CN88102311A - 人结晶胰岛素原及其生产方法 - Google Patents
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/62—Insulins
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Genetics & Genomics (AREA)
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- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Diabetes (AREA)
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- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
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Abstract
本发明提供了人结晶胰岛素原及其制备方法,该方法包括:(a)制备含约5-50mg/ml人胰岛素原、含约0.1—5mg/ml酚物质、含约0.03-0.6毫当量/ml的一种其阳离子选自锂、钙、钠、钾、铵、镁和钡的阳离子盐和含约0.2-5毫当量锌(Zn+2)的水混合物;(b)调该含水混合物PH为约5.4-6.5;(c)使晶体生成;和(d)从水混合物中回收人结晶胰岛素原。
Description
重组DNA操作技术的出现已给生产各种各样的肽和蛋白质提供了可能。人胰岛素原便是这类蛋白质中的一种,它既可用作生产人胰岛素的中间体,又可单独地作为控制糖尿病的药物。
在人胰岛素原的重组DNA生产中,产物通常在水溶液中处理并且冻干为非结晶粉。如果能用其它方法获得结晶形式的人胰岛素原,便可在贮藏性能、配方设计及其分离纯化方法等方面提供许多方便。
本发明涉及新的结晶形式的人胰岛素原及其生产方法。
因此,本发明涉及一种人结晶胰岛素原,它包含一种人胰岛素原和二价锌离子的配合物的盐,该盐的阳离子可选自锂、钙、钠、钾、铵、镁和钡这类离子。
本发明还涉及生产上述盐的方法,该方法包括:
(a)制备含约5-50毫克/毫升人胰岛素原、含约0.1-5毫克/毫升酚物质、含约0.03-0.6毫当量/毫升一种其阳离子选自锂、钙、钠、钾、铵、镁和钡的阳离子盐和含约02-5毫当量锌离子的含水混合物;
(b)调节该含水混合物的PH至约5.4-6.5;
(c)使晶体生成;和
(d)从含水混合物中回收人结晶胰岛素原。
如上所述,本发明是涉及人结晶胰岛素原及其生产方法。
本发明的人结晶胰岛素原包含一种人胰岛素原和二价锌离子的配合物的盐。通常用人胰岛素原六聚物形式来表示该配合物,该六聚物是由六个人胰岛素原分子和2个锌离子配价键联的。其阳离子是一价的阳离子或二价的阳离子,可以是锂、钙、钠、钾、铵、镁和钡离子中之一种,较好的阳离子是钙、钠、钾、铵或镁离子,更好的阳离子是钠、钙或铵离子,最好的阳离子是钙或钠离子。
生产本发明人结晶胰岛素原有许多重要的参数和条件。它们是:人胰岛素原的浓度;酚物质的存在和浓度;阳离子的存在,含量和性质;锌离子的存在和含量;及PH值。
以水为介质的人胰岛素原的浓度通常为约5-50毫克/毫升,较佳的浓度范围是低于上述浓度范围的一半,更好的浓度范围依次为约5-25毫克/毫升;约10-20毫克/毫升;及约13-17毫克/毫升,实施该方法所用的人胰岛素原的最适宜浓度约为15毫克/毫升。
本发明方法采用一种酚物质。它可以是各种通用的酚。作为这类酚的例子有酚本身、邻甲酚、间甲酚、对甲酚、间苯二酚、对羟基苯甲酸甲酯、邻苯二酚等,较佳的酚物质是酚或甲酚。将含量约为0.1-5毫克/毫升的酚物质加入水结晶混合物中,酚物质的较佳含量约为1-3毫克/毫升。
人结晶胰岛素原的阳离子可为上述各种阳离子中之任一种,它可以各种盐的形式加入含水混合物中,不过,所用的盐最好是盐酸盐。由于碳酸氢根离子的出现会抑制人胰岛素原盐的结晶等原因,因此,最好避免使用碳酸氢盐。被选用的阳离子按阳离子:人胰岛素原的摩尔比约为10∶1-1500∶1的用量加入水介质中。当然,当阳离子为二价离子(Ca++、Mg++、Ba++)时,通常加入的阳离子的量要低于阳离子为一价离子(Na+、K+、Li+、NH+ 4)时的量。一般,当阳离子为二价阳离子时,混合物中阳离子与人胰岛素原的比率约为50∶1,阳离子为一价离子时,比率为200∶1。
一般来说,过量的、高浓度的阳离子会延迟结晶,所以人胰岛素原的浓度以约13毫克/毫升或以下为宜。
本发明人结晶胰岛素原是采用可溶性锌盐形式,典型的这类可溶性盐有氯化锌、乙酸锌、硝酸锌等。本发明结晶形式的人胰岛素原所需的锌离子的化学计量为每3个人胰岛素原分子配一个锌原子。本发明方法虽可采用较低的锌离子含量,结晶虽仍会发生,但收率相应地降低。
