WO2016086860A1 - 一种β-烟酰胺单核苷酸的纯化方法 - Google Patents

一种β-烟酰胺单核苷酸的纯化方法 Download PDF

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WO2016086860A1
WO2016086860A1 PCT/CN2015/096215 CN2015096215W WO2016086860A1 WO 2016086860 A1 WO2016086860 A1 WO 2016086860A1 CN 2015096215 W CN2015096215 W CN 2015096215W WO 2016086860 A1 WO2016086860 A1 WO 2016086860A1
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nicotinamide mononucleotide
solution
nanofiltration
membrane
purifying
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PCT/CN2015/096215
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English (en)
French (fr)
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傅荣昭
戴柱
张琦
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邦泰生物工程(深圳)有限公司
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Priority to JP2016542666A priority Critical patent/JP6324511B2/ja
Priority to US15/110,005 priority patent/US10214552B2/en
Publication of WO2016086860A1 publication Critical patent/WO2016086860A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C07H1/06—Separation; Purification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/048—Pyridine radicals

Definitions

  • the present invention relates to a nucleotide coenzyme method, and more particularly to a method for purifying a ⁇ -nicotinamide mononucleotide.
  • the ⁇ -nicotinamide mononucleotide ( ⁇ ) is a synthetic substrate for coenzyme I, which becomes a coenzyme I (NAD) upon adenosine catalysis by a nicotinamide nucleotide adenosyltransferase.
  • NAD coenzyme I
  • the level of NMN in the body and the activity of nicotinamide nucleotide adenosyltransferase (NAMPT) directly affect the concentration of NAD.
  • the same NMN is directly involved in adenosine transfer in vivo, which is an important synthetic substrate and function regulation in vivo. substance.
  • NMN can be used for anti-aging, treatment of chronic diseases, etc.
  • Peer studies have shown that NMN also regulates insulin secretion and also has an effect on mRNA expression levels. Therefore, NMN has broad application prospects in medical treatment, and peers also have broad market prospects in chemical industry as a reaction substrate.
  • NMN generally uses ion exchange resin for purification. Because it is very similar to various analogs such as NAD with charge and polarity, it is very difficult to separate and purify, and it is impossible to completely remove the analog impurities therein, so ion exchange The purity of the product obtained by the method is only about 60%, the yield is only 40%, and the production efficiency is low, which is not suitable for large-scale production.
  • an object of the present invention is to provide a method for purifying a ⁇ -nicotinamide mononucleotide, which aims to solve other charges during the purification process of ⁇ -nicotinamide mononucleotide It is difficult to remove analogs with similar polarities, and the product has low purity and low yield.
  • a method for purifying a ⁇ -nicotinamide mononucleotide comprising the steps of:
  • the pretreated ⁇ -nicotinamide mononucleotide solution is subjected to microfiltration and nanofiltration using a membrane concentration device
  • the pH of the obtained crude solution is adjusted to 3-7
  • the injection column is prepared by reversed-phase high performance liquid chromatography
  • the stationary phase is octadecylsilane bonded silica gel
  • the mobile phase A is prepared by hydrochloric acid solution.
  • a solution of pH 3-7 mobile phase B is ethanol, and subjected to gradient elution purification to obtain a purified sample solution;
  • the present invention uses octadecylsilane bonded silica gel for high-performance liquid phase preparation, and purifies ⁇ -nicotinamide mononucleotide so that the purity of ⁇ -nicotinamide mononucleotide can reach 98%, yield It can reach more than 80%, and the production efficiency is more than 2 times higher than other processes. It effectively solves the problem that other analogs with similar charge and polarity are difficult to be removed during the purification of ⁇ -nicotinamide mononucleotide.
  • a preferred embodiment of the method for purifying a ⁇ -nicotinamide mononucleotide of the present invention comprises the following steps:
  • the purified sample solution was subjected to nanofiltration using a membrane concentration device, and then lyophilized by a vacuum freeze dryer to obtain a purified ⁇ -nicotinamide mononucleotide.
  • the present invention is a reverse phase high performance liquid chromatography method for the purification of the synthesis substrate ⁇ -nicotinamide mononucleotide of coenzyme I, through the stationary phase is octadecylsilane bonded silica gel, the mobile phase is hydrochloric acid
  • the prepared solution and ethanol column were purified, further concentrated and lyophilized to obtain purified ⁇ -nicotinamide mononucleotide.
  • the method for purifying the ⁇ -nicotinamide mononucleotide of the present invention is simple in operation and can effectively remove other charged and polar and similar analogs, so that the prepared ⁇ -nicotinamide mononucleotide purity High, high yield, large output, suitable for large-scale industrial production.
  • the microfiltration membrane for microfiltration in the step S100 has a pore diameter of 0.2-1 ⁇ m.
  • the pore diameter of the microfiltration membrane used for microfiltration in the step S100 of the present invention is 0.5 ⁇ m.
