WO2012152175A1 - 甘精胰岛素结晶的制备方法 - Google Patents

甘精胰岛素结晶的制备方法 Download PDF

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Publication number
WO2012152175A1
WO2012152175A1 PCT/CN2012/074392 CN2012074392W WO2012152175A1 WO 2012152175 A1 WO2012152175 A1 WO 2012152175A1 CN 2012074392 W CN2012074392 W CN 2012074392W WO 2012152175 A1 WO2012152175 A1 WO 2012152175A1
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Prior art keywords
crystallization
insulin glargine
solution
preparation
recombinant insulin
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PCT/CN2012/074392
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English (en)
French (fr)
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王大梅
李文杰
张金磊
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甘李药业股份有限公司
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Application filed by 甘李药业股份有限公司 filed Critical 甘李药业股份有限公司
Priority to US14/116,556 priority Critical patent/US9187520B2/en
Priority to EP12782610.5A priority patent/EP2708550B1/en
Publication of WO2012152175A1 publication Critical patent/WO2012152175A1/zh

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    • 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
    • C07K1/30—Extraction; Separation; Purification by precipitation
    • C07K1/306—Extraction; Separation; Purification by precipitation by crystallization
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • 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
    • C07K14/62—Insulins
    • C—CHEMISTRY; METALLURGY
    • C30—CRYSTAL GROWTH
    • C30B—SINGLE-CRYSTAL GROWTH; UNIDIRECTIONAL SOLIDIFICATION OF EUTECTIC MATERIAL OR UNIDIRECTIONAL DEMIXING OF EUTECTOID MATERIAL; REFINING BY ZONE-MELTING OF MATERIAL; PRODUCTION OF A HOMOGENEOUS POLYCRYSTALLINE MATERIAL WITH DEFINED STRUCTURE; SINGLE CRYSTALS OR HOMOGENEOUS POLYCRYSTALLINE MATERIAL WITH DEFINED STRUCTURE; AFTER-TREATMENT OF SINGLE CRYSTALS OR A HOMOGENEOUS POLYCRYSTALLINE MATERIAL WITH DEFINED STRUCTURE; APPARATUS THEREFOR
    • C30B29/00—Single crystals or homogeneous polycrystalline material with defined structure characterised by the material or by their shape
    • C30B29/54—Organic compounds
    • C30B29/58—Macromolecular compounds
    • C—CHEMISTRY; METALLURGY
    • C30—CRYSTAL GROWTH
    • C30B—SINGLE-CRYSTAL GROWTH; UNIDIRECTIONAL SOLIDIFICATION OF EUTECTIC MATERIAL OR UNIDIRECTIONAL DEMIXING OF EUTECTOID MATERIAL; REFINING BY ZONE-MELTING OF MATERIAL; PRODUCTION OF A HOMOGENEOUS POLYCRYSTALLINE MATERIAL WITH DEFINED STRUCTURE; SINGLE CRYSTALS OR HOMOGENEOUS POLYCRYSTALLINE MATERIAL WITH DEFINED STRUCTURE; AFTER-TREATMENT OF SINGLE CRYSTALS OR A HOMOGENEOUS POLYCRYSTALLINE MATERIAL WITH DEFINED STRUCTURE; APPARATUS THEREFOR
    • C30B7/00—Single-crystal growth from solutions using solvents which are liquid at normal temperature, e.g. aqueous solutions
    • C30B7/14—Single-crystal growth from solutions using solvents which are liquid at normal temperature, e.g. aqueous solutions the crystallising materials being formed by chemical reactions in the solution

Definitions

  • the present invention relates to a method for preparing a crystal of a human insulin analog, and more particularly to a method for preparing a crystal of recombinant insulin glargine (recombinant Gly ⁇ -ArgB ⁇ -ArgB 32 -human insulin) .
  • BACKGROUND OF THE INVENTION Diabetes is a common endocrine metabolic disease. In recent years, the prevalence of diabetes worldwide has grown rapidly. In China, with the changes in people's lifestyles and the acceleration of the aging process, the prevalence of diabetes has increased rapidly. In 2010, the total number of diabetic patients in China has exceeded 90 million, becoming a cardiovascular and cerebrovascular disease and tumor.
  • insulin therapy has long been regarded as an important means of treating diabetes and providing good control of blood sugar.
  • insulin analog One of the newly developed long-acting insulin analogues, insulin glargine, is increasingly being accepted and used by doctors and patients.
  • Recombinant insulin glargine (or recombinant Gly A21 -Arg B31 -Arg B32 -human insulin, or recombinant Gly A21 -human insulin-Arg B31 -Arg B31 -OH) is obtained by the asparagine at the A21 position of the human insulin A chain ( Asn) is mutated to glycine (Gly) and is obtained by adding two arginine (Arg) at the carboxy terminus of the B chain (see U.S. Patent No. 5,567,722).
  • Recombinant insulin glargine can simulate the physiological basal insulin secretion, and has no peak. It has almost no risk of hypoglycemia while controlling blood sugar, and it lasts for about 24 hours.
  • the recombinant insulin glargine is mostly present in the form of a solution or an amorphous dry powder without forming crystals.
  • the crystalline form of recombinant insulin glargine has a uniform stable solid molecular form, and has a small sedimentation volume, is easy to separate from the supernatant, has a short centrifugation time and freeze-drying time, and has high production efficiency, and is a better recombinant insulin glargine.
  • Application form is a uniform stable solid molecular form, and has a small sedimentation volume, is easy to separate from the supernatant, has a short centrifugation time and freeze-drying time, and has high production efficiency, and is a better recombinant insulin glargine.
  • Recombinant insulin glargine has two basic amino acids (Arg B31 -Arg B32 ) at the end of its B chain, resulting in its isoelectric point being more natural. Human insulin is high so that it can exist only in an amorphous state under the conditions of crystallization of existing insulin, and cannot form stable hexahedral crystals. Disclosure of the Invention An object of the present invention is to solve the above problems and to provide a method for preparing a recombinant insulin glargine crystal which can prepare a high quality and high yield of insulin glargine-zinc crystal on a large scale.
  • the present invention provides a method for preparing a recombinant insulin glargine crystal, the method comprising: crystallization from a recombinant insulin glargine, an organic solvent, a zinc compound, a phenol derivative, a salt, and an organic acid In the solution, the recombinant insulin glargine is crystallized under the conditions of pH 7.0-9.0, wherein the organic solvent has a volume percent concentration of 10-30%.
