WO2013117122A1 - 一种阿托西班的纯化方法 - Google Patents

一种阿托西班的纯化方法 Download PDF

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WO2013117122A1
WO2013117122A1 PCT/CN2013/070505 CN2013070505W WO2013117122A1 WO 2013117122 A1 WO2013117122 A1 WO 2013117122A1 CN 2013070505 W CN2013070505 W CN 2013070505W WO 2013117122 A1 WO2013117122 A1 WO 2013117122A1
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atosiban
solution
perchlorate
crude peptide
purification method
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PCT/CN2013/070505
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English (en)
French (fr)
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赵忠卫
刘建
马亚平
袁建成
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深圳翰宇药业股份有限公司
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Publication of WO2013117122A1 publication Critical patent/WO2013117122A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/16—Oxytocins; Vasopressins; Related peptides

Definitions

  • the invention belongs to the technical field of medicinal chemistry, and in particular relates to a method for purifying atosiban.
  • Atosiban English name Atosiban, chemical name: 1- (3-thiol propanol) -2- (0-ethyl-D tyrosine) -4-L-threonine-8-L - Ornithine - Oxytocin.
  • Atosiban is a synthetic cyclic peptide containing three unnatural amino acids D-Tyr ( Et ), Mpa and Orn and a pair of disulfide bonds forming a ring between Mpa and Cys.
  • the molecular weight is 994.19, and its structural formula is as follows:
  • Mpa-D-Tyr(Et)-ile-Asn-Cys-P ro-Orn-G I yN Atosiban is a breakthrough product in obstetrics and gynecology drugs. It is an oxytocin analogue and is a uterus.
  • the oxytocin competitive antagonist of the endogenous aponeurosis and the receptor on the fetal membrane can directly compete with oxytocin for oxytocin receptors, inhibit the binding of oxytocin and oxytocin receptors, thereby directly inhibiting the action of oxytocin on the uterus.
  • Atosiban can quickly suppress contractions, prolong pregnancy, delay postpartum delivery, and have no adverse effects on mothers, fetuses and babies. Therefore, atosiban has a good application prospect in clinical practice and has a high development value.
  • the amino acid resin is prepared by using Boc-protected glycine (Gly) and a chloroquinone-based resin, and then the remaining protective amino acid is coupled by cyclic condensation to obtain a peptide resin, and the peptide resin is subjected to aminolysis to obtain a fully protected peptide, which is dehydrolyzed by sodium hydrogen.
  • the side chain protection group performs iodine cyclization to obtain the target peptide.
  • Another example is the cleavage of the amide bond by TEA.
  • Aminde resin is used as the starting resin, and Fmoc solid phase synthesis method is selected.
  • the purity of the atosiban peptide obtained by the above method is low, which does not meet the industrialization requirements, and the application value is not high.
  • the present invention adopts the following technical solutions:
  • a method for purifying atosiban comprising the following steps:
  • Step 1 Dissolve the atosiban crude peptide with 50 v/v% ⁇ 100 v/v% aqueous acetic acid solution, collect the filtrate by filtration, and add the ferric ion salt according to a ratio of not less than 2% by weight of the atosiban crude peptide. And then diluting the proportion of acetic acid in the crude peptide solution to 5 ( ⁇ % or less with water;
  • Step 2 The crude peptide solution obtained in the first step is separated by high performance liquid chromatography, and the octadecylsilane or octasilane-bonded silica gel is used as a stationary phase. After the crude peptide solution obtained in the step 1 is loaded, the perchlorate aqueous solution is used.
  • the mixed solution with acetonitrile is a mobile phase, and a linear gradient elution in a range of 20% to 40% by volume of acetonitrile is carried out, and the eluate is collected to obtain an atosiban perchlorate solution, wherein the perchlorate aqueous solution is a solution having a perchlorate volume ratio of pH 2.5 to pH 3.2 of O.lv/v % to 0.8 v/v %;
  • Step 3 The reverse-performance high performance liquid chromatography is used to convert the atosiban perchlorate solution obtained in the step 2 into an acetate solution.
  • the atosiban crude peptide of the present invention refers to atosiban which has not been refined by liquid phase synthesis or solid phase synthesis and other methods, or atosiban which cannot satisfy the medicinal purity.
  • the 50v/v% ⁇ 100 v/v% aqueous solution of acetic acid in step 1 of the present invention is not more than
  • the atosiban crude peptide was dissolved at a concentration of 100 g/L.
  • the concentration of the atosiban crude peptide is 100 g/L, i.e., 1 L of 50 v/v% to 100 v/v% aqueous acetic acid solution is added per 100 g of atosiban crude peptide.
  • the atosiban crude peptide is first dissolved, and then the atosiban crude peptide solution is treated with a ferric ion salt as a reducing agent to remove unoxidized impurities therein.
  • the ferric ion salt is preferably iron chloride or iron sulfate.
  • the trivalent iron ion salt of the present invention is added in an amount of not less than 2% by weight of the atosiban crude peptide, that is, 2 g or more of the ferric ion salt is added per 100 g of the atosiban crude peptide.