  • the basic principle of microfiltration is the sieving process, filtering particles larger than ⁇ under the action of static pressure difference, the operating pressure is 0.7-7 bar, the raw material liquid is under the pressure difference, wherein the solvent penetrates the micropores on the membrane Flowing to the low pressure side of the membrane, particles larger than the pores of the membrane are trapped, thereby achieving separation of the particulates from the solvent in the raw material liquid.
  • the mechanism of the microfiltration process on the microparticles is the sieving effect.
  • the separation effect of the membrane is determined by the physical structure of the membrane and the shape and size of the pores.
  • the microfiltration membrane used in the microfiltration membrane has a pore size of 0.5 ⁇ m, which can preliminarily remove large microorganisms and large particle molecules in the crude solution of ⁇ -nicotinamide mononucleotide, and the microfiltration membrane allows macromolecules. And dissolved solids
  • the nanofiltration membrane for nanofiltration in the step S100 of the present invention has a molecular weight cut off of 200.
  • the nanofiltration membrane used in the preferred embodiment of the present invention has a pore diameter of 1.5 nm and is capable of retaining a substance having a molecular weight of 200.
  • the nanofiltration membrane for nanofiltration in the step S100 is a hollow fiber membrane.
  • Nanofiltration is a filtration method that allows the passage of solvent molecules or certain low molecular weight solutes or low-cost ions. It is characterized by high demineralization performance and molecular weight cut off at hundreds of low pressures. Substance.
  • the invention adopts a filter membrane having a pore diameter of 1.5 nm and a molecular weight cut off of 200, and can filter out a substance having a molecular weight of more than 200 to initially filter out impurities in the ⁇ -nicotinamide mononucleotide.
  • the hollow fiber membrane is used as a nanofiltration membrane, and the hollow fiber membrane has a fibrous appearance, which has a self-supporting effect and can effectively remove phosphate residues and other small molecular impurities generated in the preparation process of the ⁇ -nicotinamide mononucleotide.
  • the ⁇ -nicotinamide mononucleotide was further purified.
  • the concentrated crude solution was collected at a concentration of 20-30 g/L.
  • the mass concentration of the phosphoric acid solution or the hydrochloric acid solution in the step S200 is 2% ⁇
  • the volume of the phosphoric acid solution or the hydrochloric acid solution in the step S200 is 5-20 ml of a phosphoric acid solution or a hydrochloric acid solution per ml of the sample solution, and the volume of the ethanol solution is 100-400 per liter of the mobile phase. Million ethanol solution.
  • the mass concentration of the phosphoric acid solution or the hydrochloric acid solution is too high, which may result in elution in the gradient elution. If the mass concentration is too low, the separation and purification effect of the substance may be affected. In the present invention, the mass concentration is 2
  • the % ⁇ 5 % phosphoric acid solution or the hydrochloric acid solution can ensure the effect of highly purified ⁇ -nicotinamide mononucleotide in the case where the gradient elution is not precipitated.
  • the volume of the solution is 5 to 20 ml of phosphoric acid solution or hydrochloric acid solution per ml of the sample solution, and the volume of the mobile phase is 100-400 ml of ethanol solution per liter of the mobile phase, so that the peak shape is symmetrical and the tailing is reduced. phenomenon.
  • the step S200 uses a phosphoric acid solution or a hydrochloric acid solution to adjust the pH value of the crude ⁇ -nicotinamide mononucleotide solution purified in the previous step to 3-7, and inject the sample.
  • the column was prepared by reversed-phase high performance liquid chromatography, and the mobile phase was a solution of ⁇ 3-7 prepared by a hydrochloric acid solution, and the mobile phase was ethanol.
  • a non-polar stationary phase (eg, C18, C8) is generally used in reversed-phase high performance liquid chromatography;
  • the mobile phase is usually water or a buffer, and a water-miscible organic solvent such as methanol, ethanol, isopropanol, acetone or tetrahydrofuran is often added to adjust the retention time, which is suitable for separating non-polar and weakly polar compounds.
  • a water-miscible organic solvent such as methanol, ethanol, isopropanol, acetone or tetrahydrofuran is often added to adjust the retention time, which is suitable for separating non-polar and weakly polar compounds.
  • the pH affects the state of the sample, thus affecting the retention of the sample.
  • the PH3-7 solution and the ethanol are used as the mobile phase, which can effectively adjust the retention time of the sample, so that the sample can be optimized from the time of entering the column to the column flowing out of the column, and the sample can be made.
  • the separation effect is good. If the retention time of the sample is too long, the sensitivity of the detection will be lowered; if the retention time of the sample is too short, the separation effect of the ⁇ -nicotinamide mononucleotide and the impurity will be lowered, and the ⁇ -nicotinamide mononucleotide will be affected. purification.
