Separation and purification method of uridine diphosphate glucuronic acid

Abstract

The invention relates to the technical field of medicine purification, and discloses a method for separating and purifying uridine diphosphate glucuronic acid, which comprises the following steps: S1, loading a sample: filtering a uridine diphosphate glucuronic acid crude product, and loading the filtered uridine diphosphate glucuronic acid crude product into a chromatographic column, the filler of the chromatographic column being a monodisperse microsphere of which the matrix is polyacrylate or polystyrene-divinyl benzene; s2, elution: eluting the uridine diphosphate glucuronic acid adsorbed in the chromatographic column by using an eluent; and S3, collection and gathering: carrying out segmented collection after elution, and gathering the component liquid meeting the target peak value requirement. The separation and purification method of uridine diphosphate glucuronic acid is simple and convenient, has a short purification period, only uses a salt solution as a mobile phase in the whole purification process, does not need to use any organic solvent, and is suitable for large-scale production. The method is safe, pollution-free and very low in cost, the used stationary phase can be reused, the method can be used for large-scale production, the environmental protection pressure and the production cost are greatly reduced, and a better use prospect is brought.

Description

Technical Field

[0001] The invention relates to the technical field of drug purification, and in particular to a method for separating and purifying uridine diphosphate glucuronic acid. Background Art

[0002] Uridine diphosphate glucuronic acid (UDPGA or UDP-GlcA) is a sugar nucleotide and an important glycosyl donor in cells. It participates in a variety of metabolic pathways and is also an important glycosyl donor for glycosylation reactions in vitro. Studies have shown that there are two pathways for the synthesis of UDP-glucuronic acid: one is that 1-phospho-glucuronic acid is directly catalyzed by UDP-glucose pyrophosphorylase to generate UDP-glucuronic acid; the other pathway is more common in animals and bacteria, using nicotinamide adenine dinucleotide as a cofactor and UDP-glucose as a substrate, and UDP-glucose 6-dehydrogenase oxidizes UDP-glucose to synthesize UDP-glucuronic acid.

[0003] The structural formula of uridine diphosphate glucuronic acid is as follows:

[0004]

[0005] The main synthesis process of UDPGA is to use microbial cell enzymes to synthesize it. At present, there are few reports on the purification process of uridine diphosphate glucuronic acid in domestic and foreign patents and literature.

[0006] Therefore, in order to obtain high-purity, high-yield UDP-glucuronic acid with a short purification cycle and high stability, it is necessary to further study the fine purification process of UDP-glucuronic acid. Summary of the invention

[0007] In view of the shortcomings of the prior art, the object of the present invention is to provide a method for separating and purifying UDP-glucuronic acid, which can meet the high purity requirement of UDP-glucuronic acid by only one step of chromatography purification, with high and stable purification yield, purity of more than 99%, and yield of 85% to 95%.

[0008] To achieve this object, the present invention adopts the following technical solutions:

[0009] A method for separating and purifying uridine diphosphate glucuronic acid, the separation and purification method comprising the following steps:

[0010] S1. Loading: Filter the crude uridine diphosphate glucuronic acid product and load it into a chromatography column, wherein the chromatography column filler is a monodisperse microsphere whose matrix is ​​polyacrylate or polystyrene-divinylbenzene;

[0011] S2, elution: eluting the UDP-glucuronic acid adsorbed in the chromatography column with an eluent;

[0012] S3. Collection and aggregation: The eluted UDP-glucuronic acid solution is collected in sections, and the component solutions that meet the target peak requirements are aggregated.

[0013] The separation and purification method of the present invention can meet the high purity requirement of uridine diphosphate glucuronic acid with only one step of chromatography purification, and the purification yield is high and stable, with the purity reaching more than 99%, and the purification yield is high and stable. At the same time, the separation method of the present invention is simple and convenient, the purification cycle is short, and no organic solvent is needed in the whole purification process, so it can be used for large-scale production, which greatly reduces the production cost.

[0014] In step S1, the monodisperse microspheres are microspheres with monodisperse particle sizes and a pore structure.