  • Recombinant insulin glargine is more difficult to crystallize because its isoelectric point is different from other insulins.
  • the addition of organic solution and the adjustment of pH during crystallization are the key factors affecting the crystallization effect.
  • the organic solvent described in the present invention is one of the key factors for the formation of crystals of recombinant insulin glargine.
  • Other insulins can be crystallized in the absence of an organic solvent, but recombinant insulin glargine can only be a flocculent amorphous precipitate in the absence of an organic solvent and cannot form crystals.
  • the inventors have found through extensive experimental research that recombinant insulin glargine can begin to form crystals only when an organic solvent having a volume percentage of 10-30% is present in the crystallization liquid. This may be due to the fact that the organic solvent in the crystallization solution can reduce the solubility of the recombinant insulin glargine: (1) When an organic solvent is present in the crystallization solution, the electrolytic constant of the crystallization liquid can be lowered, and the molecular weight of the insulin glargine is increased. The mutual attraction between the charges leads to a decrease in the solubility of insulin glargine.
  • the interaction between the organic solvent and water can destroy the hydrated membrane of insulin glargine and also reduce the solubility of insulin glargine.
  • the organic solvent reduces the solubility of the recombinant insulin glargine in the crystallization liquid and promotes the formation of crystallization of insulin glargine. Therefore, the volume percentage of the organic solvent in the crystallization liquid is not less than 10% (vol/vol), otherwise it is difficult to achieve the above effects.
  • the concentration of the organic solvent is too high, the protein denaturation is easily deactivated, so the volume concentration of the organic solvent in the crystallization liquid should not exceed 30% (vol/vol).
  • the organic solvent may be any water-soluble organic solvent known to those skilled in the art such as acetonitrile, ethanol, n-propanol or isopropanol.
  • the preferred organic solvents are ethanol.
  • the crystallization pH of recombinant insulin glargine in other insulin eg pH6
  • it is only slightly turbid it is not enough to start the crystallization process. Only when the pH jumps over 7, the crystallization process can begin.
  • the conditions for crystallization in the present invention are pH 7.0-9.0, and at this pH, other insulins have been substantially clarified. crystallization.
  • the inventors have found that if the initial pH of the crystallization solution is adjusted to pH 8.0-9.0 during the crystallization of recombinant insulin glargine, the pH during crystallization is adjusted to pH 7.0-8.0, which can form A crystal having a larger particle size, a more regular structure, and a more uniform crystal.
  • the pH of the crystallizing liquid of the present invention can be adjusted by a method known to a person skilled in the art using a base or the like, and it is preferably adjusted using sodium hydroxide, potassium hydroxide or ammonium hydroxide (concentrated aqueous ammonia).
  • the concentration of recombinant insulin glargine in the crystallization liquid is 2-4 g/L, more preferably 2.8-3.2 g/L. If the concentration of insulin glargine is too low, it will increase the volume of the crystallization solution, which is not conducive to large-scale production. If the concentration is too high and exceeds the solubility of insulin glargine, the solution will be turbid.
  • the zinc compound is added in the form of a zinc salt selected from zinc salts capable of dissociating zinc ions in water.
  • a zinc salt selected from zinc salts capable of dissociating zinc ions in water.
  • representative zinc salts are zinc chloride, zinc oxide, zinc acetate, and bromine. Zinc, zinc sulfate, etc.
  • the concentration of zinc in the crystallization solution of the present invention is preferably from 20 to 200 mg/L. More preferred zinc in the present invention is zinc chloride; the concentration is 70-100 mg/L. Since the zinc compound participates in the crystallization process of insulin glargine, if the concentration of the zinc compound is too low, the crystal of insulin glargine is incomplete.
  • the phenol derivative refers to a substance capable of providing a phenolic hydroxyl group, which is selected from the group consisting of phenol, m-cresol or methylparaben or a mixture thereof; the concentration of the phenol derivative in the crystallization solution is 0.05-0.5 g/ 100ml (ie 0.05-0.5%, mass/volume).
  • a more preferred phenol derivative is phenol; a concentration of 0.1-0.2 g/100 ml (ie 0.1-0.2%, Quality / volume).
  • the phenol derivative is also involved in the crystallization of insulin glargine.
  • the salt refers to a strong electrolyte salt which is soluble in water and is selected from sodium chloride, sodium acetate or sodium citrate; and the concentration of the salt in the crystallization liquid is 0.1-0.5M.
  • a more preferred salt is sodium chloride; the concentration is from 0.3 to 0.4M.
  • the organic acid refers to a short-chain carboxylic acid which is easily soluble in water, and is selected from acetic acid, citric acid or glycine; the organic acid concentration in the crystallization process is too high, which may cause excessive heat release during crystallization and affect product stability. If the concentration is too low, the recombinant insulin glargine will not dissolve well, so the concentration of the organic acid in the crystallization solution is preferably 0.3-0.8M.
  • a more preferred organic acid is acetic acid; the concentration is from 0.4 to 0.6M.
  • the temperature of the crystallization is not critical and an acceptable temperature range is from about 1 to 30 °C.
  • the preferred initial temperature of the crystallization liquid is room temperature (10-30 ° C), which can increase the solubility of the recombinant insulin glargine before crystallization; the preferred temperature during crystallization is 2-8 ° C, which can promote recombinant glycerin The decrease in solubility of insulin at low temperatures accelerates the rate of crystallization.
  • the present invention uses the following steps to crystallize the recombinant insulin glargine: (1) preparing a crystallization liquid containing the above components and contents at room temperature, and adjusting the initial pH of the crystallization liquid to 8.0-8.5; The crystallization solution was cooled to 2-8 ° C and the pH was adjusted to 7.1-7.8 and then crystallized for at least 3 hours.
  • the manner in which the recombinant insulin glargine is dissolved in the crystallization liquid, and the order in which the components in the crystallization liquid are added, are also not critical to the present invention.
  • an organic solvent, zinc, a phenol derivative, an organic acid, and a salt may be separately added to the recombinant insulin glargine solution, or a zinc, a phenol derivative, an organic acid, and a salt may be first formulated into a solution, and then added to include a recombination.
  • a recrystallization process may be selected, and in order to reduce the content of other solvents in the crystallization, a method of washing and crystallization may also be employed.