  • the pH of the mobile phase should be controlled between 2-8, and the pH value is greater than 8 ⁇ , so that the carrier silica gel, that is, the stationary phase, can be dissolved, and the pH is less than 2 ⁇ , and the chemical bonding with the silica gel is easy to hydrolyze and fall off.
  • the pH value is 3-7, and the ⁇ -nicotinamide single nucleotide can be stably existed, and the internal structure thereof is not destroyed. Under this condition, the separation effect of the sample can be optimized. More preferably, the pH of the mobile phase in the present invention is 5, and the purification effect of this ruthenium sample is optimal.
  • the pH of the obtained crude solution is adjusted to 3-7 by using a phosphoric acid solution or a hydrochloric acid solution because the ⁇ -nicotinamide mononucleotide crude solution also contains phosphate residue, and is A solution prepared by using hydrochloric acid is used as a mobile phase.
  • a phosphoric acid solution or a hydrochloric acid solution is used to adjust the pH of the crude solution, no new impurities are introduced, and a phosphate or the like can be removed during the purification.
  • the volume ratio of the mobile phase to the mobile phase is between 2: 98-1:1.
  • the ratio of the mobile phase affects the detection effect and purification effect of the sample. If the amount of hydrochloric acid solution is increased and the amount of ethanol is lowered, the sample will grow during the retention period and can be well separated, but the peak of the sample is broadened and the peak height is lowered, which affects the sensitivity of the detection; The amount of ethanol used in the sample is higher, but the separation effect of the sample is not good.
  • a hydrochloric acid having a pH of 3-7 is prepared by mixing hydrochloric acid between 2: 98-1:1 by volume ratio, so that the sample can be separated and purified well. ⁇ Improve the sensitivity of sample detection.
  • the elution enthalpy of the gradient elution enthalpy is 40 min. Specifically 2%
  • the gradient elution method is more symmetrical and has no tailing than the isocratic elution method, and can improve the efficiency of the column. Improve the sensitivity of the test.
  • controlling the elution interval to 40 min can make the sample retention time suitable, and the separation and purification effect of ⁇ -nicotinamide single nucleotide is the best.
  • the present invention is the first to use the reversed-phase high performance liquid chromatography to purify the synthetic substrate ⁇ -nicotinamide mononucleotide of the coenzyme I, which overcomes the low purification efficiency of the conventional ion exchange resin method and cannot be separated from ⁇ - A similarity in the properties of nicotinamide mononucleotide polarities and the like, and the yield is low.
  • the present invention includes the following columns (column diameter * length): 5 cm * 30 cm, 15 cm * 30
  • the column temperature is room temperature.
  • the pretreated ⁇ -nicotinamide mononucleotide solution is subjected to microfiltration and nanofiltration by a membrane concentration device, and the microorganism is removed by microfiltration.
  • the nanofiltration is used to concentrate the crude product to 20 by using a hollow fiber membrane with a molecular weight cut off of 200. -30g/L.
  • column column diameter and length is 5 cm * 30cm
  • stationary phase octadecylsilane bonded silica gel
  • Mobile phase phase A: hydrochloric acid solution having a pH of 3; phase B: ethanol;
  • flow rate 50-80 ml / min
  • Detection wavelength 260 nm
  • the loading amount was 8-10 g.
  • Embodiment 2 is a diagrammatic representation of Embodiment 1:
  • the pretreated ⁇ -nicotinamide mononucleotide solution is subjected to microfiltration and nanofiltration using a membrane concentration device, and the microorganism is removed by microfiltration, and the hollow fiber membrane with a molecular weight cut off of 200 is used to concentrate the crude product to 20- 30g/L.
  • stationary phase octadecylsilane bonded silica gel
  • Mobile phase phase A: hydrochloric acid solution having a pH of 5; phase B: ethanol;
  • flow rate 400-500 ml / min
  • detection wavelength 260nm
  • the loading amount was 60-80 g.
  • the purified sample solution is concentrated to 100-150 g/L by a membrane concentration device (hollow fiber membrane with molecular weight cut off of 200), and then freeze-dried by a vacuum freeze dryer to obtain ⁇ -smoke having a purity greater than 98%.
  • the total yield of the amide mononucleotide can reach 82.3%.
  • the pretreated ⁇ -nicotinamide mononucleotide solution is subjected to microfiltration and nanofiltration using a membrane concentration device, and the microorganism is removed by microfiltration, and the hollow fiber membrane with a molecular weight cut off of 200 is used to concentrate the crude product to 20- by nanofiltration. 30g/L.
  • column column diameter and length is 30 cm * 30cm
  • stationary phase octadecylsilane bonded silica gel
  • phase A hydrochloric acid solution having a pH of 7
  • phase B ethanol
  • flow rate 2500-3000 ml / min
  • detection wavelength 260 nm
  • the loading amount is 350-400 g.
