WO2020114002A1 - 一种glp-1类似多肽的纯化方法 - Google Patents

一种glp-1类似多肽的纯化方法 Download PDF

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WO2020114002A1
WO2020114002A1 PCT/CN2019/101595 CN2019101595W WO2020114002A1 WO 2020114002 A1 WO2020114002 A1 WO 2020114002A1 CN 2019101595 W CN2019101595 W CN 2019101595W WO 2020114002 A1 WO2020114002 A1 WO 2020114002A1
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column
phase
pressure
acetonitrile
elution
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French (fr)
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尹传龙
宓鹏程
曾驰
陶安进
袁建成
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深圳翰宇药业股份有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/605—Glucagons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/54—Improvements relating to the production of bulk chemicals using solvents, e.g. supercritical solvents or ionic liquids

Definitions

  • the invention relates to a method for purifying polypeptides, and specifically discloses a method for purifying GLP-1 analogous polypeptides by supercritical fluid chromatography.
  • Type II diabetes is a chronic disease characterized by defects in insulin secretion and dysfunction. According to statistics from the International Diabetes Federation (IDF), it is estimated that by 2030, 500 million people will suffer from diabetes worldwide, and my country has also become a severely affected area for diabetes.
  • IDF International Diabetes Federation
  • GLP-1 receptor agonists is a peptide substance produced by gastrointestinal endocrine cells and can promote insulin Secretion, stimulation of ⁇ -cell proliferation, inhibition of glucagon release, etc.
  • the domestic marketed GLP-1 receptor agonists mainly include exenatide and liraglutide, both of which are subcutaneous injections, somalutide (semaglutide) is one of GLP-1 receptor kinetin, developed by Novo Nordisk of Denmark.
  • somalutide somalutide
  • the clinical data of semaglutide shows that the once-weekly dose has great potential for improving the treatment of patients with type 2 diabetes due to its glycemic control, weight loss, and low-level hypoglycemic drug properties.
  • the molecular formula is as follows:
  • somalutide and other GLP-1 analogs are also basically purified by reversed-phase chromatography.
  • Organic solvents are used in large amounts, industrial sewage is large, environmental pollution is heavy, and recovery and treatment are difficult and costly. Therefore, the relatively green and environmentally friendly purification process of somalutide is a difficult point in the preparation process.
  • large-scale preparation and purification to reduce environmental pollution has become one of the bottlenecks restricting the industrialization of polypeptides, and the present invention can solve this problem.
  • acetonitrile is used as the mobile phase for gradient elution.
  • a large amount of organic wastewater solution is generated, and it contains acidic substances such as hydrochloric acid. Not only is there more organic wastewater, but it is also more difficult to process.
  • a method for purifying GLP-1 analogous peptides using supercritical fluid chromatography for purification in one step Carbon dioxide with a certain concentration, temperature and pressure is used as phase A, and acetonitrile and methanol are used as entrainers as phase B. If the pressure gradient elutes, the collected solution is spin-dried and lyophilized to obtain GLP-1 analogous polypeptide.
  • the basic principle of supercritical fluid chromatography is to use supercritical fluid as the mobile phase, solid adsorbent (such as silica gel) or organic polymer bonded to the carrier (or capillary wall) as the stationary phase chromatography method.
  • the supercritical gas is carbon dioxide, mainly because when the temperature of carbon dioxide exceeds 31.05°C and the pressure exceeds 7.38mpa, it enters the state of supercritical carbon dioxide, and the carbon dioxide is stable, non-toxic, non-flammable and inexpensive, and it is favored. Since water is not used as the mobile phase, there is no industrial wastewater. Since carbon dioxide, methanol, and acetonitrile are used, the recovery is easier and the cost is lower.
  • One aspect of the present invention provides a method for purifying GLP-1 analogous polypeptides.
  • the purification method uses supercritical chromatography; characterized in that a chromatographic column using butyl-bonded silica gel as a stationary phase, and the A1 phase in the mobile phase is carbon dioxide; Phase B is an organic solvent; the organic solvent is selected from methanol, acetonitrile, or a mixture thereof;
  • the column temperature is 30-40°C and linear elution at a certain elution pressure
  • the elution pressure is a linear elution from column temperature x 0.25 column pressure to column temperature x 0.3 column pressure, where the column pressure unit is MPa.
  • the flow rate of the mobile phase is 50 ml/min-20 L/min, preferably 60-200 ml/min.
  • the flow rate of the mobile phase can be adjusted according to the length and diameter of the chromatographic column.
  • the column pressure range is 7.50-12.0 MPa.
  • the detector is an ultraviolet detector, and the detection wavelength is 220-240 nm, preferably 230 nm.
  • the volume ratio of phase B is methanol and acetonitrile is 2-10:1.
  • cleaning and equilibration are performed before elution.
  • the cleaning and equilibration is to wash the chromatography column with 50% A+50% B, pressure 10 MPa, and balance with 70% A+30% B.