  • the ferric iron The ionic salt is added in an amount of 4% to 8% by weight of the atosiban crude peptide, that is, 4 to 8 g of a ferric ion salt is added per 100 g of the atosiban crude peptide.
  • the proportion of acetic acid in the crude peptide solution is diluted to 50 v/v% or less with water.
  • the ratio of acetic acid in the diluted crude peptide solution is from 30 v/v% to 40 v/v%.
  • the crude peptide solution obtained in the step 1 is separated by high performance liquid chromatography, and the octadecylsilane-bonded silica gel is used as a stationary phase.
  • the perchlorate is used. A mixed solution of an aqueous solution and acetonitrile was eluted as a mobile phase, and the eluate was collected to obtain an atosiban perchlorate solution.
  • a person skilled in the art can select a color column for different sizes (column diameter X length) according to the amount of purified atosiban crude peptide, such as a 5 cm X 25 cm color column, a column of 15 cm 25 cm size. , 30 cm x 25 cm size column or 45 cm x 25 cm size column.
  • the column needs to be rinsed before loading, preferably with 50v/v% or more of acetonitrile in water. After the column is rinsed, the atosiban crude peptide solution obtained in the step 1 is loaded. Depending on the diameter of the purified column, choose a different amount of sample.
  • a mixed solution of a perchlorate aqueous solution and acetonitrile is used as a mobile phase, and a volume ratio of the perchlorate aqueous solution to the acetonitrile is 100%.
  • a linear gradient elution in the range of 20% to 40% by volume of acetonitrile that is, a linear gradient elution in the range of 80% to 60% by volume of the perchlorate aqueous solution.
  • the perchlorate aqueous solution is an aqueous sodium perchlorate solution, an aqueous potassium perchlorate solution or an aqueous ammonium perchlorate solution.
  • the perchlorate aqueous solution is a solution having a perchlorate volume ratio of from 0.3 v/v % to 0.6 v/v % at a pH of from 2.5 to pH 3.2. More preferably, the solution having a perchlorate volume ratio of pH 3.0 is 0.5 v/v%.
  • step 2 of the purification method of the present invention further comprises the step of concentrating the prepared atosiban perchlorate solution.
  • the concentration is preferably carried out by subjecting the obtained atosiban perchlorate solution to a concentration of about 10 to 20 mg/mL under reduced pressure at a temperature not exceeding 35 °C.
  • Reversed phase high performance liquid chromatography is a liquid phase color system consisting of a non-polar stationary phase and a polar mobile phase. It is exactly the opposite of the liquid phase color system (normal phase chromatography) consisting of a polar stationary phase and a weakly polar mobile phase.
  • RP-HPLC is the most important separation mode of liquid chromatography today, almost Used for the separation of all organics that are soluble in polar or weakly polar solvents.
  • Step 3 of the purification method of the present invention The atosiban perchlorate solution obtained in the step 2 is converted into an acetate solution by reverse-phase high performance liquid chromatography.
  • the reversed-phase high performance liquid chromatography is specifically: after the octadecylsilane or octasilane-bonded silica gel is used as a stationary phase, and the atosiban perchlorate solution obtained in the step 2 is loaded, Rinse with 0.1 v/v % ⁇ 0.8 v/v % aqueous solution of ammonium acetate containing 3 v/v% to 10 v/v % acetonitrile for 15 to 30 min, and then use 30 v/v % to 60 v/v % acetonitrile. 0.05 v/v % ⁇ 0.2 v/v % of aqueous acetic acid was eluted, and the eluate was collected and concentrated to dryness.
  • the column needs to be rinsed before loading, preferably with 50v/v% or more of acetonitrile acetic acid solution. After the column is rinsed, the atosiban perchlorate solution obtained in step 2 is loaded. Depending on the diameter of the purified column, choose a different amount of sample.
  • the concentration of acetonitrile in the aqueous acetonitrile-containing aqueous solution for rinsing after the loading is 5 v/v% to 8 v/v %, more preferably 6 v/v %, and the concentration of the aqueous ammonium acetate solution is 0.3. v/v % to 0.6 v/v %, more preferably 0.4 v/v %.
  • the acetonitrile-containing ammonium acetate aqueous solution for elution has a concentration of acetonitrile of 50 v/v% and an aqueous ammonium acetate solution of 0.1 v/v%.
  • the step of concentrating the collected eluate is further included.
  • the concentration is preferably carried out by concentrating the collected eluate under reduced pressure at a temperature not exceeding 35 ° C to about 10 to 20 mg/mL.
  • the water used in the purification method of the present invention is pure water and meets the standard of water for injection, preferably ultrapure water;
  • the acetic acid used in the present invention is analytically pure glacial acetic acid;
  • the acetonitrile used in the present invention is of analytical grade.
  • Acetonitrile glacial acetic acid preferably chromatographically pure.
  • the purification method of the present invention treats the atosiban crude peptide solution with a ferric ion salt as a reducing agent, removes the unoxidized complete impurities therein, and then purifies the atosiban crude peptide solution by high performance liquid chromatography. Then, the purified atosiban perchlorate is converted to a pure atosiban by reversed-phase high performance liquid chromatography.
