using water and second elution using an aqueous phosphate solution, collecting the effluent corresponding to the chlorinated tetrahydrate co-carboxylase in the second elution, removing water from the effluent, and obtaining a chlorinated tetrahydrate co-carboxylase pure product. The method provided by the present application can obtain a chlorinated tetrahydrate co-carboxylase pure product with a chromatographic purity of 99% or higher after only one purification, and the operation is simple. In addition, the entire separation process of the method is stable, reproducible, environmentally friendly, and has application value for further industrial production. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 Figure 1 is a spectrum of the crude chlorinated tetrahydrated co-carboxylase detected by high performance liquid chromatograph; Figure 2 Figure 2 is a spectrum of the purification of the crude chlorinated tetrahydrated co-carboxylase in Example 1; Figure 3 Figure 3 is a spectrum of the effluent collected in Example 1 detected by high performance liquid chromatograph; Figure 4 Figure 4 is a spectrum of the purification of the crude chlorinated tetrahydrated co-carboxylase in Example 2; Figure 5 Figure 5 is a spectrum of the effluent collected in Example 2 detected by high performance liquid chromatograph; Figure 6 Figure 6 is a spectrum of the purification of the crude chlorinated tetrahydrated co-carboxylase in Example 3; Figure 7 Figure 7 is a spectrum of the effluent collected in Example 3 detected by high performance liquid chromatograph; Figure 8 Figure 8 is a spectrum of the purification of the crude chlorinated tetrahydrated co-carboxylase in Comparative Example 1; Figure 9 Figure 9 is a spectrum of the effluent collected in Comparative Example 1 detected by high performance liquid chromatograph. DETAILED DESCRIPTION

[0017] The present application provides a purification method of chlorinated tetrahydrated co-carboxylase, comprising the following steps: The crude chlorinated tetrahydrated co-carboxylase is loaded on an ion exchange chromatography column, and water is used for first elution and phosphate aqueous solution is used for second elution in sequence, the effluent corresponding to the chlorinated tetrahydrated co-carboxylase in the second elution process is collected, water in the effluent is removed, and the pure chlorinated tetrahydrated co-carboxylase is obtained; the filler in the ion exchange chromatography column is ion exchange type agarose medium.

[0018] In the present application, if no special description, the raw materials used are all commercially available goods well known to those skilled in the art or prepared by methods well known to those skilled in the art.

[0019] The chlorinated tetrahydrate co-carboxylase crude product is loaded on the ion exchange chromatography column. The source of the chlorinated tetrahydrate co-carboxylase crude product is not particularly limited in the present application, and can be obtained by using commercially available products or by using methods known to those skilled in the art. The content of chlorinated tetrahydrate co-carboxylase in the chlorinated tetrahydrate co-carboxylase crude product is not particularly limited in the present application, and can be, for example, 50-65 wt%, and can be specifically 55-60 wt%. As an embodiment of the present application, the main impurities contained in the chlorinated tetrahydrate co-carboxylase crude product include thiamine trisulfate and thiamine monosulfate, wherein the content of thiamine trisulfate can be 30-35 wt%, and the content of thiamine monosulfate can be 4-6 wt%. The chlorinated tetrahydrate co-carboxylase crude product is purified by using ion exchange agarose medium as the filler, and high-purity product can be obtained by only one purification, which can avoid multiple chromatography processing operations, is beneficial to reduce the product loss rate, and improve the yield.

[0020] The filler in the ion exchange chromatography column of the present application is ion exchange agarose medium; the ion exchange agarose medium used in the examples of the present application is specifically Hedera Q FF ion exchange agarose medium. As an embodiment of the present application, the ion exchange chromatography column can specifically use a 5 mL protein preloaded column or a glass chromatography column. As an embodiment of the present application, the inner diameter of the ion exchange chromatography column can be 10-2000 mm, further can be 10-300 mm, and more further can be 10-30 mm; the effective column length can be 150-500 mm, specifically can be 150-300 mm, and further can be 150-200 mm. As an embodiment of the present application, the mass of the chlorinated tetrahydrate co-carboxylase crude product can be 0.2-1% of the mass of the filler in the ion exchange chromatography column, further can be 0.3-0.8%, more further can be 0.4-0.6%, and specifically can be 0.44%. The filler used in the examples of the present application can utilize the positive and negative charge interaction between the filler and the chlorinated tetrahydrate co-carboxylase crude product to adsorb the chlorinated tetrahydrate co-carboxylase crude product on the filler, which is beneficial to the efficient separation of other impurities and chlorinated tetrahydrate co-carboxylase; the ratio of the chlorinated tetrahydrate co-carboxylase crude product to the filler is limited in the above range in the examples of the present application, which can avoid filler overload, and is beneficial to obtain higher-purity product.