  • the chromatographic column has a diameter of 5 cm or more and a diameter of 25 cm or more.
  • the GLP-1 analogous polypeptide is a GLP-1 analogous polypeptide modified with a hydrophobic group.
  • the GLP-1 analogous polypeptide modified with a hydrophobic group is selected from the group consisting of somalutide and lirag Lutide.
  • Some peptides such as somalopeptide have long peptide chains, side chains contain long modifications, and contain amino acids that are easily isomerized during the synthesis process such as Ser, resulting in isomer impurities in the crude peptide, and the peptide sequence contains hydrophobic
  • reversed-phase chromatography uses a large amount of organic solvents to increase its elution capacity and generates a large amount of organic process wastewater.
  • the invention adopts supercritical fluid chromatography method, uses butyl bonded silica gel as the stationary phase, uses carbon dioxide as the supercritical gas, uses acetonitrile and methanol as the entraining agent, and adopts a pressure gradient elution, so that the isomers in the crude peptide can be separated at once Impurities and other difficult-to-separate impurities are well separated and removed, and effectively solve the problem of environmental pollution caused by the use of organic solvents.
  • the recycling process is easy, and the operation is simple, which is conducive to achieving large-scale preparation.
  • One aspect of the present invention provides a supercritical fluid chromatography purification method for somalutide.
  • the conditions of the supercritical fluid chromatography purification are a chromatographic column with butyl bonded silica gel as the stationary phase, and the A1 phase in the mobile phase is carbon dioxide ; Phase B is an organic solvent.
  • Flow rate 60-80ml/min.
  • Pressure gradient 8.25-9.9MPa, 60min.
  • the temperature of carbon dioxide in the mobile phase A1 of the present invention is 30-40°C, and the ratio of methanol to acetonitrile in the mobile phase B of the entrainer is 4:1.
  • the pressure range is 7.38 ⁇ 14.5MPa.
  • the pressure range is 7.50-12.0 MPa.
  • the stationary phase of the purification HPLC method of the present invention is butyl bonded silica gel.
  • the purification scale includes the following specifications of chromatographic columns: 5cm ⁇ 25cm (column diameter ⁇ length), 10cm ⁇ 25cm.
  • the invention adopts supercritical chromatography, and the carbon dioxide is stable in nature, non-toxic, non-flammable and explosive, and low in price, and is favored. Since water is not used as the mobile phase, there is no industrial wastewater. Since carbon dioxide, methanol, and acetonitrile are used, the recovery is easier and the cost is lower.
  • the method of the invention has high preparation efficiency, high purity and high yield.
  • chromatographic column a chromatographic column with butyl bonded silica gel as the stationary phase, the diameter and length of the column are: 5cm ⁇ 25cm.
  • the first step mobile phase: phase A: carbon dioxide; phase B: chromatographically pure methanol: acetonitrile (4:1). Flow rate: 60-80ml/min. Temperature 33°C, detection wavelength: 230nm. Pressure gradient: 8.25-9.9MPa, 60min.
  • the chromatographic column is washed with 50% A + 50% B, pressure 10 MPa for 10 min, and equilibrated with 70% A + 30% B for 5 min, and the sample loading is 1.5-3 g of sample solution.
  • Pressure gradient gradient elution was performed for 50-70 minutes, the target peak was collected, and the collected qualified target peptide solution was concentrated under reduced pressure by rotary evaporation at a water temperature not exceeding 32°C to about 15-50 mg/mL, and then transferred to a vial of suitable size. After freeze-drying, it can obtain somalutide with purity greater than 99.0% and meet the standard.
  • chromatographic column a chromatographic column with butyl bonded silica gel as the stationary phase, the diameter and length of the column are: 5cm ⁇ 25cm.
  • the first step mobile phase: phase A: carbon dioxide; phase B: chromatographically pure methanol nitrile: acetonitrile (9:1). Flow rate: 60-80ml/min. Temperature 38°C, detection wavelength: 230nm. Pressure gradient: 9.5-11.4MPa, 60min.
  • the chromatographic column is washed with 50% A + 50% B, pressure 10 MPa for 10 min, and equilibrated with 70% A + 30% B for 5 min, and the sample loading is 1.5-3 g of sample solution.
  • Pressure gradient gradient elution was performed for 50-70 minutes, the target peak was collected, and the collected qualified target peptide solution was concentrated under reduced pressure by rotary evaporation at a water temperature not exceeding 32°C to about 15-50 mg/mL, and then transferred to a vial of suitable size. After freeze-drying, it can obtain somalutide with purity greater than 99.0% and meet the standard.
  • chromatographic column a chromatographic column with butyl bonded silica gel as the stationary phase, the diameter and length of the column are: 5cm ⁇ 25cm.
  • the first step mobile phase: phase A: carbon dioxide; phase B: chromatographically pure methanol nitrile: acetonitrile (7:3).
