PatViewer专利搜索
中国 发明 在审

【中文】高纯度乌司他丁及其制备方法和含有乌司他丁的药物组合物
【EN】High-purity ulinastatin and preparation method thereof and pharmaceutical composition containing ulinastatin

申请(专利)号:CN201910121125.2国省代码:上海 31
申请(专利权)人:【中文】上海医药集团股份有限公司 广东天普生化医药股份有限公司【EN】Shanghai Pharmaceuticals Holding Co., Ltd.;Guangdong Tianpu Biochemical Medicine Co., Ltd.
温馨提示:Ctrl+D 请注意收藏,详细著录项请首页检索查看。 Please note the collection. For details, please search the home page.

摘要:
【中文】本发明提供一种乌司他丁及其制备方法,10万单位/ml的乌司他丁在405nm处的吸光度值不超过0.18,人尿激肽原酶的含量不超过0.0005PNAU,重金属含量不超过1.6μg/ml,细菌内毒素含量不超过6.0EU/ml。通过亚硝酸盐、硫酸铵、聚乙二醇、调节pH等方法对尿液进行预处理,同时在过柱前采用透析的方法进行初步的纯化,再依次通过凝胶柱和阳离子交换柱制备得到乌司他丁纯品,大大简化了制备工艺的同时保证成品的纯度和收率,药物活性有很大提升。
【EN】Paragraph:The present invention provides a kind of ulinastatin and preparation method thereof, absorbance value of 100000 units/ml ulinastatin at 405nm is no more than 0.18, the content of human urokinase-type peptidase is no more than 0.0005PNAU, and content of beary metal is no more than 1.6 μ g/ml, and bacteria endotoxin content is no more than 6.0EU/ml.Urine is pre-processed by the methods of nitrite, ammonium sulfate, polyethylene glycol, adjusting pH, preliminary purifying is carried out using the method for dialysis before crossing column simultaneously, gel column is passed sequentially through again and ulinastatin sterling is prepared in cation exchange column, guarantee the purity and yield of finished product while enormously simplifying preparation process, pharmaceutical activity has very big promotion.

主权项:
【中文】1.一种乌司他丁,其特征在于,所述乌司他丁浓度为10万单位/ml时,在405nm处的吸光度值不超过0.18,人尿激肽原酶的含量不超过0.0005PNAU,重金属含量不超过1.6μg/ml,细菌内毒素含量不超过6.0EU。【EN】1. a kind of ulinastatin, which is characterized in that when the ulinastatin concentration is 100,000 units/ml, the extinction at 405nm Angle value is no more than 0.18, and the content of human urokinase-type peptidase is no more than 0.0005PNAU, and content of beary metal is no more than 1.6 μ g/ml, carefully Bacterium endotoxin content is no more than 6.0EU.


相似专利
说明书

【中文】

高纯度乌司他丁及其制备方法和含有乌司他丁的药物组合物

技术领域

本发明涉及一种生物技术领域,具体涉及高纯度乌司他丁及其制备方法和含有乌司他丁的药物组合物。

背景技术

乌司他丁是从健康成年男性新鲜尿液中分离纯化出来的一种糖蛋白,由143个氨基酸组成,相对分子质量约67000。1909年,Beurer和Reich第一次报道了人尿液中存在这种胰蛋白酶抑制剂,对胰蛋白酶、α-糜蛋白酶等丝氨酸蛋白酶及粒细胞弹性蛋白酶、透明质酸酶、巯基酶、纤溶酶等多种酶有抑制作用。另具有稳定溶酶体膜,抑制溶酶体酶的释放,抑制心肌抑制因子(MDF)产生,清除氧自由基及抑制炎症介质释放的作用。乌司他丁还可改善手术刺激引起的免疫功能下降、蛋白代谢异常和肾功能降低,防止手术刺激引起的对内脏器官与细胞的损伤以及改善休克时的循环状态等。

乌司他丁多样的生物功能与其独特的分子结构相关,乌司他丁主要由3个功能域构成,包括O连接糖基化区域(Alal-Lys21)、N端Kunitz型结构域 I (Lys22-Arg77 )和具有胰蛋白酶抑制活性的 C 端Kunitz型结构域II (Thr78Leul43 )。两个Kunitz型结构域具有一定的同源性,每个结构域中都含有 6 个保守的半胱氨酸,通过形成 3 对二硫键维持一定的空间构型。乌司他丁的酶抑制活性主要存在于主体蛋白骨架中的Kunitz型结构域。

专利CN100528898C公开了高纯度乌司他丁及其制备方法和含有乌司他丁的药物组合物,其中乌司他丁粗品经过阴离子交换柱、金属螯合柱、亲和层析柱等多重柱层析制备得到。专利CN100425286C公开了纯化的乌司他丁及其制备方法和含有乌司他丁的药物组合物,同样经过阴离子交换柱、金属螯合柱、疏水柱吸附、凝胶柱层析等多种柱层析制备得到。上述专利中纯化方法经过多次柱层析,工艺过程复杂,产品收率低,生产周期长,不易放大生产。CN 107827976 A公开了一种基于疏水柱的乌司他丁纯化方法,仅仅通过一步疏水柱实现纯化,并且纯度达到了99.90%以上,比活力至少为5100U/mg,但是其疏水柱采用尿素进行保藏而实现重复利用,再次使用大大影响了乌司他丁纯品的收率。

发明内容

基于上述现有技术的缺陷,本发明提供一种乌司他丁及其制备方法与应用,该乌司他丁中杂质含量少,通过改变粗品制备方法和改进纯化工艺,进一步控制乌司他丁分子量范围,显著提高了药物活性。