  • the recombinant insulin glargine of the present invention can be obtained commercially, or can be prepared by any recognized peptide synthesis technique, such as solution method or solid phase synthesis method (J. Stewart et al., "Solid Phase Peptide Synthesis", Freeman and Co ., San Francisco, 1969), semi-synthetic method, genetic engineering method, DNA recombination method (US Patent US5656722) and the like.
  • the preparation method of the recombinant insulin glargine crystal of the invention has the following advantages: The uniform stable recombinant insulin hexahedral crystal can be crystallized, the crystal has high transparency, small sedimentation volume, short centrifugal drying time and high production efficiency.
  • FIG. 1 is a magnified view of a microscope at a magnification of 160 times after crystallization of recombinant insulin glargine of Example 1.
  • FIG. 2 is a magnified view of a microscope at a magnification of 160 times after crystallization of recombinant insulin glargine of Example 2.
  • FIG. 4 shows a magnification of 160 times under the microscope after the crystallization of the recombinant insulin glargine of Example 4;
  • FIG. 5 shows the recombination of Example 5.
  • FIG. 6 shows a magnified image of 160 times after crystallization of the recombinant insulin glargine of Example 6
  • Figure 7 shows the crystallization of the recombinant insulin glargine after the crystallization of Comparative Example 1
  • Fig. 8 shows a magnification of 160 in the microscope after the crystallization of recombinant insulin glargine in Fig. 2
  • Fig. 8 shows a magnification of 160 in the microscope after the crystallization of recombinant insulin glargine in Fig. 2; Fig.
  • FIG. 9 shows a comparison of the first recombinant insulin glargine after crystallization in the microscope
  • Figure 10 shows a magnified view of the second recombined insulin glargine in Comparative Example 3 after crystallization at 160 times
  • Figure 11 shows the third recombination of insulin glargine after crystallization in the microscope 160 times magnification DETAILED DESCRIPTION OF THE INVENTION Material insulin glargine: Provided by Gan Li Pharmaceutical Co., Ltd., batch number GLGB09001.
  • Zinc chloride was purchased from Beijing Andy Yongfu, batch number 71770; phenol was purchased from Jiangmen Hengjian Pharmaceutical Co., Ltd., batch number 081001; acetic acid was purchased from Taishan Xinning Pharmaceutical Co., Ltd., batch number 20081007; sodium chloride was purchased from Beijing Yanjing Pharmaceutical, Lot No. 080522; Concentrated ammonia water was purchased from Beijing Huateng, batch number 20080513; Ethanol was purchased from Tianjin Komiou Chemical Reagent Co., Ltd.
  • Example 1 A recombinant insulin crystallization solution was prepared, and the components and concentrations contained in the crystallization solution were: recombinant insulin glargine: 3.5 g/L, acetonitrile: 20%, acetic acid: 0.7 M, phenol: 0.4%, chlorine Zinc: 120 mg/L, sodium acetate: 0.5M.
  • the initial temperature of the above crystallization liquid was adjusted to 20-25 ° C, pH 7.8-8.2 was adjusted with ammonium hydroxide, stirred for 10 minutes until the solution was slightly mixed, and left to stand at 2-8 ° C for about 3 hours. Then, the supernatant was taken for HPLC detection, and the content of recombinant insulin glargine in the supernatant was 0.05 mg/ml, which was in compliance with the requirements. After crystallization of the suspension, the microscopic examination showed that the apparent hexahedral crystals were magnified 160 times and the transparency was high (see Figure 1).
  • Example 2 A recombinant crystallization of insulin glargine was prepared, and the components and concentrations contained in the crystallization solution were: recombinant insulin glargine: 3 g/L, ethanol: 15%, acetic acid: 0.5 M, zinc chloride: 90 mg/L , phenol: 0.2%, sodium chloride: 0.3M.
  • the pH of the acetic acid was adjusted to 7.1-7.6, stirred for about 10 minutes, and allowed to stand at 2-8 ° C for 10 hours.
  • the supernatant was then taken for HPLC detection, and the content of recombinant insulin glargine in the supernatant was as low as 0.02 mg/ml, which was satisfactory.
  • Pour out about 77L of supernatant put about 7L of the remaining suspension into a 1L centrifuge bottle, centrifuge with a centrifuge at 4000 rpm for 10 minutes, and remove the crystal precipitate from all the centrifuge bottles at a concentration of 20g/L.
  • Example 3 A recombinant insulin crystallization solution was prepared in the same manner as in Example 1, and the components and concentrations contained in the crystallization solution were: recombinant insulin glargine: 2 g/L, n-propanol: 10%, acetic acid: 0.3 M, m-cresol: 0.05%, zinc oxide: 20 mg/L, sodium acetate: 0.1 M.
  • the initial temperature of the above crystallization liquid was adjusted to 4 ° C, the pH was adjusted to 7.5 - 8.5 with ammonium hydroxide, and the crystal was allowed to stand at 4 ° C for about 10 hours.
  • the supernatant was taken for HPLC detection, and the content of recombinant insulin glargine in the supernatant was 0.05 mg/ml, which was in compliance with the requirements.
  • microscopic examination magnified 160 times, visible hexahedral crystals, high transparency (see figure
  • Example 4 A recombinant insulin crystallization solution was prepared in the same manner as in Example 1, and the components and concentrations contained in the crystallization solution were: recombinant insulin glargine: 4 g/L, methanol: 30%, citric acid: 0.8 M, hydroxybenzoic acid formazan: 0. 5%, zinc acetate: 200 mg/L, sodium citrate: 0.5M.
  • the initial temperature of the above crystallization liquid is adjusted to room temperature, the pH is adjusted to 8.5-9.0 with ammonium hydroxide, placed at 2-8 ° C for 1 hour, and then taken out, and then the pH is adjusted to 7.5-8.0 with glacial acetic acid, and then 2- Allow to stand at 8 ° C for 8 hours.
  • the supernatant was taken for HPLC detection, and the content of recombinant insulin glargine in the supernatant was 0.04 mg/ml, which was in compliance with the requirements.
  • microscopic examination magnified 160 times, visible hexahedral crystals, high transparency (see figure
  • Example 5 A recombinant insulin crystallization solution was prepared in the same manner as in Example 1, and the components and concentrations contained in the crystallization solution were: recombinant insulin glargine: 2.8 g/L, isopropanol: 15%, C Acid: 0.4 M, ethyl p-hydroxybenzoate: 0.1%, zinc bromide: 70 mg/L, sodium chloride: 0.3 M.