  • Flow rate 60-80ml/min.
  • Temperature 37°C, detection wavelength 230nm.
  • Pressure gradient 9.25-11.1MPa, 60min.
  • the chromatographic column is washed with 50% A+50% B, pressure 10 MPa for 10 minutes, and equilibrated with 70% A+30% B for 5 minutes, and the sample volume is 1.5-3 g of sample solution.
  • Pressure gradient gradient elution was performed for 50-70 minutes, the target peak was collected, and the collected qualified target peptide solution was concentrated under reduced pressure by rotary evaporation at a water temperature not exceeding 32°C to about 15-50 mg/mL, and then transferred to a vial of suitable size. After freeze-drying, it can obtain somalutide with purity greater than 99.0% and meet the standard.
  • chromatographic column a chromatographic column with butyl bonded silica gel as the stationary phase, the diameter and length of the column are: 5cm ⁇ 25cm.
  • the first step mobile phase: phase A: carbon dioxide; phase B: chromatographically pure methanol nitrile: acetonitrile (4:1). Flow rate: 60-80ml/min. Temperature 33°C, detection wavelength: 230nm. Pressure gradient: 8.25-9.9MPa, 60min.
  • the chromatographic column is washed with 50% A + 50% B, pressure 10 MPa for 10 min, and equilibrated with 70% A + 30% B for 5 min, and the sample loading is 1.5-3 g of sample solution.
  • Pressure gradient gradient elution was performed for 50-70 minutes, the target peak was collected, and the collected qualified target peptide solution was concentrated under reduced pressure by rotary evaporation at a water temperature not exceeding 32°C to about 15-50 mg/mL, and then transferred to a vial of suitable size. After freeze-drying, it can obtain somalutide with purity greater than 99.0% and meet the standard.
  • chromatographic column a chromatographic column with butyl bonded silica gel as the stationary phase, the diameter and length of the column are: 10cm ⁇ 25cm.
  • the first step mobile phase: phase A: carbon dioxide; phase B: chromatographically pure methanol nitrile: acetonitrile (4:1). Flow rate: 200-250ml/min. Temperature 31°C, detection wavelength: 230nm. Pressure gradient: 7.75-9.3MPa, 60min.
  • chromatographic column a chromatographic column with butyl bonded silica gel as the stationary phase, the diameter and length of the column are: 10cm ⁇ 25cm.
  • the first step mobile phase: phase A: carbon dioxide; phase B: chromatographically pure methanol nitrile: acetonitrile (3:1). Flow rate: 170-230ml/min. Temperature 39°C, detection wavelength: 230nm. Pressure gradient: 9.75-11.7MPa, 60min.
  • chromatographic column a chromatographic column with butyl bonded silica gel as the stationary phase, the diameter and length of the column are: 10cm ⁇ 25cm.
  • the first step mobile phase: phase A: carbon dioxide; phase B: chromatographically pure methanol: acetonitrile (9:1). Flow rate: 170-230ml/min. Temperature 38°C, detection wavelength: 230nm. Pressure gradient: 9.5-11.4MPa, 60min.
  • Sample treatment A sample containing 3 g of crude peptide of somallutide (crude peptide: 4.6 g) was dissolved in an aqueous solution of acetonitrile, and after complete dissolution, it was filtered with a 0.22 ⁇ m filter membrane. Collect the filtered aqueous solution of the crude peptide of somallutide for future use.
  • the chromatographic conditions for the first step of HPLC purification a chromatographic column with tetraalkylsilane bonded silica packing as the stationary phase (30mm ⁇ 250mm, 10 ⁇ m); 0.2% phosphoric acid (take 1000ml water, add 2ml phosphoric acid, mix well, adjust with ammonia) pH value to 2.3) is the mobile phase A; acetonitrile is the mobile phase B; the flow rate is 20mL per minute; the detection wavelength is 230nm; the single-needle sample volume is 0.6g, and the phase B elution gradient is as follows: 10%-42% ( 55min). A fraction of a sample of somalutide with a purity greater than 95% is collected.
  • the temperature of the water bath in the rotary evaporator is 30 ⁇ 35°C,
  • Vacuum is below -0.09Mpa to remove part of acetonitrile.
  • the first-step sample solution of somallutide was obtained.
  • the second step HPLC pure chromatographic conditions chromatographic column with octaalkylsilane bonded silica packing as stationary phase (30mm ⁇ 250mm, 10 ⁇ m); 20mmol/L ammonium acetate solution (take 1000ml water plus ammonium acetate 1.54g, ammonia water) Adjust the pH to 7.5) as the mobile phase A; use acetonitrile as the mobile phase B; the flow rate is 20 mL per minute; the detection wavelength is 230 nm; and the sample load is 0.43 g.
  • the elution gradient of phase B is as follows: 5%-45% (45 min).