  • the purification method of the invention is simple and feasible to operate, and the prepared atosiban pure peptide has high purity, up to 99.5% or more, and the largest single impurity is less than 0.1%.
  • FIG. 1 is a diagram showing the high performance liquid chromatography detection of the atosiban pure peptide prepared in Example 1.
  • the embodiment of the invention discloses a purification method of atosiban. Those skilled in the art can learn from the contents of this paper and appropriately improve the process parameters. It is to be noted that all such alternatives and modifications will be apparent to those skilled in the art and are considered to be included in the present invention.
  • the method of the present invention has been described by the preferred embodiments, and it is obvious that those skilled in the art can change and adapt and combine the methods described herein to implement and apply the present technology without departing from the scope of the present invention. .
  • Example 1 In order to further understand the present invention, the present invention will be described in detail below with reference to the embodiments.
  • Example 1
  • the crude peptide was dissolved in a 50% by volume aqueous solution of acetic acid at a concentration of 100 g/L, stirred, and the sample was completely dissolved, filtered through a filter, and the filtrate was collected.
  • the ferric chloride reagent was added in a proportion of 5% by weight of the crude peptide, and the volume ratio of acetic acid in the crude peptide solution was diluted to 40% with water.
  • the column is: 5cm 25cm (diameter X length). Rinse the column with 50% or more of acetonitrile and equilibrate the sample. The loading is 1.5 ⁇ 3g.
  • a mixed solution of a perchlorate aqueous solution and acetonitrile is used as a mobile phase, wherein the mobile phase A phase is a 0.4% aqueous perchloric acid solution (v/v), and the pH is adjusted to 2.5 with a 50 mmol aqueous sodium hydroxide solution; Acetonitrile.
  • the flow rate of the mobile phase was 50 ⁇ 80 mL/min, and the mobile phase B phase was 20% ⁇ 40%.
  • the linear gradient was eluted for 60 min.
  • the eluent was collected and the collected eluate was decompressed at 35 °C. Concentrate by steaming to about 10 ⁇ 20 mg/mL.
  • the column is: 5cm X 25cm (diameter X length).
  • Rinse the color column with 50v/v% or more of acetonitrile acetic acid solution, load it, load 1.5 ⁇ 3g, rinse with 0.4 v/v% ammonium acetate aqueous solution containing 6 v/v% acetonitrile for 15 ⁇ 30min. Then, it was eluted with 0.1 v/v% aqueous acetic acid solution containing 50 v/v% acetonitrile, and the eluate was collected.
  • the crude peptide was dissolved at a concentration of 100 g/L in an 80% by volume aqueous solution of acetic acid, stirred, and the sample was completely dissolved, filtered through a filter, and the filtrate was collected.
  • the ferric sulfate reagent was added in a proportion of 4% by weight of the crude peptide, and the volume ratio of acetic acid in the crude peptide solution was diluted with water to 30% for use.
  • the column is: 15 cm X 25 cm (diameter X length). Rinse the column with 50% or more of acetonitrile and equilibrate the sample. The loading is 25 ⁇ 40g.
  • a mixed solution of a perchlorate aqueous solution and acetonitrile is used as a mobile phase, wherein the mobile phase A phase is a 0.3% aqueous perchloric acid solution (v/v), and the pH value is adjusted to 3.2 by using 50 mmol of an aqueous potassium hydroxide solution; Acetonitrile.
  • the flow rate of the mobile phase is 400 ⁇ 600mL/min, and the mobile phase B phase is 20% ⁇ 40%, linear gradient elution for 60min, the eluent is collected, and the collected eluate is depressurized at 35 °C. Evaporate to about 10 ⁇ 20 mg/mL and set aside.
  • the column is: 15cm x 25cm (diameter X length).
  • Rinse the color column with 50v/v% acetonitrile acetic acid solution, load it, load 20 ⁇ 40g, rinse with 0.1 v/v % ammonium acetate aqueous solution containing 3 v/v % acetonitrile for 15 ⁇ 30min.
  • the crude peptide was dissolved at a concentration of 100 g/L in a 90% by volume aqueous solution of acetic acid, stirred, and the sample was completely dissolved, filtered through a filter, and the filtrate was collected.
  • the ferric chloride reagent was added in an amount of 8% by weight of the crude peptide, and the volume ratio of acetic acid in the crude peptide solution was diluted with water to 40% for use.
  • the column is: 30cm x 25cm (diameter X length). Rinse the column with 50% or more of acetonitrile and equilibrate the sample. The loading is 80 ⁇ 170g.
  • a mixed solution of a perchlorate aqueous solution and acetonitrile is used as a mobile phase, wherein the mobile phase A phase is 0.8% aqueous perchloric acid (v/v), adjusted to pH 3.0 with 50 mmol of potassium hydroxide aqueous solution; mobile phase B phase was acetonitrile.
  • the flow rate of the mobile phase was 2000 ⁇ 3500mL/min, and the mobile phase B phase was 20% ⁇ 40%.
  • the linear gradient was eluted for 60min.
