WO2021129016A1 - 一种多肽脱盐的方法 - Google Patents

一种多肽脱盐的方法 Download PDF

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WO2021129016A1
WO2021129016A1 PCT/CN2020/118280 CN2020118280W WO2021129016A1 WO 2021129016 A1 WO2021129016 A1 WO 2021129016A1 CN 2020118280 W CN2020118280 W CN 2020118280W WO 2021129016 A1 WO2021129016 A1 WO 2021129016A1
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sample
mobile phase
salt
phase
peptide
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French (fr)
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姜绪邦
黄嘉成
尹传龙
陶安进
余品香
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深圳翰宇药业股份有限公司
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • 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
    • 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
    • C07K1/20—Partition-, reverse-phase or hydrophobic interaction chromatography
    • 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
    • C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
    • 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
    • C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/38—Flow patterns
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G01N2030/065—Preparation using different phases to separate parts of sample
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/38—Flow patterns
    • G01N2030/388—Elution in two different directions on one stationary phase

Definitions

  • the invention belongs to the technical field of medicinal chemistry and relates to a method for desalting polypeptides.
  • Polypeptides are compounds with an amphoteric structure. Some polypeptide compounds have both acidic groups and basic groups, and some polypeptide compounds have only acidic groups or only basic groups.
  • the purification process of synthetic peptides uses a variety of buffer salts, such as trifluoroacetic acid, ammonium phosphate (or sodium, potassium), ammonium sulfate (or sodium, potassium), ammonium acetate (or sodium, potassium), ammonium formate (or sodium, potassium), Potassium), sodium perchlorate (or ammonium, potassium), ammonium bicarbonate (or sodium, potassium), or ammonium chloride (or sodium, potassium), etc.
  • buffer salts such as trifluoroacetic acid, ammonium phosphate (or sodium, potassium), ammonium sulfate (or sodium, potassium), ammonium acetate (or sodium, potassium), ammonium formate (or sodium, potassium), Potassium), sodium perchlorate (or ammonium, potassium), ammonium bicarbonate
  • the excess buffer salt in the fraction purified in the previous step needs to be removed to obtain a certain salt form of the peptide, that is, the peptide binds a certain amount of acid radicals or a certain amount
  • the cation, or the free state that does not bind to any ions can make the polypeptide drug maintain higher drug activity, better stability or good solubility.
  • the salt-forming types of peptides are acetate, trifluoroacetate, hydrochloride or salt-free, and part of the peptides will be combined with cations to prepare ammonium, sodium or potassium salts.
  • a reversed-phase high-efficiency preparative chromatography system with alkyl-bonded silica gel filler as the stationary phase is generally used.
  • Most of the salt transfer operations will also use the above-mentioned purified chromatography system to avoid the increase in the replacement of the system. Additional equipment and consumables.
  • a small number of polypeptides use reversed-phase polymer fillers, or ion exchange systems, or membrane filtration for salt transfer.
  • a reversed-phase-high-efficiency preparative chromatography system using alkyl-bonded silica gel filler as a stationary phase is the preferred method, which has the advantages of simple process flow, low production cost, and the ability to further remove impurities.
  • reverse-phase polymer fillers, ion systems, and membrane filtration methods require additional equipment and consumables, and have long processing cycles, incomplete salt transfer, reduced purity, increased impurities, and other disadvantages, so reverse-phase is preferred.
  • High-efficiency preparative chromatography system for salt conversion is the preferred method, which has the advantages of simple process flow, low production cost, and the ability to further remove impurities.
  • reverse-phase polymer fillers, ion systems, and membrane filtration methods require additional equipment and consumables, and have long processing cycles, incomplete salt transfer, reduced purity, increased impurities, and other disadvantages, so reverse-phase is preferred.
  • High-efficiency preparative chromatography system for salt conversion is the preferred method, which has the advantages of simple process flow, low production cost, and the ability
  • reversed-phase high-efficiency preparative chromatography systems generally use alkyl-bonded silica gel as the stationary phase. Due to the special properties of certain peptides, the sample cannot be eluted during the desalination process with pure water and acetonitrile. , Which will cause serious loss of samples.