  • a fraction of a sample of somalopeptide with a purity greater than 99.8% is collected. Remove part of the acetonitrile with a rotary evaporator water bath at a temperature of 30 to 35°C and a vacuum of less than -0.09Mpa. The solution contained 2.20g of somalopeptide quantitatively through the reference substance, with a yield of 73.3%. This comparative example consumes a large amount of organic solvent and generates a large amount of organic wastewater.
  • Comparative example 2 Liraglutide crude peptide purification (CN105017381A)
  • Liraglutide obtained by solid phase synthesis is dissolved with dilute ammonia water with a mass percentage concentration of about 10% (dissolved concentration is about 15mg/ml), filtered with a 0.45um pore size filter membrane, and the filtrate is collected for use;
  • the first step purification conditions chromatographic column: a chromatographic column with polystyrene divinyl phenyl as the stationary phase, the diameter and length of the column are: 3cm ⁇ 25cm.
  • Mobile phase Phase A: the ratio contains the mass concentration 0.1% aqueous ammonia solution; phase B: the ratio contains 0.1% aqueous ammonia acetonitrile solution. Flow rate: 25-30ml/min. Detection wavelength: 245nm. Gradient: mass concentration of mobile phase B: 20-65%, gradient treatment time 40-55 min.
  • the injection volume is 0.8g;
  • Purification process Rinse the column with acetonitrile with a mass concentration of 90% or more and load the sample.
  • the sample volume is the sample solution after dissolution and filtration. Linear gradient elution, collect the target peak, the purity is about 92%, place the collected peptide solution in the collection bottle for use; adjust the pH value of the sample solution to neutrality with 20% bicarbonate after purification , Concentrate under reduced pressure to a certain volume of 50-100ml to remove excess acetonitrile, prepare for the second step of purification)
  • 2Second step purification conditions chromatographic column: a column with octadecylsilane-bonded silica gel as the stationary phase, the diameter and length of the column are: 3cm ⁇ 25cm, the mobile phase: the matching ratio of phase A contains a mass concentration of 0.01% Hydrochloric acid aqueous solution; phase B: chromatographically pure acetonitrile solution; flow rate: 25-30ml/min; detection wavelength: 245nm. gradient: mass concentration of mobile phase B: 40-60%, gradient processing time 45-60min; injection The amount is the sample solution with 92% content after the first step of purification and concentration;