  • the eluent was collected and the collected eluate was depressurized at 35 °C. Evaporate to about 10 ⁇ 20 mg/mL and set aside.
  • the column is: 30cm X 25cm (diameter X length).
  • Rinse the color column with 50v/v% acetonitrile acetic acid solution, load it, load 80 ⁇ 170g, rinse with 0.8v/v% ammonium acetate aqueous solution containing 10 v/v% acetonitrile for 15 ⁇ 30min. Then, it was eluted with a 0.2 v/v% aqueous solution of acetic acid containing 60 v/v% acetonitrile, and the eluate was collected.
  • the crude peptide was dissolved in a 60% by volume aqueous solution of acetic acid at a concentration of 100 g/L, stirred, and the sample was completely dissolved, filtered through a filter, and the filtrate was collected.
  • the ferric chloride reagent was added in a proportion of 5% by weight of the crude peptide, and the volume ratio of acetic acid in the crude peptide solution was diluted with water to 30% for use.
  • the column with octadecylsilane bonded silica as the stationary phase the column is: 45cm 25cm (diameter X length). Rinse the column with 50% or more of acetonitrile and equilibrate the sample. The loading is 200 ⁇ 500g.
  • a mixed solution of a perchlorate aqueous solution and acetonitrile is used as a mobile phase, wherein the mobile phase A phase is a 0.6% aqueous perchloric acid solution (v/v), and the pH value is adjusted to 3.0 with 50 mmol of an aqueous potassium hydroxide solution; Acetonitrile.
  • the flow rate of the mobile phase was 5000 ⁇ 8500 mL/min, and the mobile phase B phase was 20% ⁇ 40%.
  • the linear gradient was eluted for 60 min.
  • the eluent was collected and the collected eluate was decompressed at 35 °C. Concentrate by steaming to about 10 ⁇ 20 mg/mL.
  • the column is: 45cm X 25 cm 3 (diameter X length).
  • Rinse the color column with 50v/v% acetonitrile acetic acid solution, load it, load 200 ⁇ 500g, rinse with 0.6v/v% ammonium acetate aqueous solution containing 8 v/v% acetonitrile for 15 ⁇ 30min. Then, it was eluted with 0.1 v/v% aqueous acetic acid solution containing 50 v/v% acetonitrile, and the eluate was collected.