[0021] As an embodiment of the present application, the loading of the crude chlorinated tetrahydrated co-factor of co-carboxylase into the ion exchange chromatography column can comprise: dissolving the crude chlorinated tetrahydrated co-factor of co-carboxylase in water, filtering, and collecting the filtrate to load into the ion exchange chromatography column. As an embodiment of the present application, the water used for dissolving can be purified water; the concentration of the solution obtained after dissolving the crude chlorinated tetrahydrated co-factor of co-carboxylase in water can be 20-50 mg / mL, further can be 30-45 mg / mL, and more further can be 35-42 mg / mL, and specifically can be 40 mg / mL; the filter membrane used for filtering can be a microporous filter membrane, and specifically can be a 0.45 μm water-based microporous filter membrane. In the embodiments of the present application, the filtrate can be uniformly pumped into the ion exchange chromatography column by using a sample pump.

[0022] After loading the crude chlorinated tetrahydrated co-factor of co-carboxylase into the ion exchange chromatography column, the present application sequentially uses water for first elution and uses a phosphate aqueous solution for second elution, and collects the effluent corresponding to the chlorinated tetrahydrated co-factor of co-carboxylase in the second elution process. As an embodiment of the present application, the water used for the first elution can be purified water; the flow rate of the water during the first elution can be 6-150 cm / h, and specifically can be 6 cm / h, 10 cm / h, 20 cm / h, 30 cm / h, 40 cm / h, 50 cm / h, 60 cm / h, 70 cm / h, 75 cm / h, 80 cm / h, 90 cm / h, 100 cm / h, 110 cm / h, 120 cm / h, 130 cm / h, 140 cm / h, or 150 cm / h; the volume of the water used for the first elution can be 3-10 column volumes, and specifically can be 3 column volumes, 3.5 column volumes, 4 column volumes, 5 column volumes, 6 column volumes, 7 column volumes, 8 column volumes, 9 column volumes, or 10 column volumes.

[0023] As an embodiment of the present application, the concentration of the aqueous phosphate solution can be 10-100 mmol / L, further can be 30-50 mmol / L, and specifically can be 30 mmol / L, 35 mmol / L, 40 mmol / L, 45 mmol / L or 50 mmol / L. As an embodiment of the present application, the second elution is specifically isocratic elution. As an embodiment of the present application, the flow rate of the aqueous phosphate solution during the second elution can be 6-150 cm / h, and specifically can be 6 cm / h, 10 cm / h, 20 cm / h, 30 cm / h, 40 cm / h, 50 cm / h, 60 cm / h, 70 cm / h, 75 cm / h, 80 cm / h, 90 cm / h, 100 cm / h, 110 cm / h, 120 cm / h, 130 cm / h, 140 cm / h or 150 cm / h; the volume of the aqueous phosphate solution used during the second elution can be 5-15 column volumes, and specifically can be 5 column volumes, 6 column volumes, 7 column volumes, 8 column volumes, 9 column volumes, 10 column volumes, 11 column volumes, 12 column volumes, 13 column volumes, 14 column volumes or 15 column volumes.

[0024] As an embodiment of the present application, the first elution and the second elution comprise: on-line monitoring the UV response value of the effluent by using a UV detector; the detection wavelength of the UV detector is 232 nm.