  • the column is washed with acetonitrile with a mass percentage of 90% or more, and then the sample is loaded.
  • the sample volume is the sample solution with a content of 92% after the first step of purification and concentration. Linear gradient elution is performed to collect the target peak. The purity is About 98%, place the collected peptide solution in the collection bottle for use;
  • 3Salt transfer 100g of anion-exchange resin Lewatit MP60 is placed in a suitable size of salt transfer glass column, washed with ultra-pure water, ethanol, alkaline wash, acid wash, alkaline wash and other series of steps to neutral before loading.
  • To use pour the concentrated peptide solution into a salt-transforming glass column, and achieve the purpose of salt-transporting by controlling the flow rate of the liquid sample, while collecting the salt-transduced peptide solution; reduce all the peptide solutions obtained after salt-transforming.
  • This comparative example consumes a large amount of organic solvent and generates a large amount of organic wastewater.

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Abstract

一种GLP-1类似多肽的纯化方法，所述纯化方法采用超临界色谱法；其特征在于，以丁基键合硅胶为固定相的色谱柱，流动相中Al相为二氧化碳；B相为有机溶剂；所述有机溶剂选自甲醇、乙睛或其混合物；所述柱温度为30-40℃，并一定洗脱压力下的线性洗脱；洗脱压力为自柱温度X0.25的柱压力至柱温度X0.3的柱压力的线性洗脱，其中柱压力单位为MPa。该方法制备环保，且效率高，纯度高，收率高。

Description

一种GLP-1类似多肽的纯化方法 技术领域
本发明涉及一种多肽的纯化方法，具体公开了超临界流体色谱法纯化GLP-1类似多肽的方法。
背景技术
Ⅱ型糖尿病，是一种胰岛素分泌缺陷和作用障碍为特征的慢性疾病。根据国际糖尿病联盟(IDF)统计，预计到2030年，全球将有5亿人罹患糖尿病，我国也已成为糖尿病的重灾区。
根据肠促胰岛素的特性，目前已研发上市的药品有2大类，即GLP-1受体激动剂和DPP-4抑制剂，GLP-1是胃肠内分泌细胞产生的肽类物质，能够促进胰岛素的分泌、刺激β细胞增生，抑制胰高血糖素的释放等等，国内已上市的GLP-1受体激动剂主要有艾塞那肽和利拉鲁肽，均为皮下注射剂，索马鲁肽(semaglutide)是GLP-1的受体激动素之一，由丹麦诺和诺德公司研制的。semaglutide的临床数据显示，每周一次的剂量，由于其血糖控制、减肥、低水平的低血糖的药物属性，在改善2型糖尿病患者治疗方面，具有巨大的潜力。
分子式如下：
Figure PCTCN2019101595-appb-000001
现有索马鲁肽等GLP-1类似物纯化也基本采用反相色谱纯化，有机溶剂使用量大，工业污水多，环境污染重、回收处理难度大成本高。因此相对绿色环保的索马鲁肽纯化工艺是制备工艺中的难点，尤其大规模制备纯化减少环境污染已 成为制约多肽产业化的瓶颈之一，本发明可以解决此问题。
专利CN 105017381 A工艺中，采用乙腈作为流动相进行梯度洗脱，洗脱过程中，产生大量的有机废水溶液，并且里面含有盐酸等酸性物质，不仅有机废水较多，并且处理难度较大。