[0015] Preferably, in step S1, the particle size of the monodisperse microspheres is 20 to 40 μm, for example, the particle size of the monodisperse microspheres is 20 μm, 21 μm, 22 μm, 23 μm, 24 μm, 25 μm, 26 μm, 27 μm, 28 μm, 29 μm, 30 μm, 31 μm, 32 μm, 33 μm, 34 μm, 35 μm, 36 μm, 37 μm, 38 μm, 39 μm, 40 μm; the pore size is For example, the aperture is

[0016] Preferably, in step S1, the monodisperse microspheres are of model NanoQ-30L, with a particle size of 30 μm and a pore size of

[0017] In the present invention, the chromatographic medium UniQ-30S / NanoQ-30L used in the purification process is a monodisperse microsphere whose matrix bonded with a quaternary amine group is polyacrylate or polystyrene-divinylbenzene. The molecular formula of uridine diphosphoglucuronic acid contains two phosphate groups and one carboxyl group. Preferably, under the selected elution conditions, the use of NanoQ-30L for UDPGA purification has achieved very good results. At the same time, the skeleton structure of polystyrene-divinylbenzene can tolerate the entire pH range, and the chromatography column can be thoroughly cleaned at a high pH in the later stage, which is convenient for repeated industrial use, has a long filler life, and reduces costs.

[0018] More preferably, in step S1, the particle size of the monodisperse microspheres is 30 μm, and the pore size is

[0019] In step S1, the HPLC purity of the crude uridine diphosphate glucuronic acid is 21.12-28.42%, for example, the HPLC purity of the crude product is 21%, 21.5%, 22%, 22.5%, 23%, 23.5%, 24%, 24.5%, 25%, 25.5%, 26%, 26.5%, 27%, 27.5%, 28%, 28.5%.

[0020] In step S1, the packing volume of the chromatography column is 4-20 mL, for example, the packing volume is 4.15 mL, 4.65 mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, 10 mL, 11 mL, 12 mL, 13 mL, 14 mL, 15 mL, 15.5 mL, 15.7 mL, 16 mL, 17 mL, 18 mL, 19 mL, 20 mL; the sample loading capacity is 5-10 g / L, for example, the sample loading capacity is 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L.

[0021] In step S2, the eluent contains 0.5 to 2 mol of salt solution, for example, the molar number of the salt solution is 0.5 mol, 0.6 mol, 0.7 mol, 0.8 mol, 0.9 mol, 1 mol, 1.1 mol, 1.2 mol, 1.3 mol, 1.4 mol, 1.5 mol, 1.6 mol, 1.7 mol, 1.8 mol, 1.9 mol, 2 mol, and does not contain an organic solvent, is safe, pollution-free and has very low cost.

[0022] Preferably, the salt solution is one of sodium chloride and ammonium chloride.

[0023] In step S2, the flow rate of the eluent is 120 to 360 cm / h, for example, the flow rate of the eluent is 120 cm / h, 140 cm / h, 160 cm / h, 180 cm / h, 200 cm / h, 220 cm / h, 240 cm / h, 260 cm / h, 280 cm / h, 300 cm / h, 320 cm / h, 340 cm / h, and 360 cm / h.

[0024] In step S2, the elution volume of the eluent is 40 to 50 CV, for example, the elution volume of the eluent is 40 CV, 41 CV, 42 CV, 43 CV, 44 CV, 45 CV, 46 CV, 47 CV, 48 CV, 49 CV, or 50 CV.

[0025] As a preferred embodiment of the present invention, the method for separating and purifying uridine diphosphate glucuronic acid comprises the following steps:

[0026] a. Loading: Filter the crude uridine diphosphate glucuronic acid with a purity of 21.12-28.42% and load it into a chromatography column, wherein the column volume of the chromatography column is 4-20 mL, the sample loading capacity is 5-10 g / L, and the chromatography column filler is a monodisperse microsphere NanoQ-30L whose matrix is ​​polystyrene-divinylbenzene;

[0027] b. Elution: eluting the UDPGA adsorbed in the chromatography column with an eluent, wherein the eluent contains 0.5 to 2 mol of a salt solution and does not contain an organic solvent, the flow rate of the eluent is 120 to 360 cm / h, and the elution volume of the eluent is 40 to 50 CV;

[0028] c. Collection and summary: The eluted UDP-glucuronic acid solution is collected in sections, and the component solutions that meet the target peak requirements are summarized.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] 1. The separation and purification method of UDP-glucuronic acid of the present invention only needs one step of chromatography purification to meet the high purity requirement of UDP-glucuronic acid, and the purification yield is high and stable, the purity is above 99%, and the yield is 88.45-94.58%.