  • the present invention provides a method for desalting polypeptides.
  • This method can be used for salt conversion on a reversed-phase-high-efficiency preparative chromatography system with alkyl-bonded silica gel filler as the stationary phase.
  • alkyl-bonded silica gel filler as the stationary phase.
  • a method for peptide desalination The peptide sample is loaded onto the preparation column, washed with mobile phases A1 and B, then washed with mobile phases A2 and B, and finally with A2 and B for gradient elution, collect the sample, and vacuum and evaporate , Desalted refined peptide is obtained after lyophilization;
  • the mobile phase A1 is selected from pure water or dilute buffer salt solution; the mobile phase A2 is pure water or aqueous carbonate solution; the mobile phase B is selected from any one of acetonitrile, methanol, ethanol or isopropanol Or mixed in any ratio of several solvents.
  • the polypeptide desalting method of the present invention uses the same preparation column for purification and salt transfer without replacing fillers or adding additional equipment, and the desalting can be completed, and the desalting operation is simple, which is beneficial to improving production efficiency.
  • the present invention before loading the sample, it also includes the step of preparing the column with the mobile phase A1 and the mobile phase B equilibrating.
  • the polypeptide desalting method of the present invention improves the yield of the desalting step by increasing the sample load in the desalting process.
  • the loading amount of the polypeptide sample is 1.0% to 10.0% of the mass of the packing of the preparation column. Further, in some embodiments, the loading amount of the polypeptide sample is 1.5% to 6.0% of the weight of the filler.
  • the polypeptide is a peptide that is difficult to be eluted on reversed-phase preparative chromatography. Such as liraglutide and somaglutide.
  • the polypeptide sample is dissolved in the mobile phase A1 and then loaded.
  • the method of the present invention further includes the step of filtering with a filter membrane after the polypeptide is dissolved.
  • the filter membrane is a 0.45 ⁇ m filter membrane.
  • the packing in the preparation column is any one of reverse phase C18, C8, C4, C1, phenyl, amino or cyano.
  • the particle diameter of the filler in the preparation column is 5-100 ⁇ m, and the pore diameter of the filler is 5-100 nm, namely
  • the salt used in the dilute buffer salt solution is at least one of ammonium acetate, sodium acetate, potassium acetate, ammonium carbonate, ammonium bicarbonate, sodium carbonate, sodium bicarbonate, potassium carbonate or potassium bicarbonate
  • the salt concentration in the dilute buffer salt solution in the method of the present invention is 5 to 500 mmol/L. In some embodiments, the concentration of the salt is 20-200 mmol/L.
  • the polypeptide desalting method of the present invention is eluted with a carbonated aqueous solution, which solves the problem that the sample is difficult to be eluted on the reversed-phase packing, and improves the yield of the sample in the desalting process.
  • the carbon dioxide concentration of the carbonic acid aqueous solution is 5 to 500 mmol/L. In some embodiments, the carbon dioxide concentration of the carbonic acid aqueous solution is 10-30 mmol/L.
  • the ratio of the mobile phases A1 to B is 90%:10%, and the flushing time is 5-15 minutes.
  • the ratio of mobile phase A2 to B is 90%:10%, and the flushing time is 5-15 minutes.
  • the ratio gradient of the mobile phase B is 10%-60%, and the elution time is 25-35 min.
  • the polypeptide desalting method of the present invention can perform salt transfer on a reversed-phase-high-efficiency preparative chromatography system using alkyl-bonded silica gel filler as the stationary phase, and solves the problem that the sample is difficult to be eluted during desalting. There is no need to add additional equipment and materials. It can further remove impurities while transferring salt, improve sample purity, reduce sample volume after salt transfer, and increase sample concentration. The entire process is simple to operate and has a short production cycle, which is conducive to large-scale industrial production. .
  • Figure 1 shows the detection spectrum of liraglutide before desalting
  • Figure 2 shows the detection map of the liraglutide desalted refined peptide of Example 1
  • Figure 3 shows the detection map of the liraglutide desalted refined peptide of Example 2.
  • Figure 4 shows the detection chromatogram of Somaglutide before desalting
  • Figure 5 shows the detection chromatogram of Somaglutide desalted refined peptide in Example 3.