本发明提供一种乌司他丁,其浓度为10万单位/ml时,在405nm处的吸光度值不超过0.18,人尿激肽原酶的含量不超过0.0005PNAU,重金属含量不超过1.6μg/ml,细菌内毒素含量不超过6.0EU。

进一步地,所述乌司他丁浓度为10万单位/ml时,在405nm处的吸光度值不超过0.16,人尿激肽原酶的含量不超过0.0004PNAU,重金属含量不超过1.0μg/ml,细菌内毒素含量不超过5.0EU。

进一步地,所述乌司他丁采用十二烷基硫酸钠聚丙烯酰胺凝胶电泳测定的分子量为37,000-43,000Da或采用高效液相色谱法测定的分子量为62,000-72,000Da。

本发明进一步提供一种上述乌司他丁的制备方法,包括以下步骤:

(1)尿液中加入亚硝酸盐,调节pH,搅拌,硫酸铵溶液进行洗脱,洗脱液进行抽滤,保留滤饼;

(2)滤饼加水溶解,调节pH,加入聚乙二醇,过滤,沉淀物干燥;

(3)步骤(2)沉淀物加水溶解,透析,干燥,即得乌司他丁粗品;

(4)乌司他丁粗品加水溶解,过凝胶柱层析,蒸馏水洗脱,收集组分,超滤;

(5)步骤(4)样品加入阳离子交换层析柱上样,用盐酸洗脱,收集洗脱液浓缩,冷冻干燥,即得乌司他丁纯品。

进一步地,所述步骤(1)中尿液与亚硝酸盐的重量比为1吨:13-16kg。

更进一步地,所述步骤(1)中调节pH至4.0-6.0。

更进一步地,所述亚硝酸盐选自亚硝酸铵、亚硝酸钠中的一种。

更进一步地,所述亚硝酸盐为亚硝酸铵。亚硝酸盐中的亚硝酸根离子可以与尿液中的尿素反应生成氮气、二氧化碳气体,有利于尿液中尿素的处理。

进一步地,所述步骤(1)中硫酸铵溶液浓度为8-12%。硫酸铵溶液的加入促使尿液中的乌司他丁蛋白类物质发生可逆性变性而沉淀下来。

进一步地,所述步骤(2)中滤饼加1-3倍水溶解,调节pH5.0-6.0,加入8-12倍聚乙二醇,搅拌20-40min,静置,板框过滤,沉淀物冷冻干燥。

进一步地,所述步骤(3)中沉淀物加水溶解,4℃透析,期间更换3-5次水,冷冻干燥,即得乌司他丁粗品。

进一步地,所述步骤(4)中凝胶柱层析填料为葡聚糖凝胶G-75,洗脱速度为0.4-0.6ml/min。

进一步地,所述步骤(4)中超滤具体为洗脱液中加入0.2-0.4gEDTA-Na/升,用分子量为3万的超滤膜超滤。

进一步地,所述步骤(5)中阳离子交换柱为732型阳离子交换树脂。

进一步地,所述步骤(5)中盐酸浓度为1.5-3mol/L。

本发明还提供了含有上述乌司他丁或上述制备方法制备得到的乌司他丁的组合物。

进一步地,所述组合物包括乌司他丁和甘露醇。

更进一步地,所述组合物制备方法为:甘露醇注射用水溶解,加入乌司他丁混合后调节pH6-7,滤膜过滤,即得。

本发明还提供了上述乌司他丁上述制备方法制备得到的乌司他丁在制备蛋白酶抑制剂药物中的应用。

本发明的有益效果:

(1)本发明采用亚硝酸盐、硫酸铵、聚乙二醇、调节pH等方法对尿液进行预处理,其中亚硝酸盐中的亚硝酸离子和尿液中尿素反应,生成氮气、二氧化碳气体,有利于尿液中尿素的处理,大大提高了预处理后乌司他丁的纯度,提高后续柱层析的效率;同时在过柱前采用透析的方法先进行初步的纯化,避免过多的杂质影响柱层析的效率。若仍有少量过量的亚硝酸根离子,也可在透析的过程中被去除。透析液中的亚硝酸根离子通过加入氧化剂即可完全去除,避免环境污染。

(2)本发明采用凝胶柱层析和阳离子交换柱层析结合的方法,对杂质进行更有效的去除,不仅提高了药物纯度,还提高了产物收率和药物活性。

具体实施方式

实施例1乌司他丁及其制备方法

(1)1吨尿液中加入14.7kg亚硝酸铵,调节pH至5.0,搅拌50min,用浓度为10%的硫酸铵溶液进行洗脱,洗脱液进行抽滤,保留滤饼;

(2)滤饼加2倍水溶解,调节pH5.0-6.0,加入8倍聚乙二醇,搅拌30min,静置,板框过滤,沉淀物冷冻干燥;

(3)步骤(2)沉淀物加水溶解,4℃透析,期间更换4次水,冷冻干燥,即得乌司他丁粗品;

(4)乌司他丁粗品加水溶解,过葡聚糖凝胶G-75,蒸馏水洗脱,流速0.6ml/min,收集洗脱组分,加入0.3gEDTA-Na/升,用分子量为3万的超滤膜超滤;

(5)步骤(4)样品加入732型阳离子交换层析柱上样,用1.5mol/L盐酸洗脱,流速1ml/min,收集洗脱液浓缩,冷冻干燥,即得乌司他丁纯品。

其中,步骤(3)中透析采用透析袋MD40(6000-8000),压平宽度40mm,直径25.5mm。

取乌司他丁纯品1亿单位,称取30g甘露醇,加500ml注射用水溶解,混合后调节pH6-7,再添加注射用水至2000ml,滤膜无菌过滤,分装于西林瓶中,冻干即得乌司他丁药物组合物。