[0025] As an embodiment of the present application, collecting the effluent corresponding to the chlorinated tetrahydrate co-carboxylase during the second elution comprises: starting to collect the effluent when the UV response value increases to 200-500 mAu during the second elution, and ending to collect the effluent when the UV response value decreases to 200-500 mAu. In the embodiment of the present application, specifically, when the UV response value increases to 200 mAu during the second elution, starting to collect the effluent, and ending to collect the effluent when the UV response value decreases to 200 mAu, based on the loading amount of the chlorinated tetrahydrate co-carboxylase crude product being 25-122 mg. In the embodiment of the present application, the purity of the effluent is detected by using a high-performance liquid chromatograph, so as to represent the purity of the chlorinated tetrahydrate co-carboxylase pure product.

[0026] After obtaining the effluent, the water in the effluent is removed to obtain the chlorinated tetrahydrate co-carboxylase pure product. As an embodiment of the present application, the way of removing the water in the effluent can comprise freeze-drying; the temperature of the freeze-drying can be -55--45℃, and specifically can be -50℃; the time can be 30-40 h, and specifically can be 36 h. In the embodiment of the present application, the freeze-drying can be performed in a freeze-drying machine. The chlorinated tetrahydrate co-carboxylase pure product of the present application can be stored in nitrogen-filled cold storage.

[0027] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0028] The chromatography system used in the following experiments of this invention is a Bio-Lab100 chromatography system, purchased from Jiangsu Hanbang Technology Co., Ltd.; the ion-exchange agarose medium used is specifically Hedera Q FF ion-exchange agarose medium, purchased from Jiangsu Hanbang Technology Co., Ltd.

[0029] The main impurities in the crude coenzyme tetrahydrate used in the following experiments of this invention are thiamine triphosphate and thiamine monophosphate. Figure 1 The chromatograms of the crude cocarboxyl chloride tetrahydrate used in this invention were obtained by high performance liquid chromatography, and the relevant data are shown in Table 1.

[0030] Table 1. Data obtained from high-performance liquid chromatography analysis of the crude cocarboxyl chloride tetrahydrate product.

[0031] Example 1 Take 25 mg of crude coenzyme tetrahydrate, dissolve it in 1 mL of purified water, and filter it through a 0.45 μm aqueous microporous membrane. Inject the filtrate into a Bio-Lab 100 chromatography system at a flow rate of 75 cm / h. The ion exchange column used in the Bio-Lab 100 chromatography system has a packed size of Φ10×150 mm, and the packing material used in the ion exchange column is Hedera Q. FF type ion-exchange agarose medium (the mass of the crude coenzyme tetrahydrate chloride is 0.44% of the packing mass in the ion-exchange chromatography column) was used. The first elution was performed with purified water at a flow rate of 75 cm / h (the volume of purified water used was 3.5 column volumes). The second elution was performed with a 50 mmol / L sodium dihydrogen phosphate aqueous solution at a flow rate of 75 cm / h (the volume of sodium dihydrogen phosphate aqueous solution used was 5 column volumes). Both elutions were detected using a UV detector (detection wavelength 232 nm). Eluent was collected when the UV response value rose to 200 mAu and when it decreased to 200 mAu. The retention time was defined as 0 min from the start of the first elution. Specifically, elutants with a retention time of 48–58 min were collected. One purification cycle (including one first elution and one second elution) was completed after 100 min.

[0032] Figure 2 The image shows the chromatogram of the purified crude tetrahydrate cocarboxylase in Example 1. The results show that the eluent with a retention time of 48-58 min has a high UV response value, indicating that the tetrahydrate cocarboxylase in the eluent has a high purity.

[0033] Figure 3 The chromatograms obtained by high-performance liquid chromatography (HPLC) of the effluent collected in Example 1 are shown in Table 2. It can be seen that the purity of coenzyme tetrahydrate (CLC4) in the effluent collected in Example 1 is above 99%.

[0034] Table 2 shows the data obtained from high-performance liquid chromatography (HPLC) analysis of the effluent collected in Example 1.