  • Figure 6 shows the detection chromatogram of Somaglutide desalted refined peptide in Example 4.
  • Figure 7 shows the detection map of the liraglutide desalted refined peptide of Comparative Example 1;
  • Figure 8 shows the detection map of the liraglutide desalted refined peptide of Comparative Example 2
  • Figure 9 shows the detection map of Somaglutide desalted refined peptide of Comparative Example 3.
  • Figure 10 shows the detection map of Somaglutide desalted refined peptide of Comparative Example 4.
  • the embodiment of the present invention discloses a method for desalting a polypeptide. Those skilled in the art can learn from the content of this article and appropriately improve the process parameters to achieve. In particular, it should be pointed out that all similar replacements and modifications are obvious to those skilled in the art, and they are all deemed to be included in the present invention.
  • the method of the present invention has been described through the preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention. .
  • the preparative column with an inner diameter of 15cm is packed with 10 ⁇ m C4 filler (filler pore size is 10nm, that is The weight is 3.0 kg), the mobile phase A1 is 200 mmol/L ammonium bicarbonate, and the mobile phase B is acetonitrile.
  • 90%A1+10%B equilibrate for 5min at 620mL/min, and then all samples are loaded, the loading amount is 1.5% of the filler weight. After loading the sample, rinse with 90% A1 + 10% B for 8 min, 620 mL/min.
  • the preparative column with an inner diameter of 15cm is packed with 10 ⁇ m C4 filler (filler pore size is 10nm, that is The weight is 3.0 kg), the mobile phase A1 is 150 mmol/L ammonium bicarbonate, and the mobile phase B is 90% ethanol + 10% isopropanol.
  • 90%A1+10%B equilibrate for 10min, 450mL/min then all samples are loaded, the loading amount is 6.0% of the filler weight. After loading the sample, rinse with 90% A1 + 10% B for 12 min, 450 mL/min.
  • the preparative column with an inner diameter of 15cm is packed with 10 ⁇ m C18 filler (filler pore diameter is 10nm, that is The weight is 3.0 kg), the mobile phase A1 is 100 mmol/L ammonium bicarbonate, and the mobile phase B is methanol.
  • 90%A1+10%B equilibrate for 10min at 550mL/min then all the samples are loaded, the loading amount is 3.3% of the filler weight. After loading the sample, rinse with 90% A1 + 10% B for 12 min, 550 mL/min.
  • the preparative column with an inner diameter of 15cm is packed with 10 ⁇ m C18 filler (filler pore diameter is 10nm, that is The weight is 3.0 kg), the mobile phase A1 is 100 mmol of ammonium bicarbonate, and the mobile phase B is 80% methanol + 20% isopropanol.
  • 90%A1+10%B equilibrate for 10min, 500mL/min then all samples are loaded, the loading amount is 5.0% of the filler weight. After loading the sample, rinse with 90% A1 + 10% B for 10 min, 500 mL/min.
  • the preparative column with an inner diameter of 15cm is filled with a 60 ⁇ m polymer filler (nanomicro UniPSN, the filler pore size is 30nm, that is The weight is 3.0kg), the sample is loaded in five times, each loading 20.0g, each loading is 0.67% of the filler weight, mobile phase A1 is 200mmol/L ammonium bicarbonate, mobile phase B is acetonitrile . 90%A1+10%B equilibrate for 5min, 620mL/min. After loading the sample, rinse with 90% A1 + 10% B for 8 min, 620 mL/min.
  • the preparative column with an inner diameter of 15cm is packed with 10 ⁇ m C4 filler (filler pore size is 10nm, that is The weight is 3.0kg), the sample is loaded in five times, 20.0g each time, the loading amount each time is 0.67% of the filler weight, the mobile phase A1 is 200mmol/L ammonium bicarbonate, and the mobile phase B is acetonitrile. 90%A1+10%B equilibrate for 5min at 620mL/min, and then all samples are loaded. After loading the sample, rinse with 90% A1 + 10% B for 8 min, 620 mL/min.