得到的乌司他丁纯品稀释至每毫升10万单位进项理化检测:

重金属检测:按照中国药典2015版第四部0821重金属检查法第二法进行检测。

分子量HPLC法:

(1)液相条件:多孔硅胶排阻色谱柱;检测波长280nm;调节流速使得牛血清白蛋白的保留时间约为36分钟。

(2)流动相制备:取16.33克磷酸二氢钾和124.15克乙二醇溶解并稀释至1000ml,必要时用磷酸调节pH至4.0。

(3)样品制备:取乌司他丁,用流动相稀释制成每1ml中约含6500单位的溶液,作为样品溶液。

(4)参照品溶液:用流动相溶解适量的γ球蛋白(分子量160,000),牛血清白蛋白(分子量67,000),肌血球蛋白(分子量17,000)并制成1mg/ml的混合溶液。

(5)测定:各取样品溶液以及参照品溶液50ul进行高效液相色谱实验,以参照品分子量的对数值为纵坐标,以保留时间为横坐标制备标准曲线。根据参照品标准曲线求得样品的分子量。

其余参照中国药典2015版第一增补本212页-213页乌司他丁及乌司他丁溶液项下检测项目进行检测,结果如下:

检查项目

检查结果

颜色A405

0.15

人尿激肽原酶的含量

0.0003PNAU

重金属

<1.0μg/ml

细菌内毒素

<5.0EU

SDS-PAGE分子量

40,312 Da

HPLC分子量

67,076Da

实施例2乌司他丁及其制备方法

(1)1吨尿液中加入16kg亚硝酸铵,调节pH至4.0,搅拌55min,用浓度为12%的硫酸铵溶液进行洗脱,洗脱液进行抽滤,保留滤饼;

(2)滤饼加1倍水溶解,调节pH5.0-6.0,加入10倍聚乙二醇,搅拌20min,静置,板框过滤,沉淀物冷冻干燥;

(3)步骤(2)沉淀物加水溶解,4℃透析,期间更换3次水,冷冻干燥,即得乌司他丁粗品;

(4)乌司他丁粗品加水溶解,过葡聚糖凝胶G-75,蒸馏水洗脱,流速0.4ml/min,收集洗脱组分,加入0.2gEDTA-Na/升,用分子量为3万的超滤膜超滤;

(5)步骤(4)样品加入732型阳离子交换层析柱上样,用2.0mol/L盐酸洗脱,流速1ml/min,收集洗脱液浓缩,冷冻干燥,即得乌司他丁纯品。

其中,步骤(3)中透析采用透析袋MD40(6000-8000),压平宽度40mm,直径25.5mm。

取乌司他丁纯品1亿单位,称取30g甘露醇,加500ml注射用水溶解,混合后调节pH6-7,再添加注射用水至2000ml,滤膜无菌过滤,分装于西林瓶中,冻干即得乌司他丁药物组合物。

得到的乌司他丁纯品稀释至每毫升10万单位进项理化检测:

重金属检测:按照中国药典2015版第四部0821重金属检查法第二法进行检测。

分子量HPLC法:

(1)液相条件:多孔硅胶排阻色谱柱;检测波长280nm;调节流速使得牛血清白蛋白的保留时间约为36分钟。

(2)流动相制备:取16.33克磷酸二氢钾和124.15克乙二醇溶解并稀释至1000ml,必要时用磷酸调节pH至4.0。

(3)样品制备:取乌司他丁,用流动相稀释制成每1ml中约含6500单位的溶液,作为样品溶液。

(4)参照品溶液:用流动相溶解适量的γ球蛋白(分子量160,000),牛血清白蛋白(分子量67,000),肌血球蛋白(分子量17,000)并制成1mg/ml的混合溶液。

(5)测定:各取样品溶液以及参照品溶液50ul进行高效液相色谱实验,以参照品分子量的对数值为纵坐标,以保留时间为横坐标制备标准曲线。根据参照品标准曲线求得样品的分子量。

其余参照中国药典2015版第一增补本212页-213页乌司他丁及乌司他丁溶液项下检测项目进行检测,结果如下:

检查项目

检查结果

颜色A405

0.14

人尿激肽原酶的含量

0.0004PNAU

重金属

<1.0μg/ml

细菌内毒素

<5.0EU

SDS-PAGE分子量

38,769Da

HPLC分子量

67,750 Da

实施例3乌司他丁及其制备方法

(1)1吨尿液中加入13kg亚硝酸铵,调节pH至6.0,搅拌50min,用浓度为8%的硫酸铵溶液进行洗脱,洗脱液进行抽滤,保留滤饼;

(2)滤饼加3倍水溶解,调节pH5.0-6.0,加入12倍聚乙二醇,搅拌40min,静置,板框过滤,沉淀物冷冻干燥;

(3)步骤(2)沉淀物加水溶解,4℃透析,期间更换5次水,冷冻干燥,即得乌司他丁粗品;

(4)乌司他丁粗品加水溶解,过葡聚糖凝胶G-75,蒸馏水洗脱,流速0.5ml/min,收集洗脱组分,加入0.4gEDTA-Na/升,用分子量为3万的超滤膜超滤;

(5)步骤(4)样品加入732型阳离子交换层析柱上样,用3mol/L盐酸洗脱,流速1ml/min,收集洗脱液浓缩,冷冻干燥,即得乌司他丁纯品。

其中,步骤(3)中透析采用透析袋MD40(6000-8000),压平宽度40mm,直径25.5mm。

取乌司他丁纯品1亿单位,称取30g甘露醇,加500ml注射用水溶解,混合后调节pH6-7,再添加注射用水至2000ml,滤膜无菌过滤,分装于西林瓶中,冻干即得乌司他丁药物组合物。

