WO2020103916A1

WO2020103916A1 - Separation and purification method for uridine triphosphate

Info

Publication number: WO2020103916A1
Application number: PCT/CN2019/120049
Authority: WO
Inventors: 江必旺; 石凌超; 邹胜; 张彦辉
Original assignee: 苏州纳微科技股份有限公司
Priority date: 2018-11-22
Filing date: 2019-11-21
Publication date: 2020-05-28
Also published as: CN109503687B; CN109503687A

Abstract

Provided is a separation and purification method for uridine triphosphate. The separation and purification method comprises the following steps: 1) loading: filtering crude uridine triphosphate and then loading same into a chromatography column, wherein the packing of the chromatography column is monodisperse microspheres with polyacrylate as a matrix; 2) eluting: eluting UTP adsorbed in the chromatography column with an eluate; and 3) collecting and gathering: collecting the eluted uridine triphosphate solution in sections, and gathering component solutions that meet the target peak requirements. The separation and purification method for uridine triphosphate of the present invention is simple and convenient, has a short purification cycle, and can satisfy the high purity requirement of uridine triphosphate by means of only one step of chromatography purification. The uridine triphosphate has a purity of 99.9% or more and a high and stable purification yield.

Description

Method for separating and purifying uridine triphosphate

Technical field

The invention relates to the technical field of drug purification, in particular to a method for separating and purifying uridine triphosphate.

Background technique

Uridine triphosphate (UTP) is a uracil nucleotide composed of a uracil, a ribose, and three phosphates, in which the phosphate group is attached to the 5 'carbon. UTP is closely related to carbohydrate metabolism. UTP and glucose 1-phosphate are catalyzed by enzymes to produce UDP-glucose and pyrophosphate. In addition, UDP-galactose, UDP-galactosamine, UDP-glucuronic acid and the like are also produced.

UTP is involved in the synthesis of pyrimidine ribonucleotides and is the raw material for RNA synthesis (transcription). In addition, UTP can also be used as an energy source, and its function is similar to ATP, but it is rarer than ATP. UTP is also involved in many energy metabolism pathways in organisms.

UTP is also involved in the activation process of certain G protein-coupled receptors (such as P2Y, P2Y2, P2Y11, etc.), thereby activating epithelial chloride channels, increasing the frequency of ciliary swings, and inducing degranulation of goblet cells in respiratory epithelial cells , Can also affect the role of inflammatory cells and vascular reactivity. In addition, UTP is also commonly used in the diagnosis and treatment of certain diseases (such as INS316, lung cancer, etc.). It also plays an important role in sinusitis, chronic bronchitis, otitis media, dry eye and gastrointestinal diseases. Its market price can reach 12,000 yuan / kg, and it has broad application prospects in the pharmaceutical market.

Among them, the structural formula of uridine triphosphate is as follows:

The main synthesis process of UTP is to use microbial cell enzyme system synthesis. At present, there are few reports on the purification process of uridine triphosphate in domestic and foreign patents and documents.

GB1167288 discloses a method for purifying uridine triphosphate. The invention provides an improved method for purifying UDP and / or UTP from its aqueous solution by using an ion exchange method, whereby a concentrated solution of UDP or UTP can be obtained. This method uses a two-step ion exchange resin to separate UTP. The purity of the sample after purification is 97% and the yield is 72.2%. The purification effect is poor and the yield is relatively low.

Xiao Mingfang et al. Used ion exchange to purify uridine triphosphate in their master thesis "Ion Exchange Separation and Purification of 5, Uridine Triphosphate". After purification, the purity of the sample was 97.17%, the yield was 92.25%, and it was used in the purification process The resin is synthesized by the laboratory itself, and it is difficult to achieve stability and industrialization.

Therefore, in order to obtain a high-purity, high-yield uridine triphosphate with a short purification cycle and high stability, it is necessary to further study the fine purification process of uridine triphosphate.

Summary of the invention

In view of the shortcomings of the prior art, the object of the present invention is to provide a method for separating and purifying uridine triphosphate. Only one step of chromatographic purification can meet the requirements of high purity of uridine triphosphate, and the purification yield is high and stable, and the purity Over 99%, the yield is 86.54 ～ 90.24.