[0035] Example 2 Take 62.5 mg of crude coenzyme tetrahydrate chloride, dissolve it in 5 mL of purified water, and filter it through a 0.45 μm aqueous microporous membrane. Inject the filtrate into a Bio-Lab 100 chromatography system at a flow rate of 75 cm / h. The ion exchange column used in the Bio-Lab 100 chromatography system has a packed size of Φ10×150 mm, and the column packing material used is Hedera Q. FF ion-exchange agarose medium (the mass of the crude coenzyme tetrahydrate chloride is 0.44% of the packing mass in the ion-exchange chromatography column) was used. The first elution was performed with purified water at a flow rate of 75 cm / h (the volume of purified water used was 4 column volumes). The second elution was performed with a 50 mmol / L sodium dihydrogen phosphate aqueous solution at a flow rate of 75 cm / h (the volume of sodium dihydrogen phosphate aqueous solution used was 8 column volumes). Both elutions were detected using a UV detector (detection wavelength 232 nm). Eluent was collected when the UV response value rose to 200 mAu and when it decreased to 200 mAu. The retention time was defined as 0 min from the start of the first elution. Specifically, elution with a retention time of 73–104 min was collected. One purification cycle (including one first elution and one second elution) was completed after 160 min.

[0036] Figure 4 The image shows the chromatogram of the crude tetrahydrate cocarboxylase purified in Example 2. The results show that the eluent with a retention time of 73-104 min has a high UV response value, indicating that the tetrahydrate cocarboxylase in the eluent has a high purity.

[0037] Figure 5The chromatograms of the effluent collected in Example 2 were obtained by high-performance liquid chromatography (HPLC), and the relevant data are shown in Table 3. It can be seen that the purity of coenzyme tetrahydrate in the effluent collected in Example 2 is above 99%.

[0038] Table 3 shows the data obtained from high-performance liquid chromatography (HPLC) analysis of the effluent collected in Example 2.

[0039] Example 3 122 mg of crude coenzyme tetrahydrate was dissolved in 10 mL of purified water and filtered through a 0.45 μm aqueous microporous membrane. The filtrate was then injected into a Bio-Lab 100 chromatography system at a flow rate of 75 cm / h. The ion-exchange column used in the Bio-Lab 100 chromatography system had a packed size of Φ10 × 150 mm, and the packing material used in the ion-exchange column was Hedera Q. FF ion-exchange agarose medium (the mass of the crude coenzyme tetrahydrate chloride is 0.44% of the packing mass in the ion-exchange chromatography column) was used. The first elution was performed with purified water at a flow rate of 75 cm / h (the volume of purified water used was 3.5 column volumes). The second elution was performed with a 30 mmol / L sodium dihydrogen phosphate aqueous solution at a flow rate of 75 cm / h (the volume of sodium dihydrogen phosphate aqueous solution used was 8 column volumes). Both elutions were detected using a UV detector (detection wavelength 232 nm). Eluent was collected when the UV response value rose to 200 mAu and when it decreased to 200 mAu. The retention time at the start of the first elution was defined as 0 min. Specifically, elution with a retention time of 45–78 min was collected. One purification cycle (including one first elution and one second elution) was completed after 140 min.

[0040] Figure 6 The image shows the chromatogram of the purification of crude coenzyme tetrahydrate in Example 3. The results show that the eluent with a retention time of 45-78 min has a high UV response value, indicating that the purity of coenzyme tetrahydrate in the eluent is high.

[0041] Figure 7 The chromatograms obtained by high-performance liquid chromatography (HPLC) of the effluent collected in Example 3 are shown in Table 4. It can be seen that the purity of coenzyme tetrahydrate (CLC4) in the effluent collected in Example 3 is above 99%.

[0042] Table 4 shows the data obtained from high-performance liquid chromatography (HPLC) analysis of the effluent collected in Example 3.

[0043] Comparative Example 1 62.5 mg of crude coenzyme tetrahydrate was dissolved in 5 mL of purified water and filtered through a 0.45 μm aqueous microporous membrane. The filtrate was injected into a Bio-Lab100 chromatography system at a flow rate of 75 cm / h. The column used in the Bio-Lab100 chromatography system had a packed size of Φ10×150 mm and was a weakly basic anion exchange resin (purchased from Tianjin Yunkai Resin Technology Co., Ltd., model D315). The mass of the crude coenzyme tetrahydrate was 0.44% of the packed mass of the column. A first elution was then performed using purified water at a flow rate of 75 cm / h (the volume of purified water used was...). (3.5 column volumes) Then, a second elution was performed using a 1.0 mol / L sodium hydroxide solution at a flow rate of 75 cm / h (the volume of sodium hydroxide solution used was 6 column volumes). During the first and second elutions, a UV detector (detection wavelength of 232 nm) was used for detection. When the UV response value increased to 200 mAu, the eluent was collected, and when the UV response value decreased to 200 mAu, the eluent was collected. The retention time at the start of the first elution was recorded as 0 min. Specifically, the eluent with a retention time of 45-57 min was collected. After 120 min, one purification cycle was completed (i.e., including one first elution and one second elution).