得到的乌司他丁纯品稀释至每毫升10万单位进项理化检测:

重金属检测:按照中国药典2015版第四部0821重金属检查法第二法进行检测。

分子量HPLC法:

(1)液相条件:多孔硅胶排阻色谱柱;检测波长280nm;调节流速使得牛血清白蛋白的保留时间约为36分钟。

(2)流动相制备:取16.33克磷酸二氢钾和124.15克乙二醇溶解并稀释至1000ml,必要时用磷酸调节pH至4.0。

(3)样品制备:取乌司他丁,用流动相稀释制成每1ml中约含6500单位的溶液,作为样品溶液。

(4)参照品溶液:用流动相溶解适量的γ球蛋白(分子量160,000),牛血清白蛋白(分子量67,000),肌血球蛋白(分子量17,000)并制成1mg/ml的混合溶液。

(5)测定:各取样品溶液以及参照品溶液50ul进行高效液相色谱实验,以参照品分子量的对数值为纵坐标,以保留时间为横坐标制备标准曲线。根据参照品标准曲线求得样品的分子量。

其余参照中国药典2015版第一增补本212页-8213页乌司他丁及乌司他丁溶液项下检测项目进行检测,结果如下:

检查项目

检查结果

颜色A405

0.16

人尿激肽原酶的含量

0.0003PNAU

重金属

<1.0μg/ml

细菌内毒素

<5.0EU

SDS-PAGE分子量

40,706Da

HPLC分子量

66,439Da

对比例1采用甲壳素代替亚硝酸盐制备得到的乌司他丁及其制备方法

(1)1吨尿液中加入500g甲壳素,搅拌50min,用浓度为10%的硫酸铵溶液进行洗脱,抽滤,保留滤饼;

(2)滤饼加2倍水溶解,调节pH5.0-6.0,加入8倍聚乙二醇,搅拌30min,静置,板框过滤,沉淀物冷冻干燥;

(3)步骤(2)沉淀物加水溶解,4℃透析,期间更换4次水,冷冻干燥,即得乌司他丁粗品;

(4)乌司他丁粗品加水溶解,过葡聚糖凝胶G-75,蒸馏水洗脱,流速0.6ml/min,收集洗脱组分,加入0.3gEDTA-Na/升,用分子量为3万的超滤膜超滤;

(5)步骤(4)样品加入732型阳离子交换层析柱上样,用1.5mol/L盐酸洗脱,流速1ml/min,收集洗脱液浓缩,冷冻干燥,即得乌司他丁纯品。

其中,步骤(3)中透析采用透析袋MD40(6000-8000),压平宽度40mm,直径25.5mm。

取乌司他丁纯品1亿单位,称取30g甘露醇,加500ml注射用水溶解,混合后调节pH6-7,再添加注射用水至2000ml,滤膜无菌过滤,分装于西林瓶中,冻干即得乌司他丁药物组合物。

得到的乌司他丁纯品稀释至每毫升10万单位进项理化检测:

重金属检测:按照中国药典2015版第四部0821重金属检查法第二法进行检测。

分子量HPLC法:

(1)液相条件:多孔硅胶排阻色谱柱;检测波长280nm;调节流速使得牛血清白蛋白的保留时间约为36分钟。

(2)流动相制备:取16.33克磷酸二氢钾和124.15克乙二醇溶解并稀释至1000ml,必要时用磷酸调节pH至4.0。

(3)样品制备:取乌司他丁,用流动相稀释制成每1ml中约含6500单位的溶液,作为样品溶液。

(4)参照品溶液:用流动相溶解适量的γ球蛋白(分子量160,000),牛血清白蛋白(分子量67,000),肌血球蛋白(分子量17,000)并制成1mg/ml的混合溶液。

(5)测定:各取样品溶液以及参照品溶液50ul进行高效液相色谱实验,以参照品分子量的对数值为纵坐标,以保留时间为横坐标制备标准曲线。根据参照品标准曲线求得样品的分子量。

其余参照中国药典2015版第一增补本212页-213页乌司他丁及乌司他丁溶液项下检测项目进行检测,结果如下:

检查项目

检查结果

颜色A405

0.19

人尿激肽原酶的含量

0.0005PNAU

重金属

<2.0μg/ml

细菌内毒素

<6.25EU

SDS-PAGE分子量

41,141Da

HPLC分子量

68,413Da

对比例2不进行透析制备得到的乌司他丁及其制备方法

(1)1吨尿液中加入14.7kg亚硝酸铵,调节pH至5.0,搅拌50min,用浓度为10%的硫酸铵溶液进行洗脱,洗脱液进行抽滤,保留滤饼;

(2)滤饼加2倍水溶解,调节pH5.0-6.0,加入8倍聚乙二醇,搅拌30min,静置,板框过滤,沉淀物冷冻干燥,得乌司他丁粗品;

(3)乌司他丁粗品加水溶解,过葡聚糖凝胶G-75,蒸馏水洗脱,流速0.6ml/min,收集洗脱组分,加入0.3gEDTA-Na/升,用分子量为3万的超滤膜超滤;

(4)步骤(3)样品加入732型阳离子交换层析柱上样,用1.5mol/L盐酸洗脱,流速1ml/min,收集洗脱液浓缩,冷冻干燥,即得乌司他丁纯品。

得到的乌司他丁纯品稀释至每毫升10万单位进项理化检测:

重金属检测:按照中国药典2015版第四部0821重金属检查法第二法进行检测。

分子量HPLC法:

(1)液相条件:多孔硅胶排阻色谱柱;检测波长280nm;调节流速使得牛血清白蛋白的保留时间约为36分钟。

(2)流动相制备:取16.33克磷酸二氢钾和124.15克乙二醇溶解并稀释至1000ml,必要时用磷酸调节pH至4.0。

(3)样品制备:取乌司他丁,用流动相稀释制成每1ml中约含6500单位的溶液,作为样品溶液。

(4)参照品溶液:用流动相溶解适量的γ球蛋白(分子量160,000),牛血清白蛋白(分子量67,000),肌血球蛋白(分子量17,000)并制成1mg/ml的混合溶液。

(5)测定:各取样品溶液以及参照品溶液50ul进行高效液相色谱实验,以参照品分子量的对数值为纵坐标,以保留时间为横坐标制备标准曲线。根据参照品标准曲线求得样品的分子量。

其余参照中国药典2015版第一增补本212页-213页乌司他丁及乌司他丁溶液项下检测项目进行检测,结果如下:

检查项目

检查结果

颜色A405

0.19

人尿激肽原酶的含量

0.0005PNAU

重金属

<2.0μg/ml

细菌内毒素

<6.25EU

SDS-PAGE分子量

41,884Da

HPLC分子量

68,435Da

对比例3采用阴离子交换柱制备得到乌司他丁及其制备方法

(1)1吨尿液中加入14.7kg亚硝酸铵,调节pH至5.0,搅拌50min,用浓度为10%的硫酸铵溶液进行洗脱,洗脱液进行抽滤,保留滤饼;

(2)滤饼加2倍水溶解,调节pH5.0-6.0,加入8倍聚乙二醇,搅拌30min,静置,板框过滤,沉淀物冷冻干燥;

(3)步骤(2)沉淀物加水溶解,4℃透析,期间更换4次水,冷冻干燥,即得乌司他丁粗品;

(4)乌司他丁粗品加水溶解,过葡聚糖凝胶G-75,蒸馏水洗脱,流速0.6ml/min,收集洗脱组分,加入0.3gEDTA-Na/升,用分子量为3万的超滤膜超滤;

(5)步骤(4)样品加入阴离子交换层析柱QAE Sephadex A-25上样,用含1mol/LNaCl和0.3mol/L的磷酸盐缓冲液洗脱,流速1ml/min,收集洗脱液浓缩,冷冻干燥,即得乌司他丁纯品。

其中,步骤(3)中透析采用透析袋MD40(6000-8000),压平宽度40mm,直径25.5mm。

取乌司他丁纯品1亿单位,称取30g甘露醇,加500ml注射用水溶解,混合后调节pH6-7,再添加注射用水至2000ml,滤膜无菌过滤,分装于西林瓶中,冻干即得乌司他丁药物组合物。

得到的乌司他丁纯品稀释至每毫升10万单位进项理化检测:

重金属检测:按照中国药典2015版第四部0821重金属检查法第二法进行检测。

分子量HPLC法:

(1)液相条件:多孔硅胶排阻色谱柱;检测波长280nm;调节流速使得牛血清白蛋白的保留时间约为36分钟。

(2)流动相制备:取16.33克磷酸二氢钾和124.15克乙二醇溶解并稀释至1000ml,必要时用磷酸调节pH至4.0。

(3)样品制备:取乌司他丁,用流动相稀释制成每1ml中约含6500单位的溶液,作为样品溶液。

(4)参照品溶液:用流动相溶解适量的γ球蛋白(分子量160,000),牛血清白蛋白(分子量67,000),肌血球蛋白(分子量17,000)并制成1mg/ml的混合溶液。

(5)测定:各取样品溶液以及参照品溶液50ul进行高效液相色谱实验,以参照品分子量的对数值为纵坐标,以保留时间为横坐标制备标准曲线。根据参照品标准曲线求得样品的分子量。

其余参照中国药典2015版第一增补本212页-213页乌司他丁及乌司他丁溶液项下检测项目进行检测,结果如下:

检查项目

检查结果

颜色A405

0.20

人尿激肽原酶的含量

0.0005PNAU

重金属

<2.0μg/ml

细菌内毒素

<6.25EU

SDS-PAGE分子量

42,351Da

HPLC分子量

68,759 Da

对比例4不使用凝胶柱层析制备得到乌司他丁及其制备方法

(1)1吨尿液中加入14.7kg亚硝酸铵,调节pH至5.0,搅拌50min,用浓度为10%的硫酸铵溶液进行洗脱,洗脱液进行抽滤,保留滤饼;

(2)滤饼加2倍水溶解,调节pH5.0-6.0,加入8倍聚乙二醇,搅拌30min,静置,板框过滤,沉淀物冷冻干燥;

(3)步骤(2)沉淀物加水溶解,4℃透析,期间更换4次水,冷冻干燥,即得乌司他丁粗品;

(4)乌司他丁粗品加水溶解,加入0.3gEDTA-Na/升,用分子量为3万的超滤膜超滤;

(5)步骤(4)样品加入732型阳离子交换层析柱上样,用2mol/L盐酸洗脱,流速1ml/min,收集洗脱液;

其中,步骤(3)中透析采用透析袋MD40(6000-8000),压平宽度40mm,直径25.5mm。

取乌司他丁纯品1亿单位,称取30g甘露醇,加500ml注射用水溶解,混合后调节pH6-7,再添加注射用水至2000ml,滤膜无菌过滤,分装于西林瓶中,冻干即得乌司他丁药物组合物。