  • the collected eluate was concentrated under reduced pressure at 35 ° C to about 50 to 200 mg / After mL, transfer to a suitable size vial. After lyophilization, the atosiban pure peptide was obtained, and the obtained atosiban pure peptide was determined by HPLC, and the purity was 99.7%.

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Abstract

本发明属于药物化学技术领域，公开了一种阿托西班的纯化方法。本发明所述纯化方法以三价铁离子盐作为还原剂对阿托西班粗肽溶液进行处理，除去其中的未氧化完全的杂质，之后利用高效液相色谱法纯化阿托西班粗肽溶液，然后利用反相高效液相色谱将纯化制得的阿托西班高氯酸盐转盐制得阿托西班纯品。本发明所述纯化方法操作简单可行，制得的阿托西班纯品肽纯度高，同时本发明所述方法阿托西班纯品肽收率高，一批次可获得1000g以上的阿托西班纯品肽，足以满足产业化要求。

Description

一种阿托西班的纯化方法 本申请要求于 2012 年 2 月 9 日提交中国专利局、 申请号为 201210028890.8、 发明名称为 "一种阿托西班的纯化方法"的中国专利申请的优 先权， 其全部内容通过引用结合在本申请中。 技术领域
本发明属于药物化学技术领域， 尤其涉及一种阿托西班的纯化方法。
背景技术
阿托西班， 英文名 Atosiban, 化学名称为： 1- ( 3-硫醇丙醇酸） -2- ( 0-乙 基 -D酪氨酸） -4-L-苏氨酸 -8-L-鸟氨酸-催产素。 阿托西班是人工合成的环状多 肽， 肽链中包含三个非天然氨基酸 D-Tyr ( Et ), Mpa和 Orn及一对在 Mpa与 Cys之间成环的二硫键， 分子式为
Figure imgf000002_0001
分子量为 994.19 , 其结构 式如下：
Mpa-D-Tyr(Et)-ile-Asn-Cys-P ro-Orn-G I y-N 阿托西班是妇产科药物中的一种突破产品， 它是一种催产素类似物，是子 宫内基蜕膜、胎膜上受体的催产素竟争性拮抗剂， 可以直接与催产素竟争催产 素受体， 抑制催产素和催产素受体结合， 从而直接抑制催产素作用于子宫， 抑 制子宫收缩； 也可以抑制磷脂酰肌醇的水解作用， 阻断第二信使的生成以及 Ca2+的活动， 从而间接抑制子宫对催产素的反应， 使子宫收缩得到抑制， 达 到保胎的目的。 在过去的几年， 许多临床试验已经评估其疗效和安全性。 阿托 西班能快速地抑制宫缩， 延长妊娠， 推迟不足月分娩， 而对母亲， 胎儿和婴儿 没有不良影响。 因此阿托西班在临床上有很好的应用前景， 具有很高的开发价 值。
目前， 国内外关于阿托西班合成工艺方法的报道有很多。如通过 Boc保护 的甘氨酸（Gly )和氯曱基树脂制备得到氨基酸树脂， 然后通过循环缩合偶联 其余的保护氨基酸得到肽树脂， 肽树脂经氨解后得到全保护肽，通过钠氢脱解 去侧链保护集团，进行碘环化得到目的肽。再如用 TEA裂解得到酰胺键的 Rink Aminde树脂作为起始树脂， 选用 Fmoc固相合成方法。 然而， 上述方法所得 阿托西班目的肽纯度均较低， 达不到产业化要求， 应用价值不高。
发明内容
有鉴于此， 本发明的目的在于提供一种阿托西班的纯化方法。
为实现本发明的目的， 本发明采用如下技术方案：
一种阿托西班的纯化方法， 包括以下步骤：
步骤 1、 以 50v/v% ~ 100 v/v %的醋酸水溶液溶解阿托西班粗肽， 过滤收 集滤液， 按照不小于阿托西班粗肽重量的 2%的比例加入三价铁离子盐， 然后 用水将粗肽溶液中的醋酸比例稀释到 5(^ %以下；
步骤 2、 高效液相色谱法分离步骤 1所得粗肽溶液， 以十八烷基硅烷或八 烷基硅烷键合硅胶为固定相， 步骤 1所得粗肽溶液上样后， 以高氯酸盐水溶液 和乙腈的混合溶液为流动相， 在乙腈体积比 20%〜40%范围内线性梯度洗脱， 收集洗脱液， 得到阿托西班高氯酸盐溶液， 其中所述高氯酸盐水溶液为 pH2.5〜pH3.2的高氯酸盐体积比为 O.lv/v %〜0.8v/v %的溶液；
步骤 3、 采用反相高效液相色谱法将步骤 2得到的阿托西班高氯酸盐溶液 转成醋酸盐溶液， 即得。
本发明所述阿托西班粗肽是指采用液相合成法或固相合成法以及其它方 法制得的、 未经过精制处理的阿托西班或者纯度不能满足药用的阿托西班。
优选的， 本发明步骤 1 所述 50v/v%〜100 v/v %的醋酸水溶液以不大于
100g/L 浓度溶解阿托西班粗肽。 在一个具体实施例中， 所述阿托西班粗肽的 浓度为 100g/L,即每 100g阿托西班粗肽加入 lL 50v/v%〜100 v/v %的醋酸水溶 液。