  • the preparative column with an inner diameter of 15cm is filled with a 60 ⁇ m polymer filler (nanomicro UniPSN, the filler pore size is 30nm, that is The weight is 3.0kg), the sample is loaded in four times, each loading 25.0g, each loading is 0.83% of the filler weight, mobile phase A1 is 200mmol/L ammonium bicarbonate, mobile phase B is methanol . 90%A1+10%B equilibrate for 10min, 550mL/min. After loading the sample, rinse with 90% A1 + 10% B for 10 min, 550 mL/min.
  • the preparative column with an inner diameter of 10cm is packed with 10 ⁇ m C18 packing (the pore diameter of the packing is 10nm, that is The weight is 1.2kg), all the samples are loaded, the loading amount is 12.5% of the filler weight, the mobile phase A1 is 200mmol/L ammonium bicarbonate, and the mobile phase B is acetonitrile.
  • 90%A1+10%B equilibrate for 5min at 620mL/min, and then all samples are loaded. After loading the sample, rinse with 90% A1 + 10% B for 8 min, 620 mL/min. A large number of samples were flushed out after 3 minutes of flushing. Collect the flushed samples and continue flushing with 90% Al+10% B for 8 minutes at 620 mL/min.

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Abstract

一种多肽脱盐的方法，将多肽样品上样至制备柱，以流动相A1和B冲洗，再以流动相A2和B冲洗，最后以A2和B进行梯度洗脱，收集样品，减压旋蒸，冻干后得脱盐精肽。本方法能够在以烷基键合的硅胶填料为固定相的反相-高效制备色谱系统上进行转盐，解决脱盐时样品难以被洗脱的问题，无需额外增加设备和物料，在转盐的同时能够进一步除去杂质，提高样品纯度，减少转盐后样品体积并增加样品浓度，整个过程操作简单，生产周期短，有利于大规模工业化生产。

Description

一种多肽脱盐的方法
本申请要求于2019年12月27日提交中国专利局、申请号为201911380280.2、发明名称为“一种多肽脱盐的方法”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，涉及一种多肽脱盐的方法。
背景技术
多肽是具有两性结构的化合物，有的多肽化合物中既有酸性基团，也有碱性基团，有的多肽化合物中仅有酸性基团或仅有碱性基团。合成多肽的纯化过程会使用多种缓冲盐，比如三氟乙酸、磷酸铵(或钠、钾)、硫酸铵(或钠、钾)、醋酸铵(或钠、钾)、甲酸铵(或钠、钾)、高氯酸钠(或铵、钾)、碳酸氢铵(或钠、钾)、或氯化铵(或钠、钾)等。当多肽的纯度符合要求，杂质降低至所需的限度以下，就需要将上一步纯化的馏分中多余的缓冲盐除去，以获得某种盐形式的肽，即肽结合一定量的酸根或一定量的阳离子，或不结合任何离子的游离状态，这样能够使多肽药物保持较高的药物活性、较优的稳定性或具有良好的溶解性。