得到的乌司他丁纯品稀释至每毫升10万单位进项理化检测:

重金属检测:按照中国药典2015版第四部0821重金属检查法第二法进行检测。

分子量HPLC法:

(1)液相条件:多孔硅胶排阻色谱柱;检测波长280nm;调节流速使得牛血清白蛋白的保留时间约为36分钟。

(2)流动相制备:取16.33克磷酸二氢钾和124.15克乙二醇溶解并稀释至1000ml,必要时用磷酸调节pH至4.0。

(3)样品制备:取乌司他丁,用流动相稀释制成每1ml中约含6500单位的溶液,作为样品溶液。

(4)参照品溶液:用流动相溶解适量的γ球蛋白(分子量160,000),牛血清白蛋白(分子量67,000),肌血球蛋白(分子量17,000)并制成1mg/ml的混合溶液。

(5)测定:各取样品溶液以及参照品溶液50ul进行高效液相色谱实验,以参照品分子量的对数值为纵坐标,以保留时间为横坐标制备标准曲线。根据参照品标准曲线求得样品的分子量。

其余参照中国药典2015版第一增补本212页-213页乌司他丁及乌司他丁溶液项下检测项目进行检测,结果如下:

检查项目

检查结果

颜色A405

0.19

人尿激肽原酶的含量

0.0005PNAU

重金属

<2.0μg/ml

细菌内毒素

<6.25EU

SDS-PAGE分子量

37,764Da

HPLC分子量

63,673Da

实施例4不同乌司他丁比较

按照药典方法测定乌司他丁比活,同时比较乌司他丁制备方法的收率及其纯度。

上述详细说明是针对本发明其中之一可行实施例的具体说明,该实施例并非用以限制本发明的专利范围,凡未脱离本发明所为的等效实施或变更,均应包含于本发明技术方案的范围内。

【EN】

High-purity ulinastatin and preparation method thereof and pharmaceutical composition containing ulinastatin

Technical field

The present invention relates to a kind of field of biotechnology, and in particular to high-purity ulinastatin and preparation method thereof and containing crow

The pharmaceutical composition of Si Tading.

Background technique

Ulinastatin is a kind of glycoprotein for isolating and purifying out from healthy adult male freshly voided urine, by 143 ammonia

Base acid composition, relative molecular mass about 67000.1909, Beurer and Reich reported in human urine that there are this for the first time

Trypsin inhibitor, to the serine proteases such as trypsase, Chymetin and granulocyte elastase, hyaluronic acid

A variety of enzymes such as enzyme, sulfydryl enzyme, fibrinolysin have inhibiting effect.It is another to inhibit the release of lysosomal enzyme with lysosome membrane is stablized, inhibit

Myocardial depressant factor (MDF) (MDF) generates, scavenging activated oxygen and the effect for inhibiting inflammatory mediator release.Ulinastatin can also improve hand

The decline of immune function caused by art stimulates, protein metabolism exception and renal function reduce, internal internal organs caused by preventing operation from stimulating

Recurrent state the etc. when damage and improvement shock of official and cell.

The biological function of ulinastatin multiplicity is related to its unique molecular structure, and ulinastatin is mainly by 3 functional domains

It constitutes, including O connection glycosylated region (Alal-Lys21), N-terminal Kunitz type structural domain I (Lys22-Arg77) and has

The end the C Kunitz type domain II (Thr78Leul43) of trypsin inhibition activity.Two Kunitz type structural domains have

Certain homology passes through containing 6 conservative cysteines in each structural domain and forms 3 pairs of disulfide bond and maintain one

Fixed steric configuration.The enzyme inhibition activity of ulinastatin is primarily present in the Kunitz type structural domain in Bulk protein skeleton.

Patent CN100528898C discloses high-purity ulinastatin and preparation method thereof and the drug containing ulinastatin

Composition, wherein ulinastatin crude product is by the multiple column chromatography preparation such as anion-exchange column, metal chelating column, affinity column

It obtains.Patent CN100425286C discloses ulinastatin of purifying and preparation method thereof and the pharmaceutical composition containing ulinastatin

Object also passes through a variety of column chromatographies such as anion-exchange column, metal chelating column, drainage column absorption, gel filtration chromatography and is prepared.

Purification process is chromatographed by multiple column in above-mentioned patent, and complex technical process, product yield is low, and the production cycle is long, is not easy to amplify

Production.107827976 A of CN discloses a kind of ulinastatin purification process based on drainage column, only by a step drainage column

Realize purifying, and purity has reached 99.90% or more, Rate activity is at least 5100U/mg, but its drainage column using urea into

Row preservation and realize recycling, reuse the yield for leveraging ulinastatin sterling.

Summary of the invention

Based on the defect of the above-mentioned prior art, the present invention provides a kind of ulinastatin and the preparation method and application thereof, the crow

Impurity content is few in Si Tading, by changing crude product preparation method and improving purifying process, further controls ulinastatin molecule

Range is measured, pharmaceutical activity is significantly improved.

The present invention provides a kind of ulinastatin, and when concentration is 100,000 units/ml, the absorbance value at 405nm does not surpass

0.18 is crossed, the content of human urokinase-type peptidase is no more than 0.0005PNAU, and content of beary metal is no more than 1.6 μ g/ml, bacterial endotoxin

Content is no more than 6.0EU.

Further, when the ulinastatin concentration is 100,000 units/ml, the absorbance value at 405nm is no more than

0.16, the content of human urokinase-type peptidase is no more than 0.0004PNAU, and content of beary metal is no more than 1.0 μ g/ml, and bacterial endotoxin contains

Amount is no more than 5.0EU.