因此,以化学计量表示的所用锌盐的量通常至少为化学计算量(1Zn++/3HPI),也可高达约每个人胰岛素原分子配2个Zn++的摩尔比。结晶过程最适宜的锌离子浓度约为每2个人胰岛素原分子配1个锌离子下进行。
欲制备含各所需量的人胰岛素原、酚物质、阳离子和锌离子的含水混合物,宜于在约PH3.5以下或最好在约6.5以上进行。
完全的混合物一旦制成,即调PH至约5.4-6.5,最好使PH维持在约5.8-6.3的范围内,约6.0-6.1更好。
维持温度于约0℃-40℃下,使晶体从混合物中生成。如所预料一样,结晶最好在上述温度范围的低限例如约5℃-25℃下进行。如先于约25℃,再于约5℃,通过搅拌混合物,更可加速结晶过程。
业已发现,通过从最初的结晶混合物中倾去母液,接着再用母液洗液洗涤晶体,便可大大提高晶体的回收效率。术语“母液洗液”意指配制成的含酚物质,所需阳离子盐和锌盐的溶液,其中每种成分的浓度均接近结晶混合物中所表示的浓度。
进一步提高人胰岛素原晶体回收效率的方法涉及把母液洗液中的晶体浓浆加入至少约10倍体积的无水乙醇中,该程序有利于使人胰岛素原保留结晶形式。
生产本发明人胰岛素原晶体的一般方法和优选方法如下:
把人胰岛素原溶解于水,使其浓度约为15毫克/毫升,PH约5.6-7.0,再将酚或甲酚(约0.3%;3毫克/毫升)加入混合物中,继之加入足量的阳离子盐以制成相当于约0.35M一价盐(NaCl KCl,LiCl,或NH4Cl)或相当于约0.077M二价盐(CaCl2·2H2O,MgCl2,或BaCl2)的溶液,调所得溶液PH至约6.5,随后加入约1.16mM氯化锌(每100毫升溶液含0.8毫升2%氯化锌)。如有需要,可通过加入稀氢氧化钠或稀盐酸调所得混合物PH至约6.0-6.1。混合物于25℃搅拌24小时,再于5℃搅拌24小时,使生成的晶体沉降,倾去母液。混合物于约2500rpm转速下离心2-3分钟,再一次倾去母液。用1-2倍体积的母液洗液使晶体成浆,再离心混合物,倾去母液。再用1-2倍体积的母液洗液使晶体成浆,之后,不断搅拌混合物,慢慢地将混合物倒入10-15倍体积的无水乙醇中。随后继续搅拌10-15分钟,当晶体沉降时,倾去乙醇洗液。离心混合物,再用无水乙醇洗涤晶体,倾去乙醇洗液。再离心,再倾去乙醇洗液。真空干燥所回收的晶体约12-24小时。
下面实施例是说明用各种阳离子生产本发明人结晶胰岛素原的方法。该实施例并非用于限制本发明的宽广范围。
实施例
把22克纯化的、冻干的人胰岛素原溶解于1000毫升纯水中,得到浓度为19.53毫克/毫升的人胰岛素原。加2毫升液态酚后,用250毫升0.2%酚稀释该溶液至15.62毫克人胰岛素原/毫升,PH为6.5。
取20毫升等分试样(312.5毫克人胰岛素原)各加入如下盐酸盐:77mM CaCl2(0.456毫升50%CaCl2溶液);75,150和300mM NaCl(87.2,175.2和350.4毫克);75,150和300mM KCl(112,224和448毫克);及75,150和300mM NH4Cl(80,160和320毫克)。各用0.16ml2%ZnCl2(76μg Zn++/ml或1.16mM),用10%盐酸调PH至6.0-6.1。于25℃搅拌混合物24小时,再于5℃搅拌24小时,然后使其沉降。24和48小时后,将结晶混合物取样、过滤、测定以检测人胰岛素原的结晶程度。倾去上清液,迅速搅拌的同时缓慢地将人胰岛素原晶体的浓浆注入10-15倍体积的冷无水乙醇中,搅拌10-15分钟后,将晶体离心短时间,倾去乙醇,再用乙醇洗涤晶体,离心,倾去乙醇,真空干燥晶体并称重,所得结果列于下表。
表
人结晶胰岛素原
母液,%HPI 干晶体
盐 浓度(M) 室温24 5℃24 mg 收率%
小时后 小时后
CaCl20.077 5.9 2.2 278 89
NaCl 0.075 23.9 10.1 250 80
″ 0.150 9.6 5.2 277 89
″ 0.300 13.7 10.9 281 90
KCl 0.075 17.8 7.8 269 86
″ 0.150 9.6 5.6 282 90
″ 0.300 8.9 6.3 256 82
NH4Cl 0.075 6.9 5.8 269 86
″ 0.150 7.8 5.3 265 85
″ 0.300 9.9 6.1 269 86
Claims (15)
1、生产人结晶胰岛素原的方法,该方法包括:
(a)制备含约5mg-50mg/ml人胰岛素原,含约0.1-5mg/ml酚物质、含约0.03-0.6毫当量/ml的一种其阳离子选自锂、钙、钠、钾、铵、镁和钡的阳离子盐和含约0.2-5毫当量锌离子(Zn+2)的含水混合物;
(b)调该含水混合物PH至约5.4-6.5;
(c)使晶体生成;和
(d)从含水混合物中回收人结晶胰岛素原。
2、权利要求1的方法,其中人胰岛素原的浓度约为5-25mg/ml。