通常，肽的成盐类型为醋酸盐、三氟乙酸盐、盐酸盐或无盐，部分多肽会与阳离子结合制备成铵盐、钠盐或钾盐。在合成多肽的纯化过程中，一般采用烷基键合的硅胶填料为固定相的反相-高效制备色谱系统，大多数的转盐操作也会使用上述纯化的色谱系统，避免更换系统而导致增加额外的设备和耗材。有少部分的多肽采用反相-聚合物填料，或采用离子交换系统，或采用膜过滤的方式进行转盐。上述四种转盐方式中，以烷基键合的硅胶填料为固定相的反相-高效制备色谱系统是优选的方式，其具有流程简便，生产成本低，并能够进一步除去杂质等优势。但反相-聚合物填料、离子系统和膜过滤的方式需要增加额外的设备和耗材，且具有处理周期长，转盐不彻底，纯度降低，杂质增多变大等不利因素，所以首选反相-高效制备色谱系统进行转盐。
但是，反相-高效制备色谱系统一般使用烷基键合的硅胶填料为固定相，而由于某些多肽的特殊性质，以纯水和乙腈进行脱盐的过程中会出现样品无法被洗脱的现象，这会导致样品严重损失。
发明内容
有鉴于此，本发明为了解决上述的难题，提供了一种多肽脱盐的方法，该方法能够在以烷基键合的硅胶填料为固定相的反相-高效制备色谱系统上进行转盐，解决脱盐时样品难以被洗脱的问题。
为实现本发明的目的，本发明采用如下技术方案：
一种多肽脱盐的方法，将多肽样品上样至制备柱，以流动相A1和B冲洗，再以流动相A2和B冲洗，最后以A2和B进行梯度洗脱，收集样品，减压旋蒸，冻干后得脱盐精肽；
其中所述流动相A1选自纯水或稀的缓冲盐溶液；所述流动相A2为纯水或碳酸水溶液；所述流动相B选自乙腈、甲醇、乙醇或异丙醇中的任意一种或几种溶剂的任意比例混合。
本发明所述多肽脱盐的方法纯化和转盐使用同一个制备柱，无需更换填料，也无需增加额外的设备，即可完成脱盐，且脱盐的操作简单，有利于提高生产效率。
本发明中，在上样前还包括以流动相A1和流动相B平衡制备柱的步骤。
本发明所述多肽脱盐的方法通过提高脱盐过程的上样量，提高脱盐步骤的收率。本发明中，所述多肽样品的上样量为所述制备柱填料质量的1.0％～10.0％。进一步的，在一些实施方案中，所述多肽样品的上样量为填料重量的1.5％～6.0％。
本发明中，所述多肽为在反相制备色谱上难以被洗脱的肽。如利拉鲁肽、索玛鲁肽。
本发明中，所述多肽样品用流动相A1溶解后上样。
进一步的，本发明所述方法在多肽溶解后还包括用滤膜过滤的步骤。在一些实施方案中，所述滤膜为0.45μm滤膜。
本发明中，所述制备柱内装填料为反相的C18、C8、C4、C1、苯基、氨基或氰基中的任意一种。
本发明中，所述制备柱内装填料的粒径为5～100μm，填料的孔径为5～100nm，即
Figure PCTCN2020118280-appb-000001
本发明中，所述稀的缓冲盐溶液所用的盐为醋酸铵、醋酸钠、醋酸钾、碳酸铵、碳酸氢铵、碳酸钠、碳酸氢钠、碳酸钾或碳酸氢钾中至少一种
进一步的，本发明所述方法中所述稀的缓冲盐溶液中盐的浓度为5～500mmol/L。在一些实施方案中，所述盐的浓度为20～200mmol/L。
本发明所述多肽脱盐的方法以碳酸水溶液进行洗脱，解决样品在反相填料上难以被洗脱的难题，提高样品在脱盐过程中的收率。
本发明中，所述的碳酸水溶液的二氧化碳浓度为5～500mmol/L。在一些实施方案中，所述的碳酸水溶液的二氧化碳浓度为10～30mmol/L。
本发明中，所述流动相A1和B冲洗时流动相A1与B比为90％：10％，冲洗时间为5～15min。
本发明中，所述流动相A2和B冲洗时流动相A2与B比为90％：10％，冲洗时间为5～15min。
本发明中，所述流动相A2和B梯度洗脱时流动相B比梯度为10％-60％，洗脱时间为25～35min。
与现有技术相比，本发明所述多肽脱盐的方法，能够在以烷基键合的硅胶填料为固定相的反相-高效制备色谱系统上进行转盐，解决脱盐时样品难以被洗脱的问题，无需额外增加设备和物料，在转盐的同时能够进一步除去杂质，提高样品纯度，减少转盐后样品体积并增加样品浓度，整个过程操作简单，生产周期短，有利于大规模工业化生产。