专利CN 103421092 A工艺中，采用酸性条件的醋酸铵和乙腈作为流动相进行洗脱，纯化过程中，同样产生大量的有机溶剂废水，污染较大，处理较困难。
现有多肽纯化方法基本采用反相色谱方法，需要用到大量的水、有机溶剂，进而会产生大量的工业废水，难处理、难回收。
发明内容
一种GLP-1类似多肽的纯化方法，采用超临界流体色谱进行纯化，一步即可完成。以一定浓度、温度和压力的二氧化碳为A相，以乙腈和甲醇作为夹带剂为B相，压力梯度洗脱的，收集溶液旋蒸冻干即得到GLP-1类似多肽。
超临界流体色谱法基本原理是以超临界流体作流动相，以固体吸附剂(如硅胶)或键合在载体(或毛细管壁)上的有机高分子聚合物作固定相的色谱方法，采用的超临界气体为二氧化碳，主要是因为当二氧化碳当温度超过31.05℃，压力超过7.38mpa，即进入超临界二氧化碳状态，并且二氧化碳性质稳定、无毒、不易燃爆和价格低廉而备受青睐。因未采用水作为流动相，所以没有工业废水，因采用的为二氧化碳和甲醇、乙腈，回收较容易，成本较低。
本发明一个方面提供了一种GLP-1类似多肽的纯化方法，所述纯化方法采用超临界色谱法；其特征在于，以丁基键合硅胶为固定相的色谱柱，流动相中A1相为二氧化碳；B相为有机溶剂；所述有机溶剂选自甲醇、乙腈或其混合物；
所述柱温度为30-40℃，并在一定洗脱压力下的线性洗脱；
洗脱压力为自柱温度×0.25的柱压力至柱温度×0.3的柱压力的线性洗脱，其中柱压力单位为MPa。
在本发明的技术方案中，流动相流速为50ml/min-20L/min，优选为60-200ml/min。所述的流动相流速可以根据色谱柱长度和直径调整。
在本发明的技术方案中，柱压力范围为7.50-12.0MPa。
在本发明的技术方案中，检测器为紫外检测器，检测波长为220-240nm，优选为230nm。
在本发明的技术方案中，B相为甲醇和乙腈的体积比例为2-10:1。
在本发明的技术方案中，在洗脱前进行清洗和平衡，所述清洗和平衡为将色谱柱用50％A+50％B，压力10MPa清洗，用70％A+30％B平衡。
在本发明的技术方案中，色谱柱直径为5cm以上，直径25cm以上。
在本发明的技术方案中，所述的GLP-1类似多肽为疏水基团修饰的GLP-1类似多肽，优选地，疏水基团修饰的GLP-1类似多肽选自索马鲁肽、利拉鲁肽。
索马鲁肽等多肽肽链较长，侧链含有较长的修饰，并且含有如Ser等合成过程中易异构化的氨基酸而导致粗肽中具有异构体杂质，同时肽序中含有疏水性较强的氨基酸，传统反相色谱制备大量使用有机溶剂，增加其洗脱能力，产生大量的有机工艺废水。本发明通过采用超临界流体色谱方法，以丁基键合硅胶为固定相，以二氧化碳为超临界气体，以乙腈和甲醇为夹带剂，采用压力梯度洗脱，可以一次性将粗肽中的异构体杂质和其他难分离杂质很好的分离而去除，且有效的解决了有机溶剂使用环境污染问题，回收处理容易，同时操作简便，有利于实现规模化的制备。
本发明一个方面提供了一种索马鲁肽的超临界流体色谱纯化方法，所述的超临界流体色谱纯化的条件为，以丁基键合硅胶为固定相的色谱柱，流动相中A1相为二氧化碳；B相为有机溶剂。流速：60-80ml/min。温度33℃，检测波长：230nm。压力梯度：8.25-9.9MPa，60min。
作为优选，本发明所述流动相A1二氧化碳的温度为30-40℃，夹带剂流动相B中甲醇和乙腈的比例为4:1。
本发明发现在30-40℃范围内，温度与压力呈0.25～0.3倍关系梯度洗脱时，纯化效果呈相关性，即纯化时洗脱超临界压力＝T(温度)*倍数。压力范围为7.38～14.5MPa。
作为优选，压力范围为7.50-12.0MPa。
作为优选，本发明所述纯化HPLC方法的固定相为丁基键合硅胶。纯化规模包括以下规格色谱柱：5cm×25cm(柱子直径×长度)、10cm×25cm。
有益效果
本发明采用超临界色谱法，二氧化碳性质稳定、无毒、不易燃爆和价格低廉而备受青睐。因未采用水作为流动相，所以没有工业废水，因采用的为二氧化碳和甲醇、乙腈，回收较容易，成本较低。本发明的方法制备效率高，纯度高，收 率高。
具体实施方式
实施例1：索马鲁肽粗肽纯化
将索马鲁肽粗肽2.0g用甲醇：乙腈(4:1)，含0.1％氨水溶解过滤，收集滤液备用。
1、纯化条件：色谱柱：以丁基键合硅胶为固定相的色谱柱，柱子直径和长度为：5cm×25cm。第一步：流动相：A 1相：二氧化碳；B相：色谱纯甲醇：乙腈(4：1)。流速：60-80ml/min。温度33℃，检测波长：230nm。压力梯度：8.25-9.9MPa，60min。
纯化过程：将色谱柱用50％A+50％B，压力10MPa清洗10min，用70％A+30％B平衡5min，上样量为1.5-3g样品溶液。压力线性梯度洗脱50-70min，收集目的峰，将收集的合格目的肽溶液于水温不超过32℃下减压旋蒸浓缩至约15-50mg/mL后转至合适大小西林瓶。冷冻干燥后即可得到纯度大于99.0％的符合标准索马鲁肽。
冻干后得白色粉末状固体精肽0.98g。纯度99.3％。纯化收率70％(以粗品中索马鲁肽含量计算)，总收率49％。
实施例2：索马鲁肽粗肽纯化
将索马鲁肽粗肽2.0g用甲醇：乙腈(9:1)，含0.1％氨水溶解过滤，收集滤液备用。