3、权利要求2的方法,其中人胰岛素原的浓度约为13-17mg/ml。
4、权利要求1至3的任一项方法,其中酚物质是酚或甲酚。
5、权利要求1至4的任一项方法,其中酚物质的含量为约1-3mg/ml。
6、权利以前1-5的任一项方法,其中盐是盐酸盐。
7、权利要求1-6的任一项方法,其中盐的阳离子是选自钠、钙、铵离子。
8、权利要求7的方法,其中盐的阳离子是钠离子。
9、权利要求7的方法,其中盐的阳离子是钙离子。
10、权利要求1至9的任一项方法,其中所加的锌离子(Zn+2)含量约为每3个胰岛素原分子配1个锌离子至每1个胰岛素原分子配2个锌离子。
11、权利要求1至10的任一项方法,其中所制备的含人胰岛素原、酚物质、阳离子和锌离子的含水混合物的PH值约为3.5以下或约为6.5以上。
12、权利要求1至11的任一项方法,其中含人胰岛素原、酚物质、阳离子和锌离子的含水混合物,在一经配制后,其PH值从约5.8延续并维持至6.3。
13、权利要求12的方法,其中最终的PH约为6.0至6.1。
14、权利要求1至13的任一项方法,其中从含水混合物中回收所生成的人结晶胰岛素原之前,倾去该混合物中的母液上清液,后用母液洗液洗涤残留的晶体。
15、权利要求14的方法,其中含人结晶胰岛素原的母液洗液被加到至少约10倍体积的无水乙醇中。
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US07/041,498 US4764592A (en) | 1987-04-23 | 1987-04-23 | Crystalline human proinsulin and process for its production |
US041,498 | 1987-04-23 |
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EP (1) | EP0288273B1 (zh) |
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CN (1) | CN1030988C (zh) |
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CA (1) | CA1339045C (zh) |
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IE (1) | IE65952B1 (zh) |
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WO2012152175A1 (zh) * | 2011-05-09 | 2012-11-15 | 甘李药业股份有限公司 | 甘精胰岛素结晶的制备方法 |
CN107029212A (zh) * | 2010-05-10 | 2017-08-11 | 诺沃—诺迪斯克有限公司 | 用于制备胰岛素‑锌复合物的方法 |
CN109957001A (zh) * | 2017-12-26 | 2019-07-02 | 甘李药业股份有限公司 | 甘赖脯胰岛素结晶的制备方法 |
US11167035B2 (en) | 2005-12-28 | 2021-11-09 | Novo Nordisk A/S | Insulin compositions and method of making a composition |
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PH23446A (en) * | 1986-10-20 | 1989-08-07 | Novo Industri As | Peptide preparations |
DE58906966D1 (de) * | 1988-06-23 | 1994-03-24 | Hoechst Ag | Mini-Proinsulin, seine Herstellung und Verwendung. |
US5130298A (en) * | 1989-05-16 | 1992-07-14 | Ethicon, Inc. | Stabilized compositions containing epidermal growth factor |
DK168790D0 (zh) * | 1990-07-13 | 1990-07-13 | Novo Nordisk As | |
US5504188A (en) * | 1994-06-16 | 1996-04-02 | Eli Lilly And Company | Preparation of stable zinc insulin analog crystals |