附图说明
图1示脱盐前的利拉鲁肽检测图谱；
图2示实施例1的利拉鲁肽脱盐精肽检测图谱；
图3示实施例2的利拉鲁肽脱盐精肽检测图谱；
图4示索玛鲁肽脱盐前的检测色谱图；
图5示实施例3的索玛鲁肽脱盐精肽检测色谱图；
图6示实施例4的索玛鲁肽脱盐精肽检测色谱图；
图7示对比例1的利拉鲁肽脱盐精肽检测图谱；
图8示对比例2的利拉鲁肽脱盐精肽检测图谱；
图9示对比例3的索玛鲁肽脱盐精肽检测图谱；
图10示对比例4的索玛鲁肽脱盐精肽检测图谱。
具体实施方式
本发明实施例公开了一种多肽脱盐的方法。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：利拉鲁肽脱盐精肽的制备
称取利拉鲁肽45.0g(纯度99.16％，色谱分析结果见图1)，TFA盐(TFA含量为2.9％)，以1L的200mmol/L碳酸氢铵水溶液溶解，而后用0.45μm滤膜过滤。
内径15cm的制备柱内装10μm的C4填料(填料孔径为10nm，即
Figure PCTCN2020118280-appb-000002
重量为3.0kg)，流动相A1为200mmol/L的碳酸氢铵，流动相B为乙腈。90％A1+10％B平衡5min，620mL/min，而后样品全部上样，上样量为填料重量的1.5％。上样后以90％A1+10％B冲洗8min，620mL/min。切换A1为A2，A2为纯水，90％A2+10％B冲洗，10min，620mL/min；而后梯度洗脱，B的梯度为10％-60％(25min)，分段收集馏分，以UPLC检测纯度，将纯度≥99.0％，单杂≤0.10％的样品合并。而后减压旋蒸，冻干得无盐的利拉鲁肽40.0g，无盐制备过程的收率为88.9％，冻干后的样品中未检出TFA。无盐精肽检测图见图2。
实施例2：利拉鲁肽脱盐精肽的制备
称取利拉鲁肽180.0g(纯度99.16％，色谱分析结果见图1)，TFA盐(TFA含量为2.9％)，以4L的200mmol/L碳酸氢铵水溶液溶解，而后用0.45μm滤膜过滤。
内径15cm的制备柱内装10μm的C4填料(填料孔径为10nm，即
Figure PCTCN2020118280-appb-000003
重量为3.0kg)，流动相A1为150mmol/L的碳酸氢铵，流动相B为90％乙醇+10％异丙醇。90％A1+10％B平衡10min，450mL/min，而后样品全部上样，上样量为填料重量的6.0％。上样后以90％A1+10％B冲洗12min，450mL/min。切换A1为A2，A2为30mmol/L的二氧化碳水溶液，90％A2+10％B冲洗12min，450mL/min；而后梯度洗脱，B的梯度为10％-60％(35min)，分段收集馏分，以UPLC检测纯度，将纯度≥99.0％，单杂≤0.10％的样品合并。而后减压旋蒸，冻干得无盐的利拉鲁肽173.7g，无盐制备过程的收率为96.5％，冻干后的样品中未检出TFA。无盐精肽检测图见图3。
实施例3：索玛鲁肽脱盐精肽的制备
称取索玛鲁肽100.0g(纯度99.23％，色谱分析结果见图4)，TFA盐(TFA含量为3.3％)，以2L的200mmol/L碳酸氢铵水溶液溶解，而后用0.45μm滤膜过滤。
内径15cm的制备柱内装10μm的C18填料(填料孔径为10nm，即
Figure PCTCN2020118280-appb-000004
重量为3.0kg)，流动相A1为100mmol/L的碳酸氢铵，流动相B为甲醇。90％A1+10％B平衡10min，550mL/min，而后样品全部上样，上样量为填料重量的3.3％。上样后以90％A1+10％B冲洗12min，550mL/min。切换A1为A2，A2为10mmol/L二氧化碳水溶液，90％A2+10％B冲洗12min，550mL/min；而后梯度洗脱，B的梯度为10％-60％(35min)，分段收集馏分，以UPLC检测纯度，将纯度≥99.0％，单杂≤0.10％的样品合并。而后减压旋蒸，冻干得无盐的索玛鲁肽95.3g，无盐制备过程的收率为95.3％，冻干后的样品中未检出TFA。无盐精肽检测图见图5。
实施例4：索玛鲁肽脱盐精肽的制备