  • the initial temperature of the above crystallization liquid was adjusted to 20-25 ° C, the pH was adjusted to 7.1-7.8 with ammonium hydroxide, stirred for 10 minutes until the solution was slightly mixed, and left to stand at 2-8 ° C for about 6 hours.
  • the supernatant was taken for HPLC detection, and the content of the recombinant insulin glargine in the supernatant was 0.03 mg/ml, which was in compliance with the requirements.
  • the microscopic examination showed that the apparent hexahedral crystals were magnified 160 times and the transparency was high (see Figure 5).
  • Example 6 A recombinant insulin crystallization solution was prepared in the same manner as in Example 1, and the components and concentrations contained in the crystallization solution were: recombinant insulin glargine: 3.2 g/L, tert-butanol: 20%, different Butyric acid: 0.6 M, phenol: 0.1%, m-cresol: 0.1%, zinc sulfate: 100 mg/L, sodium sulfate: 0.4 M. The initial temperature of the above crystallization liquid was adjusted to room temperature, pH 8.5 was adjusted with ammonium hydroxide, and the crystal was allowed to stand at room temperature for about 12 hours.
  • the supernatant was taken for HPLC detection, and the content of recombinant insulin glargine in the supernatant was 0.05 mg/ml, which was in compliance with the requirements.
  • microscopic examination magnified 160 times, visible hexahedral crystals, high transparency (see figure
  • Comparative Example 1 A recombinant insulin crystallization solution was prepared in the same manner as in Example 1, and the components and concentrations contained in the crystallization solution were: recombinant insulin glargine: lg/L, ethanol: 5%, acetic acid: 0.1 M , zinc chloride: 10 mg / L, phenol: 0.01%, sodium chloride: 0.1M.
  • the initial temperature of the above crystallization liquid is adjusted to 20-25 ° C, the pH is adjusted to 8.1-8.8 with ammonium hydroxide, placed at 2-8 ° C for 1 hour, and then taken out, and then adjusted to pH 7.1-7.8 with glacial acetic acid. It was allowed to stand at 2-8 ° C for 5 hours.
  • Comparative Example 2 A recombinant insulin crystallization solution was prepared in the same manner as in Example 1, and the components and concentrations contained in the crystallization solution were: recombinant insulin glargine: 30 g/L, ethanol: 50%, acetic acid: 3 M, Zinc chloride: 300 mg/L, phenol: 2%, sodium chloride: 1 M.
  • the initial temperature of the above crystallization liquid is adjusted to 20-25 ° C, the pH is adjusted to 8.1-8.8 with ammonium hydroxide, placed at 2-8 ° C for 1 hour, and then taken out, and then adjusted to pH 7.1-7.8 with glacial acetic acid. It was allowed to stand at 2-8 ° C for 5 hours. The supernatant was then taken for HPLC detection, and the content of recombinant insulin glargine in the supernatant was 0.2 mg/ml. After crystallization of the suspension, the microscopic examination revealed that the number of hexahedral crystals was small at 160 times and the structure was uneven (see Figure 8).