[0031] 2. The separation and purification method of uridine diphosphate glucuronic acid of the present invention is simple and convenient, with a short purification cycle. In the entire purification process, only a salt solution is used as the mobile phase, and no organic solvent is required. The method is safe, pollution-free and has a very low cost. The stationary phase used can be reused and can be used for large-scale production, which greatly reduces environmental pressure and production costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a scanning electron microscope image of NanoQ-30L polystyrene-divinylbenzene microspheres used in Example 1 of the method for separating and purifying uridine diphosphate glucuronic acid of the present invention;

[0033] Figure 2 The chromatographic purification chromatogram of Example 1 of the present invention;

[0034] Figure 3 The HPLC chromatogram of the crude uridine diphosphate glucuronic acid product before purification in Example 1 of the present invention;

[0035] Figure 4 The HPLC chromatogram of UDP-glucuronic acid after purification in Example 1 of the present invention. DETAILED DESCRIPTION

[0036] The following is combined with Figure 1-4The technical solution of the invention is further described through specific embodiments, but the present invention is not limited to these embodiments.

[0037] A method for separating and purifying uridine diphosphate glucuronic acid of the present invention comprises the following steps:

[0038] S1. Loading: Filter the crude uridine diphosphate glucuronic acid product and load it into a chromatography column, wherein the chromatography column filler is a monodisperse microsphere whose matrix is ​​polystyrene-divinylbenzene;

[0039] S2, elution: eluting the UDPGA adsorbed in the chromatography column with an eluent;

[0040] S3. Collection and aggregation: The eluted UDP-glucuronic acid solution is collected in sections, and the component solutions that meet the target peak requirements are aggregated.

[0041] Example 1

[0042] Take 45mL of crude uridine diphosphate glucuronic acid (24.82% purity, the sample was diluted 5 times with mobile phase A), filter with a pore size of 0.45μm filter membrane, and collect the filtrate for use. Use a 7.7×100mm pre-packed column and NanoQ-30L strong anion exchange microspheres as the chromatography column filler, with a column volume of 4.65mL and a sample load of 10g / L.

[0043] The column was equilibrated before loading the sample, and then the sample was loaded and eluted using a gradient elution method.

[0044] The solution of the target peak was collected in sections, and the component solutions meeting the requirements were summarized. After high performance liquid chromatography analysis, the purity of UDPG in the eluate was 99.17% and the yield was 91.23%.

[0045] Figure 1 This is a scanning electron microscope image of NanoQ-30L strong anion exchange resin microspheres. Figure 1 It can be seen that the microspheres exhibit a high degree of particle size uniformity.

[0046] Figure 2 is the HPLC chromatogram of crude UDP-glucuronic acid before purification. Figure 2 It can be seen that the impurity content in the crude UDP-glucuronic acid is high, which does not meet the high purity requirement of UDP-glucuronic acid.

[0047] Figure 3 is the HPLC chromatogram of purified uridine diphosphate glucuronic acid, Figure 3 It can be seen that the impurity content in UDPG is reduced after purification, which meets the requirement of high purity of UDPG.

[0048] Example 2

[0049] Take 300mL of crude uridine diphosphate glucuronic acid (25.23% purity, the sample was diluted 5 times with mobile phase A), filter with a pore size of 0.45μm filter membrane, and collect the filtrate for use. Use Tricorn10-200 chromatographic column and NanoQ-30L strong anion exchange microspheres as chromatography column fillers, with a column volume of 15.7mL and a sample load of 10g / L.

[0050] The column was equilibrated before loading the sample, and then the sample was loaded and eluted using a gradient elution method.

[0051] The solution of the target peak was collected in sections, and the component solutions meeting the requirements were summarized. After high performance liquid chromatography analysis, the purity of UDPG in the eluate was 99.19% and the yield was 93.24%.

[0052] Example 3

[0053] Take 25mL of crude uridine diphosphate glucuronic acid (23.82% purity, the sample was diluted 5 times with mobile phase A), filter with a pore size of 0.45μm filter membrane, and collect the filtrate for use. Use a 7.7×100mm pre-packed column and NanoQ-30L strong anion exchange microspheres as the chromatography column filler, with a column volume of 4.65mL and a sample load of 5g / L.

[0054] The column was equilibrated before loading the sample, and then the sample was loaded and eluted using a gradient elution method.

[0055] The solution of the target peak was collected in sections, and the component solutions meeting the requirements were summarized. After high performance liquid chromatography analysis, the purity of UDP-glucuronic acid in the eluate was 99.47%, and the yield was 92.75%.

[0056] Example 4

[0057] This example uses another strong anion exchange microsphere UniQ-30S as the chromatography column filler, and other purification conditions remain the same as in Example 1.