称取索玛鲁肽150.0g(纯度99.23％，色谱分析结果见图4)，TFA盐(TFA含量为3.3％)，以3L的200mmol碳酸氢铵水溶液溶解，而后用0.45μm滤膜过滤。
内径15cm的制备柱内装10μm的C18填料(填料孔径为10nm，即
Figure PCTCN2020118280-appb-000005
重量为3.0kg)，流动相A1为100mmol的碳酸氢铵，流动相B为80％甲醇+20％异丙醇。90％A1+10％B平衡10min，500mL/min，而后样品全部上样，上样量为填料重量的5.0％。上样后以90％A1+10％B冲洗10min，500mL/min。切换A1为A2，A2为20mmol/L二氧化碳水溶液，90％A2+10％B冲洗10min，500mL/min；而后梯度洗脱，B的梯度为10％-60％(35min)，分段收集馏分，以UPLC检测纯度，将纯度≥99.0％，单杂≤0.10％的样品合并。而后减压旋蒸，冻干得无盐的索玛鲁肽145.5g，无盐制备过程的收率为97.0％，冻干后的样品中未检出TFA。无盐精肽检测图见图6。
对比例1：利拉鲁肽脱盐精肽的制备
称取利拉鲁肽100.0g(纯度99.16％，色谱分析结果见图1)，TFA盐TFA含量为2.9％)，以2L的200mmol/L碳酸氢铵水溶液溶解，而后用0.45μm滤膜过滤。
内径15cm的制备柱内装60μm的聚合物填料(纳微UniPSN，填料孔径30nm，即
Figure PCTCN2020118280-appb-000006
重量为3.0kg)，样品分五次上样，每次上样20.0g，每次的上样量为填料重量的0.67％，流动相A1为200mmol/L的碳酸氢铵，流动相B为乙腈。90％A1+10％B平衡5min，620mL/min。上样后以90％A1+10％B冲洗8min，620mL/min。切换A1为A2，A2为纯水，90％A2+10％B冲洗20min，620mL/min；而后梯度洗脱，B的梯度为10％-60％(25min)，分段收集馏分，以UPLC检测纯度，将纯度≥99.0％，单杂≤0.10％的样品合并。而后减压旋蒸，冻干得无盐的利拉鲁肽80.5g，无盐制备过程的收率为80.5％，冻干后的样品中未检出TFA。无盐精肽检 测图见图7。
对比例2：利拉鲁肽脱盐精肽的制备
称取利拉鲁肽100.0g(纯度99.16％，色谱分析结果见图1)，TFA盐TFA含量为2.9％)，以2L的200mmol/L碳酸氢铵水溶液溶解，而后用0.45μm滤膜过滤。
内径15cm的制备柱内装10μm的C4填料(填料孔径为10nm，即
Figure PCTCN2020118280-appb-000007
重量为3.0kg)，样品分五次上样，每次上样20.0g，每次的上样量为填料重量的0.67％，流动相A1为200mmol/L碳酸氢铵，流动相B为乙腈。90％A1+10％B平衡5min，620mL/min，而后样品全部上样。上样后以90％A1+10％B冲洗8min，620mL/min。切换A1为A2，A2为纯水，90％A2+10％B冲洗10min，620mL/min；而后梯度洗脱，B的梯度为10％-60％(25min)，分段收集馏分，以UPLC检测纯度，将纯度≥99.0％，单杂≤0.10％的样品合并。而后减压旋蒸，冻干得无盐的利拉鲁肽68.0g，无盐制备过程的收率为68.0％，冻干后的样品中未检出TFA。无盐精肽检测图见图8。
对比例3：索玛鲁肽脱盐精肽的制备
称取索玛鲁肽100.0g(纯度99.23％，色谱分析结果见图4)，TFA盐(TFA含量为3.3％)，以2L的200mmol/L碳酸氢铵水溶液溶解，而后用0.45μm滤膜过滤。
内径15cm的制备柱内装60μm的聚合物填料(纳微UniPSN，填料孔径30nm，即
Figure PCTCN2020118280-appb-000008
重量为3.0kg)，样品分四次上样，每次上样25.0g，每次的上样量为填料重量的0.83％，流动相A1为200mmol/L的碳酸氢铵，流动相B为甲醇。90％A1+10％B平衡10min，550mL/min。上样后以90％A1+10％B冲洗10min，550mL/min。切换A1为A2，A2为纯水，90％A2+10％B冲洗20min，550mL/min；而后梯度洗脱，B的梯度为10％-60％(35min)，分段收集馏分，以UPLC检测纯度，将纯度≥99.0％，单杂≤0.10％的样品合并。而后减压旋蒸，冻干得无盐的索玛鲁肽81.0g， 无盐制备过程的收率为81.0％，冻干后的样品中未检出TFA。无盐精肽检测图见图9。