1、纯化条件：色谱柱：以丁基键合硅胶为固定相的色谱柱，柱子直径和长度为：5cm×25cm。第一步：流动相：A 1相：二氧化碳；B相：色谱纯甲醇腈：乙腈(9：1)。流速：60-80ml/min。温度38℃，检测波长：230nm。压力梯度：9.5-11.4MPa，60min。
纯化过程：将色谱柱用50％A+50％B，压力10MPa清洗10min，用70％A+30％B平衡5min，上样量为1.5-3g样品溶液。压力线性梯度洗脱50-70min，收集目的峰，将收集的合格目的肽溶液于水温不超过32℃下减压旋蒸浓缩至约15-50mg/mL后转至合适大小西林瓶。冷冻干燥后即可得到纯度大于99.0％的符合标准索马鲁肽。
冻干后得白色粉末状固体精肽0.96g。纯度99.17％。纯化收率68.5％(以 粗品中索马鲁肽含量计算)，总收率48％。
实施例3：索马鲁肽粗肽纯化
将索马鲁肽粗肽2.0g用甲醇：乙腈(4:1)，含0.1％氨水溶解过滤，收集滤液备用。
1、纯化条件：色谱柱：以丁基键合硅胶为固定相的色谱柱，柱子直径和长度为：5cm×25cm。第一步：流动相：A 1相：二氧化碳；B相：色谱纯甲醇腈：乙腈(7：3)。流速：60-80ml/min。温度37℃，检测波长：230nm。压力梯度：9.25-11.1MPa，60min。
纯化过程：将色谱柱用50％A+50％B,压力10MPa清洗10min，用70％A+30％B平衡5min，上样量为1.5-3g样品溶液。压力线性梯度洗脱50-70min，收集目的峰，将收集的合格目的肽溶液于水温不超过32℃下减压旋蒸浓缩至约15-50mg/mL后转至合适大小西林瓶。冷冻干燥后即可得到纯度大于99.0％的符合标准索马鲁肽。
冻干后得白色粉末状固体精肽0.98g。纯度99.57％。纯化收率70％(以粗品中索马鲁肽含量计算)，总收率49％。
实施例4：索马鲁肽粗肽纯化
将索马鲁肽粗肽2.0g用甲醇：乙腈(9:1)，含0.1％氨水溶解过滤，收集滤液备用。
1、纯化条件：色谱柱：以丁基键合硅胶为固定相的色谱柱，柱子直径和长度为：5cm×25cm。第一步：流动相：A 1相：二氧化碳；B相：色谱纯甲醇腈：乙腈(4：1)。流速：60-80ml/min。温度33℃，检测波长：230nm。压力梯度：8.25-9.9MPa，60min。
纯化过程：将色谱柱用50％A+50％B，压力10MPa清洗10min，用70％A+30％B平衡5min，上样量为1.5-3g样品溶液。压力线性梯度洗脱50-70min，收集目的峰，将收集的合格目的肽溶液于水温不超过32℃下减压旋蒸浓缩至约15-50mg/mL后转至合适大小西林瓶。冷冻干燥后即可得到纯度大于99.0％的符合标准索马鲁肽。
冻干后得白色粉末状固体精肽1.1g。纯度99.52％，单个杂质均小于0.15％。纯化收率78％(以粗品中索马鲁肽含量计算)，总收率55％。
实施例5：索马鲁肽粗肽纯化
将索马鲁肽粗肽15.0g用甲醇：乙腈(9:1)，含0.1％氨水溶解过滤，收集滤液备用。
1、纯化条件：色谱柱：以丁基键合硅胶为固定相的色谱柱，柱子直径和长度为：10cm×25cm。第一步：流动相：A 1相：二氧化碳；B相：色谱纯甲醇腈：乙腈(4：1)。流速：200-250ml/min。温度31℃，检测波长：230nm。压力梯度：7.75-9.3MPa，60min。
纯化过程：将色谱柱用50％A+50％B，压力10MPa清洗10min，用70％A+30％B平衡5min，上样量为15g样品溶液。压力线性梯度洗脱50-70min，收集目的峰，将收集的合格目的肽溶液于水温不超过32℃下减压旋蒸浓缩至约15-50mg/mL后转至合适大小西林瓶。冷冻干燥后即可得到纯度大于99.0％的符合标准索马鲁肽。
冻干后得白色粉末状固体精肽1.02g。纯度99.47％。纯化收率72.8％(以粗品中索马鲁肽含量计算)，总收率50％。
实施例6：利拉鲁肽粗肽纯化
将利拉鲁肽粗肽15.0g用甲醇：乙腈(8:2)，含0.1％氨水溶解过滤，收集滤液备用。
1、纯化条件：色谱柱：以丁基键合硅胶为固定相的色谱柱，柱子直径和长度为：10cm×25cm。第一步：流动相：A 1相：二氧化碳；B相：色谱纯甲醇腈：乙腈(3：1)。流速：170-230ml/min。温度39℃，检测波长：230nm。压力梯度：9.75-11.7MPa，60min。
纯化过程：将色谱柱用50％A+50％B，压力10MPa清洗10min，用70％A+30％B平衡5min，上样量为15g样品溶液。压力线性梯度洗脱50-70min，收集目的峰，将收集的合格目的肽溶液于水温不超过32℃下减压旋蒸浓缩至约15-50mg/mL后转至合适大小西林瓶。冷冻干燥后即可得到纯度大于99.0％的符合标准利拉鲁肽。
冻干后得白色粉末状固体精肽6.3g。纯度99.36％。纯化收率63％(以粗品中利拉鲁肽含量计算)，总收率42％。
实施例7：利拉鲁肽粗肽纯化
将利拉鲁肽粗肽15.0g用甲醇：乙腈(8:2)，含0.1％氨水溶解过滤，收集滤液备用。
1、纯化条件：色谱柱：以丁基键合硅胶为固定相的色谱柱，柱子直径和长度为：10cm×25cm。第一步：流动相：A 1相：二氧化碳；B相：色谱纯甲醇：乙腈(9：1)。流速：170-230ml/min。温度38℃，检测波长：230nm。压力梯度：9.5-11.4MPa，60min。
纯化过程：将色谱柱用50％A+50％B,压力10MPa清洗10，用70％A+30％B平衡5min，上样量为15g样品溶液。压力线性梯度洗脱50-70min，收集目的峰，将收集的合格目的肽溶液于水温不超过32℃下减压旋蒸浓缩至约15-50mg/mL后转至合适大小西林瓶。冷冻干燥后即可得到纯度大于99.0％的符合标准利拉鲁肽。