[0058] Take 45 mL of crude UDP-glucuronic acid (24.28% purity, the sample was diluted 5 times with mobile phase A), filter it with a 0.45 μm filter membrane, and collect the filtrate for later use. Use a 7.7×100 mm prepacked column, UniQ-30S strong anion exchange microspheres (polyacrylate matrix, particle size about 36 μm, pore size about ) was used as the chromatography column filler, with a column packing volume of 4.65 mL and a sample loading capacity of 10 g / L.

[0059] The column was equilibrated before loading the sample, and then the sample was loaded and eluted using a gradient elution method.

[0060] The solution of the target peak was collected in sections, and the component solutions meeting the requirements were summarized and analyzed by high performance liquid chromatography. The purity of the purified sample UDPGA was 99.09% and the yield was 91.82%.

[0061] In summary, the separation and purification method of UDP-glucuronic acid of the present invention can meet the high purity requirement of UDP-glucuronic acid with only one step of chromatography purification, and the purification yield is high and stable, the purity is above 99%, and the yield is 88.45-94.58%.

[0062] In addition, the separation and purification method of uridine diphosphate glucuronic acid of the present invention is simple and convenient, with a short purification cycle, and only a salt solution is used as a mobile phase in the entire purification process, without the need to use any organic solvent. It is safe, pollution-free and has very low cost. The stationary phase used can be reused and can be used for large-scale production, greatly reducing environmental pressure and production costs.

[0063] The present invention illustrates the detailed process equipment and process flow of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned detailed process equipment and process flow, that is, it does not mean that the present invention must rely on the above-mentioned detailed process equipment and process flow to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for separating and purifying uridine diphosphate glucuronic acid, characterized in that: The separation and purification method comprises the following steps: S1. Loading: Filter the crude uridine diphosphate glucuronic acid product and load it into a chromatography column, wherein the filler of the chromatography column is a monodisperse microsphere whose matrix is ​​polyacrylate or polystyrene-divinylbenzene; S2, elution: eluting the UDP-glucuronic acid adsorbed in the chromatography column with an eluent; S3. Collection and aggregation: The eluted UDP-glucuronic acid solution is collected in sections, and the component solutions that meet the target peak requirements are aggregated.

2. The method for separating and purifying UDP-glucuronic acid according to claim 1, wherein: In step S1, the monodisperse microspheres are microspheres with monodisperse particle sizes and a pore structure.

3. A method for separating and purifying UDP-glucuronic acid according to claim 1 or 2, characterized in that: In step S1, the particle size of the monodisperse microspheres is 20 to 40 μm, and the pore size is 4. A method for separating and purifying UDP-glucuronic acid according to claim 3, characterized in that: In step S1, the model of the monodisperse microspheres is UniQ-30S or NanoQ-30L.

5. A method for separating and purifying UDP-glucuronic acid according to claim 4, characterized in that: The following steps are involved: In step S1, the HPLC purity of the crude UDP-glucuronic acid is 21.12-28.42%.

6. The method for separating and purifying UDP-glucuronic acid according to claim 5, characterized in that: In step S1, the column volume of the chromatography column is 4-20 mL, and the sample loading capacity is 5-10 g / L.

7. The method for separating and purifying UDP-glucuronic acid according to claim 6, characterized in that: In step S2, the eluent contains 0.5-2 mol of a salt solution and does not contain an organic solvent.

8. The method for separating and purifying UDP-glucuronic acid according to claim 7, characterized in that: In step S2, the flow rate of the eluent is 120-360 cm / h.

9. The method for separating and purifying UDP-glucuronic acid according to claim 8, characterized in that: In step S2, the elution volume of the eluent is 30 to 60 CV.

10. The method for separating and purifying UDP-glucuronic acid according to claim 9, characterized in that: The separation and purification method comprises the following steps: a. Loading: Filter the crude uridine diphosphate glucuronic acid with a purity of 21.12-28.42% and load it into a chromatography column, wherein the column volume of the chromatography column is 4-20 mL, the sample loading capacity is 5-10 g / L, and the chromatography column filler is a monodisperse microsphere NanoQ-30L whose matrix is ​​polystyrene-divinylbenzene; b. Elution: eluting the UDP-glucuronic acid adsorbed in the chromatography column with an eluent, wherein the eluent contains 0.5 to 2 mol of a salt solution and does not contain an organic solvent, the flow rate of the eluent is 120 to 360 cm / h, and the elution volume of the eluent is 30 to 60 CV; c. Collection and summary: The eluted UDP-glucuronic acid solution is collected in sections, and the component solutions that meet the target peak requirements are summarized.

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