本发明所述纯化方法步骤 1先将阿托西班粗肽溶解，然后以三价铁离子盐 作为还原剂对阿托西班粗肽溶液进行处理， 除去其中的未氧化完全的杂质。 其 中所述三价铁离子盐优选为氯化铁或硫酸铁。
本发明所述三价铁离子盐的加入量不小于阿托西班粗肽重量的 2%, 即每 100g阿托西班粗肽加入 2g或 2g以上的三价铁离子盐。 优选的， 所述三价铁 离子盐的加入量为阿托西班粗肽重量的 4%〜8% ,即每 100g阿托西班粗肽加入 4〜8g三价铁离子盐。
本发明所述纯化方法，在三价铁离子盐还原剂对阿托西班粗肽溶液进行处 理后用水将粗肽溶液中的醋酸比例稀译到 50v/v%以下。 优选的， 所述稀译后 的粗肽溶液中醋酸的比例为 30v/v%〜40v/v%。
本发明所述纯化方法步骤 2利用高效液相色谱法分离步骤 1 所得粗肽溶 液， 以十八烷基硅烷键合硅胶为固定相， 步骤 1所得粗肽溶液上样后， 以高氯 酸盐水溶液和乙腈的混合溶液为流动相洗脱， 收集洗脱液，得到阿托西班高氯 酸盐溶液。
本领域技术人员可以根据纯化阿托西班粗肽的量，选择不同规格大小 (柱 子直径 X长度 ) 的色语柱纯化如 5 cm X 25 cm规格色 "普柱、 15 cm 25 cm规 格色谱柱、 30 cm x 25 cm规格色谱柱或 45 cm x 25 cm规格色谱柱。
在上样前， 还需要对色谱柱进行冲洗， 优选的， 用 50v/v%以上的乙腈水 溶液冲洗。 色谱柱冲洗后， 将步骤 1所得的阿托西班粗肽溶液上样。 根据纯化 色语柱的直径大小不同， 选择不同的上样量。
本发明所述纯化方法步骤 2 以高氯酸盐水溶液和乙腈的混合溶液为流动 相， 高氯酸盐水溶液和乙腈的体积和为 100%。在乙腈体积比 20%〜40%范围内 线性梯度洗脱， 即在高氯酸盐水溶液体积比 80%〜60%范围内线性梯度洗脱。
作为优选， 所述高氯酸盐水溶液为高氯酸钠水溶液、 高氯酸钾水溶液或高 氯酸铵水溶液。
优选的， 所述高氯酸盐水溶液为 pH2.5〜pH3.2的高氯酸盐体积比为 0.3v/v %〜0.6v/v %的溶液。 更优选为， pH3.0的高氯酸盐体积比为 0.5v/v %的溶液。
优选的，本发明所述纯化方法步骤 2还包括对制得的阿托西班高氯酸盐溶 液进行浓缩步骤。 所述浓缩优选为将制得的阿托西班高氯酸盐溶液于不超过 35 °C的条件下减压旋蒸浓缩至约 10〜20 mg/mL。
反相高效液相色 i普, 英文名 reversed phase high performance liquid chromatography, 简称， RP-HPLC , 是由非极性固定相和极性流动相所组成的 液相色语体系。它正好与由极性固定相和弱极性流动相所组成的液相色语体系 (正相色谱）相反。 RP-HPLC是当今液相色谱的最主要的分离模式， 几乎可 用于所有能溶于极性或弱极性溶剂中的有机物的分离。本发明所述纯化方法步 骤 3 采用反相高效液相色谱法将步骤 2得到的阿托西班高氯酸盐溶液转成醋 酸盐溶液。
优选的， 所述反相高效液相色谱法具体为： 以十八烷基硅烷或八烷基硅烷 键合硅胶为固定相， 步骤 2 所得阿托西班高氯酸盐溶液上样后， 先用含 3v/v%〜10 v/v %乙腈的 0.1 v/v %〜0.8 v/v %的醋酸铵水溶液冲洗 15〜30min, 然 后用含 30 v/v %〜60 v/v %乙腈的 0.05 v/v %〜0.2 v/v %的醋酸水溶液洗脱，收集 洗脱液， 浓缩干燥即得。
在上样前， 还需要对色谱柱进行冲洗， 优选的， 用 50v/v%以上的乙腈醋 酸溶液冲洗。 色谱柱冲洗后， 将步骤 2所得阿托西班高氯酸盐溶液上样。 根据 纯化色语柱的直径大小不同， 选择不同的上样量。
优选的， 所述上样后用于冲洗的含乙腈的醋酸铵水溶液中乙腈的浓度为 5v/v%〜8 v/v %,更优选为 6 v/v %,醋酸铵水溶液的浓度为 0.3 v/v %〜0.6 v/v %, 更优选为 0.4 v/v %。
优选的，所述用于洗脱的含乙腈的醋酸铵水溶液中乙腈的浓度为 50v/v %, 醋酸铵水溶液的浓度为 0.1 v/v %。
优选的，本发明所述纯化方法步骤 3所述干燥前还包括对收集的洗脱液进 行浓缩步骤。 所述浓缩优选为将收集的洗脱液于不超过 35 °C的条件下减压旋 蒸浓缩至约 10〜20 mg/mL。
本发明所述纯化方法所使用的水均为纯净水， 并符合注射用水标准，优选 为超纯水； 本发明所使用的醋酸为分析纯的冰醋酸； 本发明所使用的乙腈为分 析纯的乙腈冰醋酸， 优选为色谱纯。
本发明所述纯化方法以三价铁离子盐作为还原剂对阿托西班粗肽溶液进 行处理， 除去其中的未氧化完全的杂质，之后利用高效液相色谱法纯化阿托西 班粗肽溶液，然后利用反相高效液相色谱将纯化制得的阿托西班高氯酸盐转盐 制得阿托西班纯品。本发明所述纯化方法操作简单可行， 制得的阿托西班纯品 肽纯度高， 可达 99.5%以上， 且最大单杂小于 0.1%。 同时本发明所述方法阿 托西班纯品肽收率可达 85%以上， 总收率可达 50%以上， 一批次可获得 1000g 以上的阿托西班纯品肽， 足以满足产业化要求。 附图说明 图 1示实施例 1制得的阿托西班纯品肽的高效液相色语检测图。