  • Comparative Example 3 9 g of recombinant insulin glargine was weighed and dissolved in 1.8 L of purified water to prepare a recombinant insulin glargine solution. The solution was aliquoted into three portions, and insulin glargine crystals were prepared according to the methods of human insulin, insulin lispro and insulin glargine, respectively. That is, glacial acetic acid, zinc chloride and water are added to the first solution to make the acetic acid concentration 0.25M, the insulin glargine concentration is 1.6-2.1g/L, the zinc content is 63.5mg/L, and then 4M ammonium hydroxide is used.

Description

甘精胰岛素结晶的制备方法 技术领域 本发明涉及人胰岛素类似物结晶的制备方法， 具体地讲， 涉及一种重组甘精胰岛 素 （重组 Gly^-ArgB^-ArgB32-人胰岛素） 结晶的制备方法。 背景技术 糖尿病是一种常见的内分泌代谢疾病。 近年来， 全世界糖尿病的患病率都在迅猛 增长。 而在中国， 随着人民生活方式的改变和老龄化进程的加速， 糖尿病的患病率呈 快速上升趋势， 在 2010年中国糖尿病患者总数已突破 9000万人， 成为继心脑血管疾 病、 肿瘤之后的另一个严重危害人民健康的重要慢性非传染性疾病。 糖尿病的急、 慢 性并发症， 尤其是慢性病并发症累及多个器官， 致残、 致死率高， 严重影响患者的身 心健康， 并给个人、 家庭和社会带来沉重的负担。 胰岛素治疗一直被当作是治疗糖尿病并使血糖得到良好控制的重要手段。近年来， 随着胰岛素技术的不断发展， 人们开发出了具有不同作用时间的新一代胰岛素， 即胰 岛素类似物。 其中一种新开发的长效胰岛素类似物一甘精胰岛素， 正在越来越多被医 生和患者所接受和使用。 重组甘精胰岛素（或重组 GlyA21-ArgB31-ArgB32-人胰岛素， 或重组 GlyA21-人胰岛素 -ArgB31-ArgB31-OH), 是通过将人胰岛素 A链 A21 位的天冬酰胺 (Asn)突变为甘氨酸 (Gly) , 并且在 B 链羧基末端增加两个精氨酸 (Arg)而得到的 （参见美国专利 US5656722)。 重组甘精胰岛素由于能够模拟生理基础胰岛素分泌， 平稳无峰， 在控制 血糖轻易达标的同时几乎没有低血糖风险， 且持续时间为 24小时左右，符合人类生活 作息周期， 每天只需注射一次， 而被患者和医生所青睐。 在现有的重组甘精胰岛素产品中， 重组甘精胰岛素大多以溶液形式或无定形干粉 形式存在， 而没有形成结晶。 但是， 结晶形式的重组甘精胰岛素由于具有均一稳定的 固体分子形式， 并且沉降体积小， 易与上清分离， 离心时间及冻干时间短， 生产效率 高， 是更好的重组甘精胰岛素的应用形式。 因此， 现在亟需将重组甘精胰岛素制备成 结晶， 再应用到胰岛素药物制剂中。 现有技术中有很多胰岛素的结晶方法， 例如在 Abel J. J, PNAS, 12: 132(1926), 美 国专利 US2920104, 中国专利 CN95106555.6等中所描述的那些方法。 但是， 发明人 通过对各类胰岛素及类似物的结晶工艺的广泛研究发现，应用现有的胰岛素结晶方法， 并不能使重组甘精胰岛素形成结晶： 1 )在人胰岛素的结晶条件下： 0.25M乙酸、 1.6-2.1 克/ L人胰岛素、 2%锌、 pH5.95-6.05， 甘精胰岛素不能形成六面体结晶， 只以无定形沉 淀形式存在； 2) 在重组赖脯胰岛素 （LyS B28-PiOB29-人胰岛素） 的结晶条件下： 1M 乙 酸、 1.8-2.5克 /重组赖脯胰岛素、 100-300mg锌、 0.2%苯酚、 pH5.9-6.2， 甘精胰岛素仍 不能形成六面体结晶， 仍以无定形沉淀形式存在。 用现有的胰岛素结晶方法不能使重组甘精胰岛素结晶的原因可能是由于： 重组甘 精胰岛素在其 B链末端多两个碱性氨基酸（ArgB31-ArgB32)， 导致其等电点较天然人胰 岛素高， 从而使其在现有胰岛素结晶的条件下， 仅能以无定形状态存在， 不能形成稳 定的六面体结晶。 发明内容 本发明的目的是解决现有上述问题， 提供一种重组甘精胰岛素晶体的制备方法， 该方法可大规模地制备高质量和高收率的甘精胰岛素-锌结晶。该晶体具有稳定的固体 分子形式， 易于大批生产操作， 沉降体积小， 易与上清分离， 离心时间及冻干时间短， 生产效率高。 因此， 为了实现本发明的目的，本发明提供一种重组甘精胰岛素晶体的制备方法， 该方法包括： 从含有重组甘精胰岛素、 有机溶剂、 锌化合物、 酚衍生物、 盐以及有机 酸的结晶液中，在 pH7.0-9.0的条件下使重组甘精胰岛素结晶，其中所述有机溶剂的体 积百分浓度为 10-30%。 重组甘精胰岛素因其等电点与其他胰岛素差异较大， 所以结晶较为困难， 结晶过 程中有机溶液剂的加入以及 pH值的调节是影响结晶效果的关键因素。 本发明中所述的有机溶剂是重组甘精胰岛素形成结晶的关键因素之一。 其他胰岛 素可以在没有有机溶剂的条件下结晶， 但重组甘精胰岛素在没有有机溶剂的条件下， 只能是絮状的无定形沉淀， 无法形成结晶。 