对比例4：索玛鲁肽脱盐精肽的制备
称取索玛鲁肽150.0g(纯度99.23％，色谱分析结果见图4)，TFA盐(TFA含量为3.3％)，以3L的200mmol/L碳酸氢铵水溶液溶解，而后用0.45μm滤膜过滤。
内径10cm的制备柱内装10μm的C18填料(填料孔径为10nm，即
Figure PCTCN2020118280-appb-000009
重量为1.2kg)，样品全部上样，上样量为填料重量的12.5％，流动相A1为200mmol/L的碳酸氢铵，流动相B为乙腈。90％A1+10％B平衡5min，620mL/min，而后样品全部上样。上样后以90％A1+10％B冲洗8min，620mL/min。冲洗至3min时有大量样品被冲出，收集冲出的样品，继续以90％A1+10％B冲洗8min，620mL/min。切换A1为A2，A2为纯水，90％A2+10％B冲洗8min，620mL/min；而后梯度洗脱，B的梯度为10％-60％(25min)，分段收集馏分，以UPLC检测纯度，将纯度≥99.0％，单杂≤0.10％的样品合并。而后减压旋蒸，冻干得无盐的索玛鲁肽65.0g，无盐制备过程的收率为43.3％，冻干后的样品中未检出TFA。无盐精肽检测图见图10。
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 (10)

  1. 一种多肽脱盐的方法，将多肽样品上样至制备柱，以流动相A1和B冲洗，再以流动相A2和B冲洗，最后以A2和B进行梯度洗脱，收集样品，减压旋蒸，冻干后得脱盐精肽；
    其中所述流动相A1选自纯水或稀的缓冲盐溶液；所述流动相A2为纯水或碳酸水溶液；所述流动相B选自乙腈、甲醇、乙醇或异丙醇中的任意一种或几种溶剂的任意比例混合。
  2. 根据权利要求1所述的方法，在上样前还包括以流动相A1和流动相B平衡制备柱的步骤。
  3. 根据权利要求1或2所述的方法，所述多肽样品的上样量为所述制备柱填料质量的1.0％～10.0％。
  4. 根据权利要求1-3任一项所述的方法，所述多肽为在反相制备色谱上难以被洗脱的肽。
  5. 根据权利要求1-4任一项所述的方法，所述多肽样品用流动相A1溶解后上样。
  6. 根据权利要求1-5任一项所述的方法，所述制备柱内装填料为反相的C18、C8、C4、C1、苯基、氨基或氰基中的任意一种；所述制备柱内装填料的粒径为5～100μm，填料的孔径为5～100nm。
  7. 根据权利要求1-6任一项所述的方法，所述稀的缓冲盐溶液所用的盐为醋酸铵、醋酸钠、醋酸钾、碳酸铵、碳酸氢铵、碳酸钠、碳酸氢钠、碳酸钾或碳酸氢钾中至少一种；所述稀的缓冲盐溶液中盐的浓度为5～500mmol/L。
  8. 根据权利要求1-7任一项所述的方法，所述的碳酸水溶液的二氧化碳浓度为5～500mmol/L。
  9. 根据权利要求1-8任一项所述的方法，所述流动相A1和B冲洗时流动相A1与B比为90％：10％，冲洗时间为5～15min；所述流动相A2和B冲洗时流动相A2与B比为90％：10％，冲洗时间为5～15min。
  10. 根据权利要求1-9任一项所述的方法，所述流动相A2和B梯度洗脱时流动相B比梯度为10％-60％，洗脱时间为25～35min。
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CN114478750A (zh) * 2021-12-28 2022-05-13 深圳翰宇药业股份有限公司 一种特立帕肽的纯化方法
CN114478750B (zh) * 2021-12-28 2024-04-02 深圳翰宇药业股份有限公司 一种特立帕肽的纯化方法

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