  • Purification process The concentrated crude solution is adjusted to pH 3-7 with a phosphoric acid solution or a hydrochloric acid solution, and the column is rinsed with 30% or more of ethanol and equilibrated to load, and the sample loading is 350-400 g. A linear gradient eluted at 40 mi n to collect the target peak.
  • the purified sample solution is concentrated to 100-150 g/L by a membrane concentration device (hollow fiber membrane with molecular weight cut off of 200), and then freeze-dried by a vacuum freeze dryer to obtain ⁇ -smoke having a purity greater than 98%.
  • the total yield of the amide mononucleotide can reach 80.9%.
  • the present invention by collecting the target peak of the sample, when it reaches the standard enthalpy, it is subjected to nanofiltration using a membrane concentration device, and then lyophilized by a vacuum freeze dryer to obtain a purified ⁇ -nicotinamide mononucleotide. .
  • the freeze-dried product ⁇ -nicotinamide mononucleotide prepared by the method of the invention has a purity of up to 98%, and the total yield can reach above 80%.
  • the invention since the invention is simple in operation, the production efficiency is improved compared with other processes. More than twice, it effectively solves the problem that the phosphate residue in the prior art is difficult to solve and the prepared product has low purity and low yield.
  • the invention has good market prospects and is suitable for industrial production of purified ⁇ -nicotinamide mononucleotides.

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Abstract

本发明公开了一种β-烟酰胺单核苷酸的纯化方法，其包括以下步骤：a、将经过预处理的β-烟酰胺单核苷酸溶液用膜浓缩设备先后进行微滤和纳滤，收集浓缩的粗品溶液；b、将获得的粗品溶液pH值调至3-7，进样上反相高效液相色谱制备柱，固定相为十八烷基硅烷键合硅胶，流动相A为盐酸溶液配成的pH3-7的溶液，流动相B为乙醇，进行梯度洗脱纯化，得到纯化的样品溶液；c、将纯化的样品溶液用膜浓缩设备进行纳滤后，用真空冷冻干燥机冻干，获得纯化的β-烟酰胺单核苷酸。

Description

说明书 发明名称：一种 β_烟酰胺单核苷酸的纯化方法 技术领域
[0001] 本发明涉及核苷酸类辅酶方法， 尤其涉及一种 β-烟酰胺单核苷酸的纯化方法。
背景技术