US5631347A (en) * | 1995-06-07 | 1997-05-20 | Eli Lilly And Company | Reducing gelation of a fatty acid-acylated protein |
UA91512C2 (ru) * | 2004-07-19 | 2010-08-10 | Биокон Лимитед | Коньюгати олигомеров инсулина, их композиция (варианты) и применение |
US9241908B2 (en) | 2007-10-16 | 2016-01-26 | Biocon Limited | Orally administrable solid pharmaceutical composition and a process thereof |
CN103459417B (zh) * | 2011-02-01 | 2018-04-10 | 诺沃—诺迪斯克有限公司 | 胰岛素的纯化 |
CN111304271A (zh) * | 2020-02-28 | 2020-06-19 | 东莞市东阳光生物药研发有限公司 | 一种含脂肪酸侧链的胰岛素类似物的制备方法 |
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US2143590A (en) * | 1936-09-26 | 1939-01-10 | Univ Alberta | Insulin preparation and process of producing crystals of insulin |
US2174862A (en) * | 1938-12-30 | 1939-10-03 | Frederick Stearns & Company | Process of producing crystalline insulin |
US2626228A (en) * | 1945-05-17 | 1953-01-20 | Novo Terapeutisk Labor As | Method of producing crystalline insulin |
US2663666A (en) * | 1949-03-31 | 1953-12-22 | Organon | Process of preparing crystalline insulin from acid alcoholic extracts of pancreas glands |
US2836542A (en) * | 1952-02-13 | 1958-05-27 | Novo Terapeutisk Labor As | Process of production of crystalline insulin |
DK78069C (da) * | 1952-06-23 | 1954-09-06 | Novo Terapeutisk Labor As | Fremgangsmåde til fremstilling af krystallinsk insulin. |
US3102077A (en) * | 1953-08-19 | 1963-08-27 | Christensen Henry Marinus | Preparation of insulin containing 2.75 to 8 percent zinc content |
GB786635A (en) * | 1953-08-19 | 1957-11-20 | Roskilde Medical Company Ltd R | Method for the production of insulin with increased zinc content |
US4430266A (en) * | 1980-11-28 | 1984-02-07 | Eli Lilly And Company | Process for producing an insulin precursor |
US4654324A (en) * | 1981-08-27 | 1987-03-31 | Eli Lilly And Company | Human proinsulin pharmaceutical formulations |
DE3326473A1 (de) * | 1983-07-22 | 1985-01-31 | Hoechst Ag, 6230 Frankfurt | Pharmazeutisches mittel zur behandlung des diabetes mellitus |