Further, the ulinastatin uses the molecular weight of sodium dodecyl sulfate polyacrylamide gel electrophoresis measurement

For 37,000-43,000Da or use the molecular weight of high effective liquid chromatography for measuring for 62,000-72,000Da.

The present invention further provides a kind of preparation methods of above-mentioned ulinastatin, comprising the following steps:

(1) nitrite is added in urine, adjusts pH, stirring, ammonium sulfate is eluted, and eluent is filtered, and is retained

Filter cake;

(2) filter cake is dissolved in water, and adjusts pH, and polyethylene glycol, filtering, drying precipitate is added;

(3) step (2) sediment is dissolved in water, dialysis, dry to get ulinastatin crude product;

(4) ulinastatin crude product is dissolved in water, and crosses gel filtration chromatography, distills water elution, collects component, ultrafiltration;

(5) cation-exchange chromatography post loading is added in step (4) sample, is eluted with hydrochloric acid, collects eluent concentration, and freezing is dry

It is dry to get ulinastatin sterling.

Further, urine and the weight ratio of nitrite are 1 ton: 13-16kg in the step (1).

Further, pH to 4.0-6.0 is adjusted in the step (1).

Further, the nitrite is selected from one of ammonium nilrite, sodium nitrite.

Further, the nitrite is ammonium nilrite.Nitrite ion in nitrite can be with urine

In urea reaction generate nitrogen, carbon dioxide gas, be conducive to the processing of urea in urine.

Further, ammonium sulfate concentration is 8-12% in the step (1).The addition of ammonium sulfate promotes urine

In ulinastatin protein matter occur invertibity denaturation and precipitate.

Further, filter cake adds 1-3 times of water dissolution in the step (2), adjusts pH5.0-6.0,8-12 times of poly- second is added

Glycol stirs 20-40min, stands, plate-frame filtering, sediment freeze-drying.

Further, sediment is dissolved in water in the step (3), 4 DEG C of dialysis, during which replaces 3-5 water, and freezing is dry

It is dry to get ulinastatin crude product.

Further, gel filtration chromatography filler is sephadex G -75, elution speed 0.4- in the step (4)

0.6ml/min。

Further, ultrafiltration is specially addition 0.2-0.4gEDTA-Na/ liter in eluent in the step (4), uses molecule

The ultrafiltration membrane ultrafiltration that amount is 30,000.

Further, cation exchange column is 732 type cation exchange resins in the step (5).

Further, concentration of hydrochloric acid is 1.5-3mol/L in the step (5).

The present invention also provides the combinations for the ulinastatin being prepared containing above-mentioned ulinastatin or above-mentioned preparation method

Object.

Further, the composition includes ulinastatin and mannitol.

Further, the preparation method of composition are as follows: the dissolution of mannitol water for injection, after ulinastatin mixing is added

Adjust pH6-7, membrane filtration to get.

The ulinastatin being prepared the present invention also provides the above-mentioned preparation method of above-mentioned ulinastatin is preparing protease

Application in inhibitor medicaments.

Beneficial effects of the present invention:

(1) present invention pre-processes urine using the methods of nitrite, ammonium sulfate, polyethylene glycol, adjusting pH, the Central Asia

Urea reaction in nitrite ion and urine in nitrate generates nitrogen, carbon dioxide gas, is conducive to urea in urine

Processing substantially increases the purity of ulinastatin after pretreatment, improves the efficiency of subsequent columns chromatography;Simultaneously using saturating before crossing column

The method of analysis first carries out preliminary purifying, the efficiency for avoiding excessive impurity effect column from chromatographing.If still there is a small amount of excessive nitrous

Acid ion can also be removed during dialysis.Nitrite ion in dialyzate can be complete by the way that oxidant is added

Full removal, avoids environmental pollution.

(2) method that the present invention is combined using gel filtration chromatography and cation exchange column chromatography carries out impurity more effective

Removal, not only increase pharmaceutical purity, also improve product yield and pharmaceutical activity.

Specific embodiment

1 ulinastatin of embodiment and preparation method thereof

14.7kg ammonium nilrite is added in (1) 1 ton of urine, adjusts pH to 5.0, stirs 50min, the ammonium sulfate for being 10% with concentration

Solution is eluted, and eluent is filtered, and retains filter cake;

(2) filter cake adds 2 times of water dissolutions, adjusts pH5.0-6.0,8 times of polyethylene glycol are added, stir 30min, stand, plate-frame filtering,

Sediment freeze-drying;

(3) step (2) sediment is dissolved in water, during which 4 DEG C of dialysis are replaced 4 water, are freeze-dried to get ulinastatin crude product;

(4) ulinastatin crude product is dissolved in water, and crosses sephadex G -75, distills water elution, flow velocity 0.6ml/min, and collection is washed

De- component, is added 0.3gEDTA-Na/ liter, the ultrafiltration membrane ultrafiltration for being 30,000 with molecular weight;

(5) 732 type cation-exchange chromatography post loadings are added in step (4) sample, are eluted with 1.5mol/L hydrochloric acid, flow velocity 1ml/

Min collects eluent concentration, is freeze-dried to get ulinastatin sterling.

Wherein, dialysis uses bag filter MD40(6000-8000 in step (3)), flatten width 40mm, diameter 25.5mm.

100,000,000 unit of ulinastatin sterling is taken, 30g mannitol is weighed, adds 500ml water for injection to dissolve, is adjusted after mixing

PH6-7, then add and inject water to 2000ml, filter membrane is sterile filtered, and is sub-packed in cillin bottle, is lyophilized up to ulinastatin drug

Composition.