To achieve this goal, the present invention adopts the following technical solutions:

A method for separating and purifying uridine triphosphate. The method includes the following steps:

1) Loading: The crude uridine triphosphate is filtered and loaded into a chromatography column, and the packing material of the chromatography column is monodisperse microspheres with a matrix of polyacrylate;

2) Elution: elute the UTP adsorbed in the chromatography column with the eluent;

3) Collection and collection: The eluted uridine triphosphate solution is collected in stages, and the component liquids that meet the target peak requirements are collected.

The separation and purification method of the present invention can meet the requirements of high purity of uridine triphosphate in only one step of chromatography purification. The purification yield is high and stable, the purity is more than 99%, and the purification yield is high and stable. At the same time, the separation method of the present invention Simple and convenient, the purification cycle is short, and no organic solvent is used in the entire purification process, which can be used for large-scale production, which greatly reduces the production cost.

In step 1), the monodisperse microspheres are microspheres with a monodisperse particle size and a pore structure.

Preferably, in step 1), the particle diameter of the monodisperse microspheres is 40-60 μm, for example, the particle diameter of the monodisperse microspheres is 40 μm, 41 μm, 42 μm, 43 μm, 44 μm, 45 μm, 46 μm, 47 μm, 48 μm, 49 μm , 50μm, 51μm, 52μm, 53μm, 54μm, 55μm, 56μm, 57μm, 58μm, 59μm, 60μm; the pore size is

For example, the aperture is

Preferably, in step 1), the model of the monodisperse microspheres is UniQ-50XS, produced by Suzhou Nanomicro Technology Co., Ltd., with a particle size of 55 μm and a pore size of

In the present invention, the chromatography medium UniQ-50XS used in the purification process is a monodisperse microsphere in which the matrix bonded to the quaternary amine group is polyacrylate. The molecular formula of uridine triphosphate contains three phosphate groups. Preferably, under the selected elution conditions, UniQ-50XS produced by Suzhou Nanomicro Technology Co., Ltd. is used for UTP purification to obtain very good results. At the same time, the framework structure of the polyacrylate can tolerate the full pH range, and the column can be thoroughly cleaned at a high pH in the later stage, which is convenient for industrial repeated use, the filler has a long life, and reduces the cost.

More preferably, in step 1), the particle diameter of the monodisperse microspheres is 55 μm, and the pore diameter is

In step 1), the HPLC purity of the crude uridine triphosphate is 64-66%, for example, the HPLC purity of the crude product is 64%, 64.1%, 64.2%, 64.3%, 64.4%, 64.5%, 64.6%, 64.7% , 64.8%, 64.9%, 65%, 65.1%, 65.2%, 65.23%, 65.3%, 65.417%, 65.5%, 65.6%, 65.7%, 65.8%, 65.9%, 66%.

In step 1), 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, 15mL, 15.5mL, 15.7mL, 16mL, 17mL, 18mL, 19mL, 20mL; loading capacity is 10-30g / L, for example loading capacity is 10g / L, 11g / L, 12g / L, 13g / L , 14g / L, 15g / L, 16g / L, 17g / L, 18g / L, 19g / L, 20g / L, 21g / L, 22g / L, 23g / L, 24g / L, 25g / L, 26g / L, 27g / L, 28g / L, 29g / L, 30g / L.

In step 2), the eluent contains 0.5 to 2 mol of salt solution, for example, the mole 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 , 1.4mol, 1.5mol, 1.6mol, 1.7mol, 1.8mol, 1.9mol, 2mol, and does not contain organic solvents, safe, pollution-free and low cost.

Preferably, the salt solution is one of sodium chloride, ammonium chloride and sodium acetate.

In step 2), the flow rate of the eluent is 200-300 cm / h, for example, the flow rate of the eluent is 200 cm / h, 210 cm / h, 220 cm / h, 230 cm / h, 240 cm / h, 250 cm / h, 260cm / h, 270cm / h, 280cm / h, 290cm / h, 300cm / h.

In step 2), the elution amount of the eluent is 60-80CV, for example, the elution amount of the eluent is 60CV, 61CV, 62CV, 63CV, 64CV, 65CV, 66CV, 67CV, 68CV, 69CV, 70CV, 71CV, 72CV, 73CV, 74CV, 75CV, 76CV, 77CV, 78CV, 79CV, 80CV.