发明人经大量实验研究发现， 只有当结晶 液中存在有体积百分浓度为 10-30%的有机溶剂时， 重组甘精胰岛素才能开始形成结 晶。这可能是由于结晶液中的有机溶剂能够降低重组甘精胰岛素的溶解度的缘故： （1 ) 当结晶液中存在有机溶剂时， 能降低结晶液的电解常数， 增加甘精胰岛素分子上不同 电荷之间的相互引力， 导致甘精胰岛素溶解度的降低； （2) 有机溶剂与水之间的相互 作用能够破坏甘精胰岛素的水化膜， 也可以降低甘精胰岛素的溶解度。 有机溶剂降低了结晶液中重组甘精胰岛素的溶解度，促使甘精胰岛素结晶的形成， 因此结晶液中有机溶剂的体积百分浓度不宜低于 10% (体积 /体积）， 否则难以达到上 述效果。 同时， 如果有机溶剂的浓度过高， 又会容易引起蛋白质变性失活， 因此结晶 液中有机溶剂的体积百分浓度也不宜超过 30% (体积 /体积）。 在本发明中， 所述有机 溶剂可以为乙腈、 乙醇、 正丙醇、 异丙醇等任何本领域技术人员所公知的易溶于水的 有机溶剂。 其中优选的有机溶剂为乙醇。 另外， 由于重组甘精胰岛素的等电点较高在 pH6-8之间， 而其他胰岛素的等点电 通常在 pH5-5.5之间， 所以重组甘精胰岛素在其他胰岛素的结晶 pH值（例如 pH6)时 只是略显混浊， 不足以开始结晶过程， 只有当 pH跃过 7以后结晶过程才能开始， 因 此本发明中结晶的条件是 pH7.0-9.0， 而在此 pH值其他胰岛素已基本澄清无法结晶。 此外， 发明人经研究发现， 如果在重组甘精胰岛素的结晶过程中， 将结晶液的初始 pH 值调节至 pH8.0-9.0， 结晶过程中的 pH值调节至 pH7.0-8.0， 能形成粒径更大、 结构更 规则且更为均一的结晶。 本发明结晶液的 pH值可通过使用碱等本领域技术人员所公 知的方法进行调节， 优选使用氢氧化钠、 氢氧化钾、 氢氧化铵 （浓氨水） 进行调节。 优选地， 所述结晶液中重组甘精胰岛素的浓度为 2-4 g/L， 更优选为 2.8-3.2 g/L。 甘精胰岛素浓度太低， 会加大结晶液的体积， 不利于大规模生产， 如果浓度过高超过 甘精胰岛素的溶解度， 会使溶液混浊。 优选地， 所述锌化合物是以锌盐的形式加入的， 选自能够在水中解离出锌离子的 锌盐， 具有代表性的锌盐的实例有氯化锌、 氧化锌、 乙酸锌、 溴化锌、 硫酸锌等。 本 领域技术人员应该知道， 还有许多其他的锌盐也可以用于本发明的方法； 本发明结晶 液中锌的浓度优选为 20-200mg/L。 本发明更优选的锌为氯化锌； 浓度为 70-100mg/L。 由于锌化合物参与甘精胰岛素结晶过程， 如果锌化合物的浓度过低会使甘精胰岛素结 晶不完全， 如果锌化合物浓度过高， 会使结晶初期的晶核增多， 从而使结晶的颗粒变 小。 优选地， 所述酚衍生物是指能够提供酚羟基的物质， 选自苯酚、 间甲苯酚或对羟 基苯甲酸甲酯或它们的混合物； 结晶液中酚衍生物的浓度为 0.05-0.5g/100ml (即 0.05-0.5%,质量 /体积）。更优选的酚衍生物为苯酚；浓度为 0.1-0.2g/100ml (即 0.1-0.2%， 质量 /体积）。 酚衍生物也参与甘精胰岛素结晶过程， 如果酚衍生物浓度过低则不利于 晶体生成，酚衍生物浓度过高虽然不对结晶过程本身造成影响，但是会增加生产成本。 优选地， 所述盐是指易溶于水的强电解质盐， 选自氯化钠、 乙酸钠或柠檬酸钠； 结晶液中盐的浓度为 0.1-0.5M。 更优选的盐为氯化钠； 浓度为 0.3-0.4M。 盐浓度低于 0.1M时， 结晶液在 pH7.0-9.0的条件下为混浊溶液， 不能有效形成晶体， 而盐浓度高 于 0.5M时，结晶液在 pH7.0-9.0的条件下始终是澄清液体，这样也不能析出形成晶体。 优选地， 所述有机酸是指易溶于水的短链羧酸， 选自乙酸、 柠檬酸或甘氨酸； 结 晶过程中有机酸浓度过高易造成结晶过程中放热过多而影响产品的稳定性， 浓度过低 会使重组甘精胰岛素溶解效果不好， 因此结晶液中有机酸的浓度优选为 0.3-0.8M。 更 优选的有机酸为乙酸； 浓度为 0.4-0.6M。 结晶的温度不是关键性的， 可以接受的温度范围为约 1-30°C。 优选的结晶液初始 温度为室温 （10-30°C )， 该温度能够增加重组甘精胰岛素结晶前的溶解度； 优选的结 晶过程中的温度为 2-8°C，该温度能够促进重组甘精胰岛素在低温下溶解度的降低，加 快结晶速度。 优选的， 本发明采用以下步骤对重组甘精胰岛素进行结晶： （1 ) 在室温下制备含 有上述组分和含量的结晶液， 并调节结晶液初始 pH值至 8.0-8.5; (2) 将上述结晶液 降温至 2-8°C， 并调节 pH值至 7.1-7.8， 然后结晶至少 3小时。 重组甘精胰岛素溶于结晶液中的方式， 以及结晶液中各组分加入的顺序对本发明 来说也不是关键性的。 例如， 可以分别将有机溶剂、 锌、 酚衍生物、 有机酸以及盐加 入到重组甘精胰岛素溶液中， 也可以先将锌、 酚衍生物、 有机酸和盐配制成溶液， 再 加入到包含重组甘精胰岛素和有机溶剂的溶液中。 在工业化生产过程中， 为了使结晶胰岛素更纯， 可以选择重结晶工艺， 为了降低 结晶中的其他溶剂含量， 也可以采取水洗结晶的方法。 本发明的重组甘精胰岛素可以通过商购获得， 也可以采用任何一种公认的肽合成 技术制备，例如溶液法、固相合成法（J. Stewart等,《固相肽合成》， Freeman and Co., San Francisco, 1969)、 半合成法、 基因工程法、 DNA重组法 (美国专利 US5656722)等。 本发明重组甘精胰岛素结晶的制备方法具有以下优点： 可以结晶出均一稳定的重 组甘精胰岛素六面体结晶， 晶体的透明度高， 沉降体积小， 离心干燥时间短， 生产效 率高。 附图说明 图 1示出了实施例 1重组甘精胰岛素结晶后在显微镜下 160倍放大图； 图 2示出了实施例 2重组甘精胰岛素结晶后在显微镜下 160倍放大图； 图 3示出了实施例 3重组甘精胰岛素结晶后在显微镜下 160倍放大图； 图 4示出了实施例 4重组甘精胰岛素结晶后在显微镜下 160倍放大图； 图 5示出了实施例 5重组甘精胰岛素结晶后在显微镜下 160倍放大图； 图 6示出了实施例 6重组甘精胰岛素结晶后在显微镜下 160倍放大图； 图 7示出了对比例 1重组甘精胰岛素结晶后在显微镜下 160倍放大图； 图 8示出了对比例 2重组甘精胰岛素结晶后在显微镜下 160倍放大图； 图 9示出了对比例 3第一份重组甘精胰岛素结晶后在显微镜下 160倍放大图； 图 10示出了对比例 3第二份重组甘精胰岛素结晶后在显微镜下 160倍放大图；以 及 图 11示出了对比例 3第三份重组甘精胰岛素结晶后在显微镜下 160倍放大图。 