[0002] β-烟酰胺单核苷酸 （ΝΜΝ) 是辅酶 I的合成底物， 在被烟酰胺核苷酸腺苷转移 酶腺苷化后即变成辅酶 I (NAD) 。 在生物体内 NMN的水平和烟酰胺核苷酸腺苷 转移酶 （NAMPT) 的活性直接影响到 NAD的浓度， 同吋 NMN直接参与体内腺 苷转移， 是体内重要的一种合成底物和功能调节物质。 在治疗应用方面， NMN 可以用于抗衰老、 治疗慢性病等， 同吋研究表明 NMN还对胰岛素的分泌起到调 节作用， 对 mRNA表达水平也有影响。 因此， NMN在医药治疗方面有着广泛的 应用前景， 同吋也作为一种反应底物在化工方面有着广泛的市场前景。
[0003] NMN—般应用离子交换树脂进行纯化， 由于其与多种类似物如 NAD带电荷和 极性极为相近， 分离纯化有很大困难， 无法将其中的类似物杂质完全去除， 所 以离子交换法获得的产品纯度只有 60%左右， 收率只有 40%， 生产效率低下， 不 适合规模化生产。
[0004] 因此， 现有技术还有待于改进和发展。
技术问题
[0005] 鉴于上述现有技术的不足之处， 本发明的目的在于提供一种 β-烟酰胺单核苷酸 的纯化方法， 旨在解决 β-烟酰胺单核苷酸的纯化过程中其他电荷和极性相近的类 似物难以除尽， 且产品纯度低、 收率低的问题。
问题的解决方案
技术解决方案
[0006] 为了达到上述目的， 本发明采取了以下技术方案：
[0007] 一种 β-烟酰胺单核苷酸的纯化方法， 其中， 包括以下步骤：
[0008] a、 将经过预处理的 β-烟酰胺单核苷酸溶液用膜浓缩设备先后进行微滤和纳滤
， 收集浓缩的粗品溶液； [0009] b、 将获得的粗品溶液 pH值调至 3-7， 进样上反相高效液相色谱制备柱， 固定相 为十八烷基硅烷键合硅胶， 流动相 A为盐酸溶液配成的 pH3-7的溶液， 流动相 B为 乙醇， 进行梯度洗脱纯化， 得到纯化的样品溶液；
[0010] c、 将纯化的样品溶液用膜浓缩设备进行纳滤后， 用真空冷冻干燥机冻干， 获 得纯化的 β-烟酰胺单核苷酸。
[0011] 所述的 β-烟酰胺单核苷酸的纯化方法， 其中 ， 所述步骤 a中用于微滤的微滤膜 孔径为 0.2-1 μηι。
[0012] 所述的 β-烟酰胺单核苷酸的纯化方法， 其中， 所述步骤 a中用于纳滤的纳滤膜
， 其截留分子量为 200。
[0013] 所述的 β-烟酰胺单核苷酸的纯化方法， 其中， 所述步骤 a中用于纳滤的纳滤膜 为中空纤维膜。
[0014] 所述的 β-烟酰胺单核苷酸的纯化方法， 其中， 所述步骤 a中浓缩的粗品溶液的 浓度为 20-30g/L。
[0015] 所述的 β-烟酰胺单核苷酸的纯化方法， 其中， 所述步骤 c中纯化的样品溶液用 膜浓缩设备进行纳滤后的浓度为 100~150g/L。
[0016] 所述的 β-烟酰胺单核苷酸的纯化方法， 其中， 所述步骤 b中按体积比计， 流动 相 Α: 流动相 Β为 2: 98-1： 1之间。
[0017] 所述的 β-烟酰胺单核苷酸的纯化方法， 其中， 所述步骤 c中用于纳滤的纳滤膜 为截留分子量为 200的中空纤维膜。
[0018] 所述的 β-烟酰胺单核苷酸的纯化方法， 其中， 所述步骤 b中， 梯度洗脱吋的洗 脱吋间为 40min。
发明的有益效果
有益效果
[0019] 本发明应用十八烷基硅烷键合硅胶进行高效液相制备， 对 β-烟酰胺单核苷酸进 行纯化， 使 β-烟酰胺单核苷酸的纯度可以达到 98%， 收率可以到达 80%以上， 生 产效率也比其他工艺提高了 2倍以上。 有效解决了 β-烟酰胺单核苷酸的纯化过程 中其他电荷和极性相近的类似物难以除尽的问题。
实施该发明的最佳实施例 本发明的最佳实施方式
[0020] 本发明一种 β-烟酰胺单核苷酸的纯化方法的较佳实施例， 其包括以下步骤：
[0021] S100、 将经过预处理的 β-烟酰胺单核苷酸溶液用膜浓缩设备先后进行微滤和纳 滤， 收集浓缩的粗品溶液；
[0022] S200、 将获得的粗品溶液 pH值调至 3-7， 进样上反相高效液相色谱制备柱， 固 定相为十八烷基硅烷键合硅胶， 流动相 A为盐酸溶液配成的 pH3-7的溶液， 流动 相 B为乙醇， 进行梯度洗脱纯化， 得到纯化的样品溶液；
[0023] S300、 将纯化的样品溶液用膜浓缩设备进行纳滤后， 用真空冷冻干燥机冻干， 获得纯化的 β-烟酰胺单核苷酸。
[0024] 本发明是采用反相高效液相色谱法对辅酶 I的合成底物 β-烟酰胺单核苷酸进行纯 化， 通过以固定相为十八烷基硅烷键合硅胶， 流动相为盐酸配制而成的溶液和 乙醇的色谱柱进行纯化， 再进一步浓缩及冻干， 获得纯化的 β-烟酰胺单核苷酸。 本发明所述的 β-烟酰胺单核苷酸的纯化方法操作简单， 且能有效除去其他带电荷 和极性及其相似的类似物， 使所制备出的 β-烟酰胺单核苷酸纯度高、 收率大、 产 量大， 适用于大规模工业化生产。