DE3326472A1 (de) * | 1983-07-22 | 1985-02-14 | Hoechst Ag, 6230 Frankfurt | Neue insulin-derivate, verfahren zu deren herstellung und deren verwendung sowie pharmazeutische mittel zur behandlung des diabetes mellitus |
US4616078A (en) * | 1985-04-08 | 1986-10-07 | Eli Lilly And Company | Process for purifying proinsulin-like materials |
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US11167035B2 (en) | 2005-12-28 | 2021-11-09 | Novo Nordisk A/S | Insulin compositions and method of making a composition |
CN107029212A (zh) * | 2010-05-10 | 2017-08-11 | 诺沃—诺迪斯克有限公司 | 用于制备胰岛素‑锌复合物的方法 |
WO2012152175A1 (zh) * | 2011-05-09 | 2012-11-15 | 甘李药业股份有限公司 | 甘精胰岛素结晶的制备方法 |
US9187520B2 (en) | 2011-05-09 | 2015-11-17 | Gan & Lee Pharmaceuticals | Method for preparing insulin glargine crystal |
CN109957001A (zh) * | 2017-12-26 | 2019-07-02 | 甘李药业股份有限公司 | 甘赖脯胰岛素结晶的制备方法 |
CN109957001B (zh) * | 2017-12-26 | 2022-11-18 | 甘李药业股份有限公司 | 甘赖脯胰岛素结晶的制备方法 |
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Publication number | Publication date |
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CN1030988C (zh) | 1996-02-14 |
DK210188A (da) | 1989-01-03 |
PT87265B (pt) | 1992-08-31 |
JP2602529B2 (ja) | 1997-04-23 |
HU202555B (en) | 1991-03-28 |
GR3015662T3 (en) | 1995-07-31 |
EP0288273A3 (en) | 1990-08-08 |
KR0126477B1 (ko) | 1997-12-19 |
ZA882714B (en) | 1989-12-27 |
DK210188D0 (da) | 1988-04-18 |
PT87265A (pt) | 1988-05-01 |
PH24261A (en) | 1990-05-04 |
MX166069B (es) | 1992-12-17 |
DE3852963D1 (de) | 1995-03-23 |
IE881183L (en) | 1988-10-23 |
EP0288273A2 (en) | 1988-10-26 |
NZ224281A (en) | 1990-06-26 |
US4764592A (en) | 1988-08-16 |
ES2068826T3 (es) | 1995-05-01 |
IE65952B1 (en) | 1995-11-29 |
AU1474788A (en) | 1988-11-10 |
AU609798B2 (en) | 1991-05-09 |
CA1339045C (en) | 1997-04-01 |
HUT46705A (en) | 1988-11-28 |
KR880012642A (ko) | 1988-11-28 |
JPS63280100A (ja) | 1988-11-17 |
EP0288273B1 (en) | 1995-02-08 |
IL86094A (en) | 1993-06-10 |
DE3852963T2 (de) | 1995-06-29 |
AR244242A1 (es) | 1993-10-29 |
IL86094A0 (en) | 1988-09-30 |
ATE118220T1 (de) | 1995-02-15 |
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