具体实施方式 材料 甘精胰岛素： 甘李药业有限公司提供， 批号 GLGB09001。 氯化锌购自北京安迪永富， 批号 71770; 苯酚购自江门市恒建药业， 批号 081001 ; 乙酸购自台山新宁制药有限公司， 批号 20081007; 氯化钠购自北京燕京药业， 批号 080522； 浓氨水购自北京华腾， 批号 20080513 ; 乙醇购自天津市科密欧化学试剂有限 公司， 批号 091005， 醋酸铵购于上海科彤化工有限公司， 批号 20081101 ; 乙腈购自上 海星可， 批号 20091005; 乙酸钠购自天津市科密欧化学试剂有限公司， 批号 090504。 实施例 1 制备重组甘精胰岛素结晶液， 该结晶液中所包含的组分和浓度为： 重组甘精胰岛 素： 3.5g/L， 乙腈： 20%， 乙酸： 0.7M, 苯酚： 0.4%， 氯化锌： 120mg/L， 乙酸钠： 0.5M。 称取 6.3g重组甘精胰岛素溶于 1L纯净水中， 制成重组甘精胰岛素混悬液； 另将 乙腈 360ml， 冰乙酸 72.09ml， 10%的苯酚溶液 72ml， 5.08 g/100ml (即 5.08%， 质量 / 体积） 的氯化锌溶液 4.25ml， 4M的乙酸钠溶液 225ml， 混合后加水至终体积 0.6L。 将上述 0.6 L溶液加入到上述 1L重组甘精胰岛素混悬液中， 并补水至终体积 1.8L, 制 成结晶液。 将上述结晶液初始温度调到 20-25°C， 用氢氧化铵调节 pH7.8-8.2， 搅拌 10分钟至 溶液微混， 放入 2-8°C静置结晶约 3小时。 然后取上清液进行 HPLC检测， 上清液中 重组甘精胰岛素的含量为 0.05mg/ml， 符合要求。 搅起结晶混悬液后镜检， 放大 160倍可见明显的六面体结晶， 透明度较高 （见图 1 )。 实施例 2 制备重组甘精胰岛素结晶液， 该结晶液中所包含的组分和浓度为： 重组甘精胰岛 素： 3g/L， 乙醇： 15%， 乙酸： 0.5M, 氯化锌： 90mg/L， 苯酚： 0.2%， 氯化钠： 0.3M。 称取 251.8g重组甘精胰岛素装在已清洁的不锈钢桶中， 加入 96%乙醇 13.1L， 纯 冰乙酸 2.4L, 5.08 g/100ml (即 5.08%， 质量 /体积） 的氯化锌溶液 148.6ml， 10%的苯 酚溶液 1678ml， 氯化钠 1472g， 加水至终体积 83.9L。 将上述溶液的初始温度调到 23±2°C， 用浓氨水调节 pH至 8.0-8.5， 搅拌约 10分 钟至溶液微混， 然后于 2-8°C放置 1小时降温后取出， 再用冰乙酸调 pH至 7.1-7.6， 搅 拌约 10分钟， 再于 2-8°C静置 10小时。 然后取上清液进行 HPLC检测， 上清液中重 组甘精胰岛素的含量已低至 0.02mg/ml， 符合要求。 倾出上清液约 77L， 将剩余约 7L混悬液分别放入 1L离心瓶中， 用离心机 4000 转 /分钟离心 10分钟，将所有离心瓶中的结晶沉淀取出，按 20g/L的浓度混悬于 0.02M 的醋酸铵溶液中， 调 pH值 7.8洗涤， 再次离心收集沉淀， 重复洗涤一次， 再次离心去 除上清， 进行冷冻干燥， 40小时后得到冻干粉末 245.6克， 含水量 2.3%。 将冻干粉末 加水制成悬浮液后镜检， 放大 160倍可见大量均一的、 规则的六面体结晶， 透明度很 高 （见图 2)。 实施例 3 应用与实施例 1相同的方法制备重组甘精胰岛素结晶液， 该结晶液中所包含的组 分和浓度为： 重组甘精胰岛素： 2g/L， 正丙醇： 10%， 乙酸： 0.3M, 间甲酚： 0.05%, 氧化锌： 20mg/L， 乙酸钠： 0.1M。 将上述结晶液初始温度调到 4°C， 用氢氧化铵调节 pH7.5-8.5， 继续在 4°C下静置 结晶约 10小时。 然后取上清液进行 HPLC检测， 上清液中重组甘精胰岛素的含量为 0.05mg/ml， 符合要求。 搅起结晶混悬液后镜检， 放大 160倍可见明显的六面体结晶， 透明度较高 （见图
3 )。 实施例 4 应用与实施例 1相同的方法制备重组甘精胰岛素结晶液， 该结晶液中所包含的组 分和浓度为： 重组甘精胰岛素： 4g/L， 甲醇： 30%， 柠檬酸： 0.8M, 对羟基苯甲酸甲 酉旨： 0. 5%, 乙酸锌： 200mg/L, 柠檬酸钠： 0.5M。 将上述结晶液初始温度调到室温， 用氢氧化铵调节 pH8.5-9.0， 放入 2-8°C放置 1 小时降温后取出， 再用冰乙酸调 pH至 7.5-8.0， 再于 2-8°C静置 8小时。 然后取上清液 进行 HPLC检测， 上清液中重组甘精胰岛素的含量为 0.04mg/ml， 符合要求。 搅起结晶混悬液后镜检， 放大 160倍可见明显的六面体结晶， 透明度较高 （见图
4)。 实施例 5 应用与实施例 1相同的方法制备重组甘精胰岛素结晶液， 该结晶液中所包含的组 分和浓度为： 重组甘精胰岛素： 2.8g/L， 异丙醇： 15%， 丙酸： 0.4M, 对羟基苯甲酸 乙酯： 0. 1%, 溴化锌： 70mg/L， 氯化钠： 0.3M。 将上述结晶液初始温度调到 20-25 °C， 用氢氧化铵调节 pH7.1-7.8， 搅拌 10分钟至 溶液微混， 放入 2-8°C静置结晶约 6小时。 然后取上清液进行 HPLC检测， 上清液中 重组甘精胰岛素的含量为 0.03mg/ml， 符合要求。 搅起结晶混悬液后镜检， 放大 160倍可见明显的六面体结晶， 透明度较高 （见图 5 )。 实施例 6 应用与实施例 1相同的方法制备重组甘精胰岛素结晶液， 该结晶液中所包含的组 分和浓度为： 重组甘精胰岛素： 3.2g/L， 叔丁醇： 20%， 异丁酸： 0.6M, 苯酚： 0.1%， 间甲酚： 0.1%, 硫酸锌： 100mg/L， 硫酸钠： 0.4M。 将上述结晶液初始温度调到室温， 用氢氧化铵调节 pH8.5， 继续在室温下静置结晶约 12小时。 然后取上清液进行 HPLC 检测， 上清液中重组甘精胰岛素的含量为 0.05mg/ml， 符合要求。 搅起结晶混悬液后镜检， 放大 160倍可见明显的六面体结晶， 透明度较高 （见图