具体实施方式
本发明实施例公开了一种阿托西班的纯化方法。本领域技术人员可以借鉴 本文内容， 适当改进工艺参数实现。 特别需要指出的是， 所有类似的替换和改 动对本领域技术人员来说是显而易见的， 它们都被视为包括在本发明。本发明 的方法已经通过较佳实施例进行了描述， 相关人员明显能在不脱离本发明内 容、精神和范围内对本文所述的方法进行改动或适当变更与组合， 来实现和应 用本发明技术。
为了进一步理解本发明， 下面结合实施例对本发明进行详细说明。 实施例 1 :
用体积比 50%的醋酸水溶液按照 100g/L的浓度溶解粗肽， 搅拌使样品完 全溶解后用滤膜过滤，收集滤液。按照粗肽重量的 5%的比例加入氯化铁试剂， 用水将粗肽溶液中的醋酸的体积比例稀释到 40%备用。
以十八烷基硅烷键合硅胶为固定相的色谱柱， 色谱柱为： 5cm 25cm (直 径 X长度）。 将色谱柱用 50%以上的乙腈冲洗干净后平衡上样， 上样量为 1.5〜3g。 以高氯酸盐水溶液和乙腈的混合溶液为流动相， 其中流动相 A相为 0.4%高氯酸水溶液 (v/v), 用 50mmol的氢氧化钠水溶液调 pH值 2.5; 流动相 B 相为乙腈。 流动相的流速为 50〜80 mL/min, 以流动相 B相为 20% ~ 40%, 线 性梯度洗脱 60min,收集洗脱液，将收集的洗脱液于 35 °C的条件下减压旋蒸浓 缩至约 10〜20 mg/mL后备用。
以八烷基硅烷键合硅胶为固定相的色谱柱， 色谱柱为： 5cm X 25cm (直 径 X长度）。 将色语柱用 50v/v%以上的乙腈醋酸溶液冲洗干净后上样， 上样量 1.5〜3g, 用含 6 v/v %乙腈的 0.4 v/v %的醋酸铵水溶液冲洗 15〜30min, 然后用 含 50 v/v %乙腈的 0.1 v/v%的醋酸水溶液洗脱， 收集洗脱液， 将收集的洗脱液 于 35 °C条件下减压旋蒸浓缩至约 50〜200 mg/mL后转至合适大小西林瓶。 冷 冻干燥后即得阿托西班纯品肽， HPLC检测制得的阿托西班纯品肽， 纯度为 99.7%。 实施例 2:
用体积比 80%的醋酸水溶液按照 100g/L的浓度溶解粗肽， 搅拌使样品完 全溶解后用滤膜过滤，收集滤液。按照粗肽重量的 4%的比例加入硫酸铁试剂， 用水将粗肽溶液中的醋酸的体积比例比例稀释到 30%备用。
以八烷基硅烷键合硅胶为固定相的色谱柱， 色谱柱为： 15 cm X 25cm (直 径 X长度）。 将色谱柱用 50%以上的乙腈冲洗干净后平衡上样， 上样量为 25〜40g。 以高氯酸盐水溶液和乙腈的混合溶液为流动相， 其中流动相 A相为 0.3%高氯酸水溶液 (v/v), 用 50mmol的氢氧化钾水溶液调 pH值 3.2; 流动相 B 相为乙腈。 流动相的流速为 400〜600mL/min, 以流动相 B相为 20% ~ 40%, 线 性梯度洗脱 60min,收集洗脱液，将收集的洗脱液于 35 °C的条件下减压旋蒸浓 缩至约 10〜20 mg/mL后备用。
以十八烷基硅烷键合硅胶为固定相的色谱柱， 色谱柱为： 15cm x 25cm (直 径 X长度）。 将色语柱用 50v/v%以上的乙腈醋酸溶液冲洗干净后上样， 上样量 20〜40g, 用含 3 v/v %乙腈的 0.1 v/v %的醋酸铵水溶液冲洗 15〜30min, 然后用 含 30 v/v %乙腈的 0.05v/v%的醋酸水溶液洗脱， 收集洗脱液， 将收集的洗脱液 于 35 °C条件下减压旋蒸浓缩至约 50〜200 mg/mL后转至合适大小西林瓶。 冷 冻干燥后即得阿托西班纯品肽， HPLC检测制得的阿托西班纯品肽， 纯度为 99.6%。 实施例 3:
用体积比 90%的醋酸水溶液按照 100g/L的浓度溶解粗肽， 搅拌使样品完 全溶解后用滤膜过滤，收集滤液。按照粗肽重量的 8%的比例加入氯化铁试剂， 用水将粗肽溶液中的醋酸的体积比例比例稀释到 40%备用。
以十八烷基硅烷键合硅胶为固定相的色谱柱， 色谱柱为： 30cm x 25cm (直 径 X长度）。 将色谱柱用 50%以上的乙腈冲洗干净后平衡上样， 上样量为 80〜 170g。 以高氯酸盐水溶液和乙腈的混合溶液为流动相， 其中流动相 A相为 0.8%高氯酸水溶液 (v/v), 用 50mmol的氢氧化钾水溶液调 pH值 3.0; 流动相 B 相为乙腈。 流动相的流速为 2000〜3500mL/min, 以流动相 B相为 20% ~ 40%, 线性梯度洗脱 60min, 收集洗脱液，将收集的洗脱液于 35 °C的条件下减压旋蒸 浓缩至约 10〜20 mg/mL后备用。