As a preferred solution of the present invention, the separation and purification method of uridine triphosphate includes the following steps:

1) Sample loading: The crude uridine triphosphate with a purity of 64-66% is filtered and loaded onto a chromatography column, wherein the column packing volume of the chromatography column is 4-20 mL, and the sample loading capacity is 10 ~ 30g / L, the chromatography column packing is a monodisperse microsphere UniQ-50XS whose matrix is polyacrylate;

2) Elution: The UTP adsorbed in the chromatography column is eluted with an eluent, wherein the eluent contains 0.5 to 2 mol of salt solution and contains no organic solvent, and the flow rate of the eluent 200-300cm / h, the elution amount of the eluent is 60-80CV;

Compared with the prior art, the beneficial effects of the present invention are:

(1) The separation and purification method of uridine triphosphate of the present invention can meet the requirements of high purity of uridine triphosphate in only one step of chromatographic purification. The purification yield is high and stable, with a purity of more than 99% and a yield of 86.54 ~ 90.24%.

(2) The separation and purification method of uridine triphosphate of the present invention is simple and convenient, and the purification cycle is short, and only the salt solution is used as the mobile phase in the entire purification process, without using any organic solvent, safe, non-polluting, and low cost, The used stationary phase can be reused and can be used for large-scale production, which greatly reduces environmental protection pressure and production costs.

BRIEF DESCRIPTION

1 is a scanning electron micrograph of UniQ-50XS polyacrylate microspheres used in Example 1 of the method for separating and purifying uridine triphosphate of the present invention;

2 is a high performance liquid chromatogram of the purified crude uridine triphosphate of Example 1 of the present invention;

3 is a high performance liquid chromatogram of uridine triphosphate after purification in Example 1 of the present invention.

detailed description

The technical solutions of the invention will be further described below with reference to the accompanying drawings 1-3, and specific embodiments, but the invention is not limited to these embodiments.

The method for separating and purifying uridine triphosphate of the present invention includes the following steps:

Example 1

Take 47.5 mL of crude uridine triphosphate (65.417% purity, the sample is diluted 5 times with mobile phase A), filter with a 0.45 μm filter membrane, and collect the filtrate for use. A pre-packed column of 7.7 × 100mm was used. UniQ-50XS strong anion exchange microspheres (produced by Suzhou Nanomicro Technology Co., Ltd.) were used as the packing material for the chromatography column. The packed volume was 4.65mL, and the loading capacity was 20g / L.

The chromatographic column is equilibrated before loading, and then loaded, and then eluted by gradient elution.

The solution of the target peak is collected in stages, and the component solutions that meet the requirements are summarized. After high performance liquid chromatography analysis, the purity of uridine triphosphate in the eluent is 99.05%, and the yield is 90.20%.

Figure 1 is a scanning electron micrograph of UniQ-50XS strong anion exchange resin microspheres. As can be seen from Figure 1, the microspheres exhibit a high degree of particle size uniformity.

Figure 2 is a high-performance liquid chromatogram of crude uridine triphosphate before purification. As can be seen from Figure 2, the crude uridine triphosphate has high impurity content, which does not meet the high purity requirements of uridine triphosphate.

Figure 3 is a high-performance liquid chromatogram of uridine triphosphate after purification. As can be seen from Figure 3, the impurity content in uridine triphosphate after purification is reduced, which meets the high purity requirements of uridine triphosphate.

Example 2

Take 320 mL of crude uridine triphosphate (65.23% purity, the sample is diluted 5 times with mobile phase A), filter with a 0.45 μm filter membrane, and collect the filtrate for use. A Tricorn 10-200 chromatographic column and UniQ-50XS strong anion exchange microspheres (manufactured by Suzhou Nanomicro Technology Co., Ltd.) were used as the packing material for the chromatographic column. The packed volume was 15.7 mL, and the loading capacity was 20 g / L.

Collect the solution of the target peak in stages, summarize the components that meet the requirements, and analyze by high performance liquid chromatography. The purity of uridine triphosphate in the eluent is 99.089%, and the yield is 90.24%.

Example 3

Take 45mL of crude uridine triphosphate (63.947% purity, the sample is diluted 5 times with mobile phase A), filter with a 0.45μm filter membrane, and collect the filtrate for use. A pre-packed column of 7.7 × 100mm was used. UniQ-50XS strong anion exchange microspheres (manufactured by Suzhou Nano Micro Technology Co., Ltd.) were used as the packing material for the chromatography column. The packed column volume was 4.65mL and the loading capacity was 18g / L.