6)。 对比例 1 应用与实施例 1相同的方法制备重组甘精胰岛素结晶液， 该结晶液中所包含的组 分和浓度为： 重组甘精胰岛素： lg/L， 乙醇： 5%， 乙酸： 0.1M, 氯化锌： 10mg/L， 苯 酚： 0.01%， 氯化钠： 0.1M。 将上述结晶液初始温度调到 20-25 °C，用氢氧化铵调节 pH8.1-8.8，放入 2-8°C放置 1小时降温后取出， 再用冰乙酸调 pH至 7.1-7.8， 再于 2-8°C静置 5小时。 然后取上清 液进行 HPLC检测， 上清液中重组甘精胰岛素的含量为 0.2mg/ml。 搅起结晶混悬液后镜检， 放大 160倍可见六面体结晶数量很少， 且结构不均 （见 图 7)。 对比例 2 应用与实施例 1相同的方法制备重组甘精胰岛素结晶液， 该结晶液中所包含的组 分和浓度为： 重组甘精胰岛素： 30g/L， 乙醇： 50%， 乙酸： 3M， 氯化锌： 300mg/L， 苯酚： 2%， 氯化钠： 1M。 将上述结晶液初始温度调到 20-25°C，用氢氧化铵调节 pH8.1-8.8，放入 2-8°C放置 1小时降温后取出， 再用冰乙酸调 pH至 7.1-7.8， 再于 2-8°C静置 5小时。 然后取上清 液进行 HPLC检测， 上清液中重组甘精胰岛素的含量为 0.2mg/ml。 搅起结晶混悬液后镜检， 放大 160倍可见六面体结晶数量很少， 且结构不均 （见 图 8)。 对比例 3 称取 9g重组甘精胰岛素溶于 1.8L纯净水中， 制成重组甘精胰岛素溶液。 将该溶 液等分成三份， 分别按人胰岛素、 赖脯胰岛素和甘精胰岛素的方法制备甘精胰岛素结 晶。 即在第一份溶液中加入冰乙酸、 氯化锌和水， 使乙酸浓度为 0.25M, 甘精胰岛素 浓度为 1.6-2.1g/L， 锌含量达到 63.5mg/L， 然后用 4M氢氧化铵调 pH至 5.95-6.05; 第 二份加入冰乙酸、氯化锌、苯酚和水，使乙酸浓度为 1M，甘精胰岛素浓度为 1.8-2.5g/L， 含锌量为 200mg/L， 苯酚含量为 0.2%， 然后用浓氢氧化铵调 pH至 5.9-6.2; 第三份按 实施例 2的结晶液的组分和含量配制。 结果第一、 二份甘精胰岛素溶液只形成了絮状 沉淀， 未能形成正六面体结晶 （见图 9、 图 10)， 第三份甘精胰岛素形成了正六面体结 晶 （见图 11 )。

Claims

权 利 要 求 书 一种重组甘精胰岛素晶体的制备方法， 其特征在于， 所述方法包括： 从含有重 组甘精胰岛素、 有机溶剂、 锌化合物、 酚衍生物、 盐以及有机酸的结晶液中， 在 pH7.0-9.0 的条件下使所述重组甘精胰岛素结晶， 其中， 所述有机溶剂的体 积百分浓度为 10-30%。 根据权利要求 1所述的制备方法， 其特征在于， 所述有机溶剂选自乙腈、 乙醇、 正丙醇或异丙醇。 根据权利要求 1或 2所述的制备方法， 其特征在于， 所述结晶液的初始 pH值 为 8.0-9.0， 在结晶过程中所述结晶液的 pH值为 7.0-8.0。 根据权利要求 3 所述的制备方法， 其特征在于， 所述结晶液的初始 pH值为 8.0-8.5， 在结晶过程中所述结晶液的 pH值为 7.1-7.8。 根据权利要求 1所述的制备方法， 其特征在于， 所述结晶的温度为 1-30°C。 根据权利要求 5 所述的制备方法， 其特征在于， 所述结晶液的初始温度为 10-30°C， 所述结晶过程中的温度为 2-8°C。 根据权利要求 1， 2和 4-6中任一项所述的制备方法， 其特征在于， 所述结晶液 中所述重组甘精胰岛素的浓度为 2-4 g/L。 根据权利要求 1， 2和 4-6中任一项所述的制备方法， 其特征在于， 所述酚衍生 物选自苯酚、 间甲苯酚和对羟基苯甲酸甲酯或它们的混合物， 所述结晶液中所 述酚衍生物的浓度为 0.05-0.5g/100ml。 根据权利要求 1， 2和 4-6中任一项所述的制备方法， 其特征在于， 所述锌化合 物选自氯化锌、 氧化锌、 乙酸锌、 溴化锌或硫酸锌， 所述结晶液中所述锌化合 物的浓度为 20-200mg/L。 根据权利要求 1， 2和 4-6中任一项所述的制备方法， 其特征在于， 所述盐选自 氯化钠、 乙酸钠或柠檬酸钠， 所述结晶液中所述盐的浓度为 0.1-0.5M。
11. 根据权利要求 1， 2和 4-6中任一项所述的制备方法， 其特征在于， 所述有机酸 选自乙酸、 柠檬酸或甘氨酸， 所述结晶液中所述盐的浓度为 0.3-0.8M。
12. 根据权利要求 1所述的制备方法， 其特征在于， 所述制备方法包括以下步骤：
1 ) 在室温下， 制备含 2.8-3.2 g/L重组甘精胰岛素、 乙醇、 0.1-0.2 g/lOOml 苯酚、 0.4-0.6M乙酸、 70-100mg/L氯化锌、 0.3-0.4M氯化钠的结晶液， 其中， 所述乙醇的体积百分浓度为 10-30%;
2) 将所述结晶液的 pH值调节至 8.0-8.5;
3 ) 将所述结晶液降温至 2-8°C， 并调节 pH值至 7.1-7.8， 然后结晶至少 3 小时， 制得所述重组甘精胰岛素晶体。
13. 根据权利要求 1所述的制备方法， 其特征在于， 所述制备方法进一步包括： 将所述重组甘精胰岛素晶体以 20g/L 的浓度混悬于 0.02M 的醋酸铵溶液 中， 调节 pH值至 7.8， 离心收集沉淀物得到精制后的重组甘精胰岛素晶体。
14. 根据权利要求 13所述的制备方法， 其中， 包括以下步骤：
称取 251.8g重组甘精胰岛素， 加入 96%乙醇 13.1L， 纯冰乙酸 2.4L, 5.08 g/100ml的氯化锌溶液， 148.6ml 10%的苯酚溶液 1678ml， 氯化钠 1472g， 加水 至终体积 83.9L;
将上述溶液的温度调到 23±2°C， 用浓氨水调节 pH至 8.0-8.5， 搅拌约 10 分钟至溶液微混， 然后于 2-8°C放置 1 小时降温后取出， 再用冰乙酸调 pH至 7.1-7.6, 搅拌约 10分钟， 再于 2-8°C静置 10小时；
倾出上清液约 77L， 将剩余约 7L混悬液分别放入 1L离心瓶中， 用离心机 4000转 /分钟离心 10分钟， 将所有离心瓶中的结晶沉淀取出， 按 20g/L的浓度 混悬于 0.02M的醋酸铵溶液中， 调 pH值 7.8洗涤， 再次离心收集沉淀， 重复 洗涤一次， 再次离心去除上清， 进行冷冻干燥 40小时。
PCT/CN2012/074392 2011-05-09 2012-04-19 甘精胰岛素结晶的制备方法 WO2012152175A1 (zh)

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