以八烷基硅烷键合硅胶为固定相的色谱柱， 色谱柱为： 30cm X 25cm (直 径 X长度）。 将色语柱用 50v/v%以上的乙腈醋酸溶液冲洗干净后上样， 上样量 80〜170g, 用含 10 v/v %乙腈的 0.8v/v %的醋酸铵水溶液冲洗 15〜30min, 然后 用含 60 v/v %乙腈的 0.2v/v%的醋酸水溶液洗脱， 收集洗脱液， 将收集的洗脱 液于 35 °C条件下减压旋蒸浓缩至约 50〜200 mg/mL后转至合适大小西林瓶。 冷冻干燥后即得阿托西班纯品肽， HPLC检测制得的阿托西班纯品肽， 纯度为 99.8%。 实施例 4:
用体积比 60%的醋酸水溶液按照 100g/L的浓度溶解粗肽， 搅拌使样品完 全溶解后用滤膜过滤，收集滤液。按照粗肽重量的 5%的比例加入氯化铁试剂， 用水将粗肽溶液中的醋酸的体积比例比例稀释到 30%备用。
以十八烷基硅烷键合硅胶为固定相的色谱柱， 色谱柱为： 45cm 25cm (直 径 X长度）。 将色谱柱用 50%以上的乙腈冲洗干净后平衡上样， 上样量为 200〜500g。 以高氯酸盐水溶液和乙腈的混合溶液为流动相， 其中流动相 A相 为 0.6%高氯酸水溶液 (v/v), 用 50mmol的氢氧化钾水溶液调 pH值 3.0; 流动 相 B相为乙腈。 流动相的流速为 5000〜8500 mL/min, 以流动相 B相为 20% ~ 40%, 线性梯度洗脱 60min, 收集洗脱液， 将收集的洗脱液于 35 °C的条件下减 压旋蒸浓缩至约 10〜20 mg/mL后备用。
以八烷基硅烷键合硅胶为固定相的色谱柱， 色谱柱为： 45cm X 25 cm 3 (直 径 X长度）。 将色语柱用 50v/v%以上的乙腈醋酸溶液冲洗干净后上样， 上样量 200〜500g, 用含 8 v/v %乙腈的 0.6v/v %的醋酸铵水溶液冲洗 15〜30min, 然后 用含 50 v/v %乙腈的 0.1 v/v%的醋酸水溶液洗脱， 收集洗脱液， 将收集的洗脱 液于 35 °C条件下减压旋蒸浓缩至约 50〜200 mg/mL后转至合适大小西林瓶。 冷冻干燥后即得阿托西班纯品肽， HPLC检测制得的阿托西班纯品肽， 纯度为 99.7%。 以上实施例的说明只是用于帮助理解本发明的方法及其核心思想。应当指 出， 对于本技术领域的普通技术人员来说， 在不脱离本发明原理的前提下， 还 可以对本发明进行若干改进和修饰，这些改进和修饰也落入本发明权利要求的 保护范围内。

Claims

权 利 要 求
1、 一种阿托西班的纯化方法， 其特征在于， 包括以下步骤：
步骤 1、 以 50v/v% ~ 100 v/v %的醋酸水溶液溶解阿托西班粗肽， 过滤收 集滤液， 按照不小于阿托西班粗肽重量的 2%的比例加入三价铁离子盐， 然后 用水将粗肽溶液中的醋酸比例稀释到 5(^ %以下；
步骤 2、 高效液相色谱法分离步骤 1所得粗肽溶液， 以十八烷基硅烷或八 烷基硅烷键合硅胶为固定相， 步骤 1所得粗肽溶液上样后， 以高氯酸盐水溶液 和乙腈的混合溶液为流动相， 在乙腈体积比 20%〜40%范围内线性梯度洗脱， 收集洗脱液， 得到阿托西班高氯酸盐溶液， 其中所述高氯酸盐水溶液为 pH2.5〜pH3.2的高氯酸盐体积比为 O.lv/v %〜0.8v/v %的溶液；
步骤 3、 采用反相高效液相色谱法将步骤 2得到的阿托西班高氯酸盐溶液 转成醋酸盐溶液， 即得。
2、 根据权利要求 1所述纯化方法， 其特征在于， 步骤 1所述阿托西班粗 肽的浓度为 100g/L。
3、 根据权利要求 1所述纯化方法， 其特征在于， 步骤 1所述三价铁离子 盐为氯化铁或硫酸铁。
4、 根据权利要求 1所述纯化方法， 其特征在于， 步骤 1所述三价铁离子 盐为阿托西班粗肽重量的 4%〜8%。
5、 根据权利要求 1所述纯化方法， 其特征在于， 步骤 1所述稀译后的粗 肽溶液中醋酸的比例为 30v/v%〜40v/v%。
6、 根据权利要求 1所述纯化方法， 其特征在于， 步骤 2所述高氯酸盐水 溶液为高氯酸钠水溶液、 高氯酸钾水溶液或高氯酸铵水溶液。
7、 根据权利要求 1所述纯化方法， 其特征在于， 步骤 2还包括对制得的 阿托西班高氯酸盐溶液进行浓缩步骤。
8、 根据权利要求 7所述纯化方法， 其特征在于， 所述浓缩为将制得的阿 托西班高氯酸盐溶液于不超过 35 °C的条件下减压旋蒸浓缩至约 10〜20 mg/mL。
9、 根据权利要求 1所述纯化方法， 其特征在于， 步骤 3所述反相高效液 相色谱法具体为： 以十八烷基硅烷或八烷基硅烷键合硅胶为固定相， 步骤 2 所得阿托西班高氯酸盐溶液上样后， 先用含 3 v/v%〜10 v/v%乙腈的 0.1v/v %〜0.8 v/v %的醋酸铵水溶液冲洗 15〜30min, 然后用含 30 v/v %〜60 v/v %乙腈 的 0.05 v/v %〜0.2 v/v %的醋酸水溶液洗脱， 收集洗脱液， 浓缩干燥即得。
10、 根据权利要求 1所述纯化方法， 其特征在于， 步骤 3所述干燥前还包 括对收集的洗脱液进行浓缩步骤。
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