[0025] 优选地， 所述步骤 S100中用于微滤的微滤膜孔径为 0.2-1μηι。 具体而言， 本发 明步骤 S100中用于微滤的微滤膜孔径为 0.5μηι。
[0026] 微滤的基本原理是筛分过程， 在静压差作用下滤除大于 ΙΟμηι的微粒， 操作压 力为 0.7-7bar,原料液在压差作用下， 其中溶剂透过膜上的微孔流到膜的低压侧， 大于膜孔的微粒被截留， 从而实现原料液中的微粒与溶剂的分离。 微滤过程对 微粒的截留机理是筛分作用， 决定膜的分离效果是膜的物理结构、 孔的形状和 大小。
[0027] 本发明实施例中微滤吋采用的微滤膜孔径为 0.5μηι， 能初步清除 β-烟酰胺单核 苷酸粗品溶液中较大的微生物及大颗粒分子， 微滤膜允许大分子和溶解性固体
(无机盐） 等通过， 但会截留住悬浮物、 细菌及大分子量胶体等物质， 对 β-烟酰 胺单核苷酸粗品溶液进行初步纯化。 微滤膜的孔径过大， 会使较大微生物和大 颗粒分子透过微滤膜， 影响初步过滤的效果； 孔径过小， 会导致 β-烟酰胺单核苷 酸也无法透过微滤膜， 造成产品的损失。 [0028] 另外， 本发明所述步骤 S100中用于纳滤的纳滤膜， 其截留分子量为 200。 进一 步地， 本发明较佳实施例中采用的纳滤膜孔径为 1.5nm， 能够截留分子量为 200 的物质。
[0029] 更优选地， 所述步骤 S100中用于纳滤的纳滤膜为中空纤维膜。
[0030] 纳滤是一种允许溶剂分子或某些低分子量溶质或低价离子透过的过滤方法， 其 特点在于能在很低的压力下具有较高的除盐性能和截留分子量为数百的物质。 本发明采用孔径为 1.5nm的滤膜， 其截留分子量为 200， 可以将分子量大于 200的 物质过滤出来， 初步过滤掉 β-烟酰胺单核苷酸中的杂质。 并且， 将中空纤维膜作 为纳滤膜， 中空纤维膜外表为纤维状， 其具有自支撑作用， 可以有效清除 β-烟酰 胺单核苷酸制备工艺中所产生的磷酸根残留和其他小分子杂质， 使 β-烟酰胺单核 苷酸得到进一步纯化。
[0031] 经过微滤和纳滤后， 收集浓缩的粗品溶液， 其浓度为 20-30g/L。
[0032] 本发明较佳实施例中， 所述步骤 S200中磷酸溶液或盐酸溶液的质量浓度为 2%~
5% , 乙醇溶液的质量浓度为 S^ SOy^
[0033] 更优选地， 所述步骤 S200中磷酸溶液或盐酸溶液的体积用量为每毫升样品溶液 加 5~20毫升磷酸溶液或盐酸溶液， 乙醇溶液的体积用量为每升流动相加 100~400 毫升乙醇溶液。
[0034] 具体而言， 磷酸溶液或盐酸溶液的质量浓度过高， 会导致在梯度洗脱吋洗出， 若质量浓度过低， 会影响物质的分离纯化效果， 本发明中采用质量浓度为 2%~5 %的磷酸溶液或盐酸溶液， 可以保证其在梯度洗脱吋不被析出的情况下达到高度 纯化 β-烟酰胺单核苷酸的效果。
[0035] 另外， 改变酸的用量会影响峰形， 从而影响到检测效果。 本发明实施例中采用 体积用量为每毫升样品溶液加 5~20毫升的磷酸溶液或盐酸溶液， 体积用量为每 升流动相加 100~400毫升的乙醇溶液， 可以使峰形对称， 减少拖尾现象。
[0036] 本发明较佳实施例中， 所述步骤 S200用磷酸溶液或盐酸溶液将上一步骤所初步 纯化的 β-烟酰胺单核苷酸粗品溶液的 ρΗ值调至 3-7， 进样上反相高效液相色谱制 备柱， 其流动相 Α为盐酸溶液配成的 ρΗ3-7的溶液， 流动相 Β为乙醇。
[0037] 具体而言， 反相高效液相色谱法中一般用非极性固定相 （如 C18、 C8) ； 而其 流动相常为水或缓冲液， 常加入甲醇、 乙醇、 异丙醇、 丙酮、 四氢呋喃等与水 互溶的有机溶剂以调节保留吋间， 其适用于分离非极性和极性较弱的化合物， 并且 pH会影响样品存在的状态， 因而影响样品的保留吋间。 本发明实施例中采 用盐酸配成的 PH3-7的溶液和乙醇作为流动相， 可以有效调节样品的保留吋间， 使样品从进入色谱柱到流出色谱柱的吋间达到最优化， 能使样品的分离效果良 好。 样品的保留吋间过长， 会使检测的灵敏度降低； 样品的保留吋间过短， 会 使 β-烟酰胺单核苷酸与杂质的分离效果降低， 影响 β-烟酰胺单核苷酸的纯化。
[0038] 流动相的 pH值应该控制在 2-8之间， pH值大于 8吋， 可使载体硅胶即固定相溶 解， pH小于 2吋， 与硅胶化学键合相易水解脱落。 本发明实施例中， pH值为 3-7 ， 此吋 β-烟酰胺单核苷酸能稳定存在， 其内部结构不会被破坏， 在此条件下， 可 以使样品的分离效果达到最优化。 更优选地， 本发明中的流动相的 pH值为 5， 此 吋样品的纯化效果最佳。