The solution of the target peak was collected in stages, and the component liquids meeting the requirements were summarized. After high performance liquid chromatography analysis, the purity of uridine triphosphate in the eluent was 99.408%, and the yield was 86.54%.

Example 4

In this example, another strong anion exchange microsphere UniQ-30S (manufactured by Suzhou Nano Micro Technology Co., Ltd.) was used as a chromatography column packing, and other purification conditions were unchanged, as in Example 1.

Take 47.5 mL of crude uridine triphosphate (65.417% purity, the sample is diluted 5 times with mobile phase A), filter with a 0.45 μm filter membrane, and collect the filtrate for use. Using pre-packed columns of 7.7 × 100mm, UniQ-30S strong anion exchange microspheres (particle size about 36μm, pore size about

) As the packing material of the chromatography column, the packing volume is 4.65mL, and the loading capacity is 20g / L.

Collect the peak solution of interest in stages, summarize the components that meet the requirements, and analyze them by high performance liquid chromatography. The purity of the sample UTP after purification was 98.9%, and the yield was 85.2%.

In summary, the separation and purification method of uridine triphosphate of the present invention can meet the requirements of high purity of uridine triphosphate in only one step of chromatography purification, and the purification yield is high and stable, with a purity of more than 99% and a yield of 86.54 ~ 90.24%.

In addition, the separation and purification method of uridine triphosphate of the present invention is simple and convenient, and the purification cycle is short, and only the salt solution is used as the mobile phase in the entire purification process, without using any organic solvent, which is safe, non-polluting, and low in cost. The stationary phase can be reused and can be used for large-scale production, which greatly reduces environmental protection pressure and production costs.

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

Claims

A method for separating and purifying uridine triphosphate, characterized in that the method for separating and purifying comprises the following steps:

1) Loading: The crude uridine triphosphate is filtered and loaded into a chromatography column, and the packing material of the chromatography column is monodisperse microspheres with a matrix of polyacrylate;

2) Elution: elute the UTP adsorbed in the chromatography column with the eluent;

3) Collection and collection: The eluted uridine triphosphate solution is collected in stages, and the component liquids that meet the target peak requirements are collected.
The method for separation and purification according to claim 1, wherein in step 1), the monodisperse microspheres are microspheres with a monodisperse particle size and a pore structure.
The separation and purification method according to claim 1 or 2, wherein in step 1), the particle size of the monodisperse microspheres is 40-60 μm, and the pore size is
The separation and purification method according to any one of claims 1 to 3, wherein in step 1), the model of the monodisperse microsphere is UniQ-50XS.
The method for separation and purification according to any one of claims 1 to 4, wherein in step 1), the HPLC purity of the crude uridine triphosphate is 64 to 66%.
The separation and purification method according to any one of claims 1 to 5, wherein in step 1), the packed volume of the chromatography column is 4 to 20 mL, and the loading capacity of the sample is 10 to 30 g / L.
The method for separation and purification according to any one of claims 1 to 6, characterized in that, in step 2), the eluent contains 0.5 to 2 mol of salt solution and no organic solvent;

Preferably, the salt solution is one of sodium chloride, ammonium chloride and sodium acetate.
The separation and purification method according to any one of claims 1-7, wherein in step 2), the flow rate of the eluent is 200-300 cm / h.
The separation and purification method according to any one of claims 1 to 8, wherein in step 2), the elution amount of the eluent is 60 to 80 CV.
The separation and purification method according to any one of claims 1 to 9, wherein the separation and purification method comprises the following steps:

1) Sample loading: The crude uridine triphosphate with a purity of 64-66% is filtered and loaded onto a chromatography column, wherein the column packing volume of the chromatography column is 4-20 mL, and the sample loading capacity is 10 ~ 30g / L, the chromatography column packing is a monodisperse microsphere UniQ-50XS whose matrix is polyacrylate;

2) Elution: The UTP adsorbed in the chromatography column is eluted with an eluent, wherein the eluent contains 0.5 to 2 mol of salt solution and contains no organic solvent, and the flow rate of the eluent 200-300cm / h, the elution amount of the eluent is 60-80CV;

3) Collection and collection: The eluted uridine triphosphate solution is collected in stages, and the component liquids that meet the target peak requirements are collected.