[0044] Figure 8 The image shows the chromatogram of the crude coenzyme tetrahydrate in Comparative Example 1. The results show that the crude coenzyme tetrahydrate purified using this method has a relatively large number of residual impurities.

[0045] Figure 9 The chromatograms of the effluent collected in Comparative Example 1 were obtained by high-performance liquid chromatography (HPLC), and the relevant data are shown in Table 5. It can be seen that the purity of coenzyme tetrahydrate in the effluent collected in Comparative Example 1 was only 90.63%, indicating that the purification of crude coenzyme tetrahydrate using this method resulted in a relatively high amount of residual impurities.

[0046] Table 5 shows the data obtained from the high-performance liquid chromatography (HPLC) analysis of the effluent collected in Comparative Example 1.

[0047] The results above show that the present invention uses ion-exchange agarose medium as a packing material, which has better hydrophilicity, higher biocompatibility, stability and high resolution compared with resin packing material. Pure cocarboxyl chloride tetrahydrate with a purity of over 99% can be obtained after one separation and purification, with a total separation yield of ≥90%. Moreover, the operation is simple and easy to control, without the need for multi-step separation and purification, and is suitable for industrial-scale production.

[0048] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for purifying chlorinated tetrahydrate coenzyme comprising the following steps: loading a chlorinated tetrahydrate coenzyme crude product into an ion exchange chromatography column, sequentially performing a first elution with water and a second elution with a phosphate aqueous solution, collecting an effluent corresponding to the chlorinated tetrahydrate coenzyme during the second elution, removing water from the effluent, and obtaining a chlorinated tetrahydrate coenzyme pure product; and a filler in the ion exchange chromatography column is an ion exchange type agarose medium. The ion exchange chromatography column has an inner diameter of 10-2000 mm and an effective column length of 150-500 mm.

2. The purification method of claim 1, wherein, The mass of the chlorinated tetrahydrate coenzyme crude product is 0.2-1% of the mass of the filler in the ion exchange chromatography column.

3. The purification method of claim 1, wherein, The loading of the chlorinated tetrahydrate coenzyme crude product into the ion exchange chromatography column comprises dissolving the chlorinated tetrahydrate coenzyme crude product in water, filtering, and collecting the filtrate and loading it into the ion exchange chromatography column.

4. The purification method of claim 1, wherein, The concentration of the solution obtained after dissolving the chlorinated tetrahydrate coenzyme crude product in water is 20-50 mg / mL; and a filter membrane with a pore size of 0.45 μm is used for the filtering.

5. The purification method of claim 4, wherein, The flow rate of the water during the first elution is 6-150 cm / h, and the volume of the water used during the first elution is 3-10 column volumes.

6. The purification method according to any one of claims 1 to 5, characterized in that, The concentration of the phosphate aqueous solution is 10-100 mmol / L; the flow rate of the phosphate aqueous solution during the second elution is 6-150 cm / h, and the volume of the phosphate aqueous solution used during the second elution is 5-15 column volumes.

7. The purification method of claim 6, wherein, The first elution and the second elution comprise on-line monitoring of the ultraviolet response value of the effluent using an ultraviolet detector; and the detection wavelength of the ultraviolet detector is 232 nm.

8. The purification method of claim 7, wherein, The collection of the effluent corresponding to the chlorinated tetrahydrate coenzyme during the second elution comprises starting the collection of the effluent when the ultraviolet response value increases to 200-500 mAu and ending the collection of the effluent when the ultraviolet response value decreases to 200-500 mAu.

9. The purification method of claim 8, wherein, The method for removing water from the effluent comprises freeze-drying.

10. The purification method according to claim 1 or 9, characterized by, ​

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