[0039] 本发明所述步骤 S200中采用磷酸溶液或盐酸溶液将获得的粗品溶液 pH值调至 3- 7， 是因为 β-烟酰胺单核苷酸粗品溶液中也含有磷酸根残留， 且是采用盐酸配制 成的溶液作为流动相 Α， 本发明中采用磷酸溶液或盐酸溶液来调节粗品溶液的 pH 值， 不会引进新的杂质， 并且可以在纯化过程中除去磷酸根等物质。
[0040] 优选地， 本发明实施例中步骤 S200中， 其流动相 Α相与流动相 Β相的体积比为 2 ： 98-1： 1之间。
[0041] 流动相的比例会对样品的检测效果和纯化效果造成影响。 若提高盐酸溶液的用 量， 降低乙醇的用量， 则样品保留吋间会增长， 可以得到很好的分离， 但所测 的样品的峰变宽， 峰高降低， 影响检测的灵敏度； 而降低盐酸溶液的用量， 提 高乙醇的用量， 则样品的检测灵敏度较高， 但样品的分离效果不佳。 通过结合 本发明的样品性质和纯化条件， 选择按体积比计为 2: 98-1： 1之间的盐酸配制成 pH为 3-7的溶液与乙醇， 可以使样品在分离纯化效果良好的同吋提高样品检测的 灵敏度。
[0042] 另外， 本发明较佳实施例中， 梯度洗脱吋的洗脱吋间为 40min。 具体可进行 2%
B〜12%B梯度洗脱纯化。
[0043] 采用梯度洗脱法比等度洗脱法能使出峰效果更加对称无拖尾， 且能提高柱效， 改善检测的灵敏度。 另外将洗脱吋间控制为 40min可以使样品的保留吋间合适， β-烟酰胺单核苷酸的分离纯化效果最佳。
[0044] 本发明首次采用反相高效液相色谱法对辅酶 I的合成底物 β-烟酰胺单核苷酸进行 纯化， 克服了传统的离子交换树脂法的纯化效率低， 无法分离与 β-烟酰胺单核苷 酸极性等性质相近的类似物， 且收率低的问题。
本发明的实施方式
[0045] 下列通过具体实施例对本发明进一步阐释：
[0046] 本发明包括以下规格色谱柱 （柱子直径 *长度） ： 5 cm*30cm、 15 cm*30
cm、 30 cm*30cm。
[0047] 柱温为室温。
[0048] 实施例一：
[0049] 1.粗品浓缩：
[0050] 将经过预处理的 β-烟酰胺单核苷酸溶液用膜浓缩设备先后进行微滤和纳滤， 微 滤除掉微生物， 纳滤采用截留分子量 200的中空纤维膜将粗品浓缩至 20-30g/L。
[0051] 2.纯化：
[0052] 纯化条件：
[0053] 色谱柱： 柱子直径和长度为 5 cm*30cm；
[0054] 固定相： 十八烷基硅烷键合硅胶；
[0055] 流动相： A相： pH为 3的盐酸溶液； B相： 乙醇；
[0056] 流速： 50-80 ml/min;
[0057] 检测波长： 260 nm;
[0058] 梯度： B<¾: 2<¾〜12<¾ (40 min) ；
[0059] 上样量为 8-10g。
[0060] 纯化过程： 将浓缩后的粗品溶液用磷酸溶液或盐酸溶液调 pH至 3-7， 将色谱柱 用 30%以上的乙醇冲洗干净后平衡上样， 上样量为 8-10g， 线性梯度洗脱 40min， 收集目的峰。
[0061] 3.浓缩及冻干： [0062] 将纯化后的样品溶液用膜浓缩设备 （截留分子量 200的中空纤维膜） 纳滤浓缩 至 100-150g/L， 然后用真空冷冻干燥机冻干， 得到纯度大于 98%的 β-烟酰胺单核 苷酸， 总收率可以达到 81.3%。
[0063] 实施例二：
[0064] 1.粗品浓缩：
[0065] 将经过预处理的 β-烟酰胺单核苷酸溶液用膜浓缩设备进行微滤和纳滤， 微滤除 掉微生物， 纳滤采用截留分子量 200的中空纤维膜将粗品浓缩至 20-30g/L。
[0066] 2.纯化：
[0067] 纯化条件：
[0068] 色谱柱： 柱子直径和长度为 15 cm*30cm;
[0069] 固定相： 十八烷基硅烷键合硅胶；
[0070] 流动相： A相： pH为 5的盐酸溶液； B相： 乙醇；
[0071] 流速： 400-500 ml/min;
[0072] 检测波长： 260nm;
[0073] 梯度： B<¾: 2<¾〜12<¾ (40 min) ；
[0074] 上样量为 60-80g。
[0075] 纯化过程： 将浓缩后的粗品溶液用磷酸溶液或盐酸溶液调 pH至 3-7， 将色谱柱 用 30%以上的乙醇冲洗干净后平衡上样， 上样量为 60-80g， 线性梯度洗脱 40min ， 收集目的峰。
[0076] 3.浓缩及冻干：