冻干后得白色粉末状固体精肽6.25g。纯度99.36％。纯化收率62.5％(以粗品中利拉鲁肽含量计算)，总收率41.6％。
对比例1：索马鲁肽粗肽纯化(201810663478.0)
样品处理：将含3g索玛鲁肽粗肽(粗肽：4.6克)样品溶于乙腈水溶液，完全溶解后用0.22μm滤膜过滤。收集过滤后的索玛鲁肽粗肽水溶液备用。
第一步HPLC纯化色谱条件：以四烷基硅烷键合硅胶填料为固定相(30mm×250mm，10μm)的色谱柱；以0.2％磷酸(取1000ml水，加2ml磷酸，混合均匀，用氨水调节pH值至2.3)为流动相A；以乙腈为流动相B；流速为20mL每分钟；检测波长为230nm；单针上样量为0.6g，B相洗脱梯度如下：10％-42％(55min)。收取纯度大于95％的索玛鲁肽样品的馏分。用旋转蒸发器水浴温度在30～35℃，
真空度在-0.09Mpa以下除去部分乙腈。得到索玛鲁肽第一步样品溶液。
第二步HPLC纯色谱条件：以八烷基硅烷键合硅胶填料为固定相(30mm×250mm，10μm)的色谱柱；以20mmol/L的乙酸铵溶液(取1000ml水加乙酸铵1.54g，氨水调节pH至7.5)为流动相A；以乙腈为流动相B；流速为20mL每分钟；检测波长为230nm；上样量为0.43g。B相洗脱梯度如下：5％-45％(45min)。
收取纯度大于99.8％的索玛鲁肽样品的馏分。用旋转蒸发器水浴温度在 30～35℃，真空度在-0.09Mpa以下除去部分乙腈。溶液经对照品定量含索玛鲁肽2.20g，收率达73.3％。本对比例需要消耗大量的有机溶剂，产生大量的有机废水。
对比例2：利拉鲁肽粗肽纯化(CN 105017381 A)
样品处理：将固相合成所得的利拉鲁肽用质量百分浓度为10％左右的稀氨水来溶解(溶解浓度约为15mg/ml)，用孔径为0.45um滤膜过滤后收集滤液备用；
①第一步纯化条件：色谱柱：以聚苯乙烯二乙烯苯基质的填料为固定相的色谱柱，柱子直径和长度为：3cm×25cm.流动相：A相：配比含有质量百分浓度为0.1％的氨水水溶液；B相：配比含有质量百分浓度为0.1％的氨水乙腈溶液。流速：25-30ml/min。检测波长：245nm。梯度：流动相B的质量百分浓度：20-65％，梯度处理时间40-55min。进样量为0.8g；
纯化过程：将色谱柱用质量百分浓度90％以上的乙腈冲洗干净后上样，上样量为溶解过滤之后的样品溶液。线性梯度洗脱，收集目的峰，纯度为92％左右，将收集好的肽溶液放置收集瓶中备用；将纯化后收集的肽溶液用20％的碳酸氢胺调样品溶液PH值至偏中性，减压浓缩至一定体积50-100ml除过多的乙腈，为了第二步纯化做准备)
②第二步纯化条件：色谱柱：以十八烷基硅烷键合硅胶为固定相的色谱柱，柱子直径和长度为：3cm×25cm,流动相：A相配比含有质量百分浓度为0.01％的盐酸水溶液；B相：色谱纯乙腈溶液；流速：25-30ml/min；检测波长：245nm.梯度：流动相B的质量百分浓度：40-60％，梯度处理时间45-60min；进样量为第一步纯化浓缩之后含量92％的样品溶液；
纯化过程：将色谱柱用质量百分浓度90％以上的乙腈冲洗干净后上样，上样量为第一步纯化浓缩之后含量92％的样品溶液，线性梯度洗脱，收集目的峰，纯度为98％左右，将收集好的肽溶液放置收集瓶中备用；
③转盐：取100g阴离子交换树脂Lewatit MP60置于大小合适的转盐玻璃柱中，用超纯水，乙醇，碱洗，酸洗，再碱洗等一系列步骤冲洗至中性后再上样使用，将浓缩后的肽溶液倒入转盐玻璃柱中，通过控制液体样品的流速来达到转盐的目的，同时收集转盐后的肽溶液；将转盐后所得的所有肽溶液，进行减压浓 缩至1g/50ml，浓缩温度不超过40℃,然后冷冻干燥得纯度大于98.0％的利拉鲁肽，纯化收率可得60％以上。本对比例需要消耗大量的有机溶剂，产生大量的有机废水。

Claims (6)

  1. 一种GLP-1类似多肽的纯化方法，所述纯化方法采用超临界色谱法；其特征在于，以丁基键合硅胶为固定相的色谱柱，流动相中A1相为二氧化碳；B相为有机溶剂；所述有机溶剂选自甲醇、乙腈或其混合物；
    所述柱温度为30-40℃，并在一定洗脱压力下的线性洗脱；
    洗脱压力为自柱温度×0.25的柱压力至柱温度×0.3的柱压力的线性洗脱，其中柱压力单位为MPa。
  2. 根据权利要求1所述的纯化方法，柱压力范围为7.50-12.0MPa。
  3. 根据权利要求1所述的纯化方法，有机溶剂包括甲醇、乙腈、异丙醇、乙醇和四氢呋喃中的一种或几种的组合，优选甲醇、乙腈，并且B相为甲醇和乙腈的体积比例为2-10:1。
  4. 根据权利要求1所述的纯化方法，在洗脱前进行清洗和平衡，所述清洗和平衡为将色谱柱用50％A+50％B，压力10MPa清洗10min，用70％A+30％B平衡5min。
  5. 根据权利要求1所述的纯化方法，色谱柱直径为5cm以上，直径25cm左右。
  6. 根据权利要求1所述的纯化方法，所述的GLP-1类似多肽为进行了疏水基团修饰的GLP-1类似多肽，优选地，疏水基团修饰的GLP-1类似多肽选自索马鲁肽、利拉鲁肽。
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