[0077] 将纯化后的样品溶液用膜浓缩设备 （截留分子量 200的中空纤维膜） 纳滤浓缩 至 100-150g/L， 然后用真空冷冻干燥机冻干， 得到纯度大于 98%的 β-烟酰胺单核 苷酸， 总收率可以达到 82.3%。
[0078] 实施例三：
[0079] 1.粗品浓缩：
[0080] 将经过预处理的 β-烟酰胺单核苷酸溶液用膜浓缩设备进行微滤和纳滤， 微滤除 掉微生物， 纳滤采用截留分子量 200的中空纤维膜将粗品浓缩至 20-30g/L。
[0081] 2.纯化： [0082] 纯化条件：
[0083] 色谱柱： 柱子直径和长度为 30 cm*30cm;
[0084] 固定相： 十八烷基硅烷键合硅胶；
[0085] 流动相： A相： pH为 7的盐酸溶液； B相： 乙醇；
[0086] 流速： 2500-3000 ml/min;
[0087] 检测波长： 260 nm;
[0088] 梯度： B<¾: 2<¾〜12<¾ (40 min) ；
[0089] 上样量为 350-400g。
[0090] 纯化过程： 将浓缩后的粗品溶液用磷酸溶液或盐酸溶液调 pH至 3-7， 将色谱柱 用 30%以上的乙醇冲洗干净后平衡上样， 上样量为 350-400g。 线性梯度洗脱 40mi n， 收集目的峰。
[0091] 3.浓缩及冻干：
[0092] 将纯化后的样品溶液用膜浓缩设备 （截留分子量 200的中空纤维膜） 纳滤浓缩 至 100-150g/L， 然后用真空冷冻干燥机冻干， 得到纯度大于 98%的 β-烟酰胺单核 苷酸， 总收率可以达到 80.9%。
[0093] 本发明中通过收集样品的目的峰， 当其达到标准吋， 则将其用膜浓缩设备进行 纳滤， 再用真空冷冻干燥机冻干， 获得纯化的 β-烟酰胺单核苷酸。 由本发明所述 方法制备的冻干产品 β-烟酰胺单核苷酸， 其纯度可达 98%， 总收率可达 80%以上 ， 另外由于本发明操作简单， 其生产效率也比其它工艺提高了 2倍以上， 有效解 决了现有技术中磷酸根残留不易解决和制备出的产品纯度低、 收率低的问题。 本发明具有良好的市场前景， 适用于工业化生产纯化的 β-烟酰胺单核苷酸。
[0094] 应当理解的是， 本发明的应用不限于上述的举例， 对本领域普通技术人员来说 ， 可以根据上述说明加以改进或变换， 所有这些改进和变换都应属于本发明所 附权利要求的保护范围。

Claims

权利要求书
[权利要求 1] 一种 β-烟酰胺单核苷酸的纯化方法， 其特征在于， 包括以下步骤： a、 将经过预处理的 β-烟酰胺单核苷酸溶液用膜浓缩设备先后进行微 滤和纳滤， 收集浓缩的粗品溶液；
b、 将获得的粗品溶液 pH值调至 3-7， 进样上反相高效液相色谱制备柱 ， 固定相为十八烷基硅烷键合硅胶， 流动相 A为盐酸溶液配成的 pH3- 7的溶液， 流动相 B为乙醇， 进行梯度洗脱纯化， 得到纯化的样品溶 液；
c、 将纯化的样品溶液用膜浓缩设备进行纳滤后， 用真空冷冻干燥机 冻干， 获得纯化的 β-烟酰胺单核苷酸。
[权利要求 2] 根据权利要求 1所述的 β-烟酰胺单核苷酸的纯化方法， 其特征在于， 所述步骤 a中用于微滤的微滤膜孔径为 0.2-1μηι。
[权利要求 3] 根据权利要求 1所述的 β-烟酰胺单核苷酸的纯化方法， 其特征在于， 所述步骤 a中用于纳滤的纳滤膜， 其截留分子量为 200。
[权利要求 4] 根据权利要求 1所述的 β-烟酰胺单核苷酸的纯化方法， 其特征在于， 所述步骤 a中用于纳滤的纳滤膜为中空纤维膜。
[权利要求 5] 根据权利要求 1所述的 β-烟酰胺单核苷酸的纯化方法， 其特征在于， 所述步骤 a中浓缩的粗品溶液的浓度为 20-30g/L。
[权利要求 6] 根据权利要求 1所述的 β-烟酰胺单核苷酸的纯化方法， 其特征在于， 所述步骤 c中纯化的样品溶液用膜浓缩设备进行纳滤后的浓度为 100~1
50g/L。
[权利要求 7] 根据权利要求 1所述的 β-烟酰胺单核苷酸的纯化方法， 其特征在于， 所述步骤 b中按体积比计， 流动相 A: 流动相 B为 2: 98-1： 1之间。
[权利要求 8] 根据权利要求 1所述的 β-烟酰胺单核苷酸的纯化方法， 其特征在于， 所述步骤 c中， 用于纳滤的纳滤膜为截留分子量为 200的中空纤维膜。
[权利要求 9] 根据权利要求 1所述的 β-烟酰胺单核苷酸的纯化方法， 其特征在于， 所述步骤 b中， 梯度洗脱吋的洗脱吋间为 40min。
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