Obtained ulinastatin sterling is diluted to every milliliter of 100,000 unit income Physico-chemical tests:

Heavy metal analysis: it is detected according to 2015 editions the 4th 0821 the second method of heavy metal inspection technique of Chinese Pharmacopoeia.

Molecular weight HPLC method:

(1) liquid-phase condition: Bio-sil exclusion chromatography column;Detection wavelength 280nm;Adjust the guarantor that flow velocity makes bovine serum albumin(BSA)

Staying the time is about 36 minutes.

(2) prepared by mobile phase: take 16.33 grams of potassium dihydrogen phosphates and 124.15 grams of ethylene glycol to dissolve and be diluted to 1000ml,

When necessary with phosphorus acid for adjusting pH to 4.0.

(3) sample preparation: taking ulinastatin, is made in every 1ml with flowing phase dilution containing about the solution of 6500 units, as

Sample solution.

(4) referring to product solution: with the flowing suitable gamma Globulin of phased soln (molecular weight 160,000), bovine serum albumin(BSA)

(molecular weight 67,000), flesh hyperglobulinemia (molecular weight 17,000) and the mixed solution that 1mg/ml is made.

(5) it measures: respectively taking sample solution and carry out high performance liquid chromatography experiment referring to product solution 50ul, referring to product point

The logarithm of son amount is ordinate, prepares standard curve by abscissa of retention time.Sample is acquired according to referring to product standard curve

The molecular weight of product.

Remaining is referring under Chinese Pharmacopoeia 2015 editions the first enlarged editions ulinastatin of page -213 of page 212 and ulinastatin solution item

Detection project is detected, as a result as follows:

Inspection item

Inspection result

Color A405

0.15

The content of human urokinase-type peptidase

0.0003PNAU

Heavy metal

1.0 μ g/ml of <

Bacterial endotoxin

< 5.0EU

SDS-PAGE molecular weight

40,312 Da

HPLC molecular weight

67,076Da

2 ulinastatin of embodiment and preparation method thereof

16kg ammonium nilrite is added in (1) 1 ton of urine, adjusts pH to 4.0, stirs 55min, the ammonium sulfate for being 12% with concentration is molten

Liquid is eluted, and eluent is filtered, and retains filter cake;

(2) filter cake adds 1 times of water dissolution, adjusts pH5.0-6.0,10 times of polyethylene glycol are added, stir 20min, stand, sheet frame mistake

Filter, sediment freeze-drying;

(3) step (2) sediment is dissolved in water, during which 4 DEG C of dialysis are replaced 3 water, are freeze-dried to get ulinastatin crude product;

(4) ulinastatin crude product is dissolved in water, and crosses sephadex G -75, distills water elution, flow velocity 0.4ml/min, and collection is washed

De- component, is added 0.2gEDTA-Na/ liter, the ultrafiltration membrane ultrafiltration for being 30,000 with molecular weight;

(5) 732 type cation-exchange chromatography post loadings are added in step (4) sample, are eluted with 2.0mol/L hydrochloric acid, flow velocity 1ml/

Min collects eluent concentration, is freeze-dried to get ulinastatin sterling.

Wherein, dialysis uses bag filter MD40(6000-8000 in step (3)), flatten width 40mm, diameter 25.5mm.

100,000,000 unit of ulinastatin sterling is taken, 30g mannitol is weighed, adds 500ml water for injection to dissolve, is adjusted after mixing

PH6-7, then add and inject water to 2000ml, filter membrane is sterile filtered, and is sub-packed in cillin bottle, is lyophilized up to ulinastatin drug

Composition.

Obtained ulinastatin sterling is diluted to every milliliter of 100,000 unit income Physico-chemical tests:

Heavy metal analysis: it is detected according to 2015 editions the 4th 0821 the second method of heavy metal inspection technique of Chinese Pharmacopoeia.

Molecular weight HPLC method:

(1) liquid-phase condition: Bio-sil exclusion chromatography column;Detection wavelength 280nm;Adjust the guarantor that flow velocity makes bovine serum albumin(BSA)

Staying the time is about 36 minutes.

(2) prepared by mobile phase: take 16.33 grams of potassium dihydrogen phosphates and 124.15 grams of ethylene glycol to dissolve and be diluted to 1000ml,

When necessary with phosphorus acid for adjusting pH to 4.0.

(3) sample preparation: taking ulinastatin, is made in every 1ml with flowing phase dilution containing about the solution of 6500 units, as

Sample solution.

(4) referring to product solution: with the flowing suitable gamma Globulin of phased soln (molecular weight 160,000), bovine serum albumin(BSA)

(molecular weight 67,000), flesh hyperglobulinemia (molecular weight 17,000) and the mixed solution that 1mg/ml is made.

(5) it measures: respectively taking sample solution and carry out high performance liquid chromatography experiment referring to product solution 50ul, referring to product point

The logarithm of son amount is ordinate, prepares standard curve by abscissa of retention time.Sample is acquired according to referring to product standard curve

The molecular weight of product.

Remaining is referring under Chinese Pharmacopoeia 2015 editions the first enlarged editions ulinastatin of page -213 of page 212 and ulinastatin solution item

Detection project is detected, as a result as follows:

Inspection item

Inspection result

Color A405

0.14

The content of human urokinase-type peptidase

0.0004PNAU

Heavy metal

1.0 μ g/ml of <

Bacterial endotoxin

< 5.0EU

SDS-PAGE molecular weight

38,769Da

HPLC molecular weight

67,750 Da

3 ulinastatin of embodiment and preparation method thereof

13kg ammonium nilrite is added in (1) 1 ton of urine, adjusts pH to 6.0, stirs 50min, the ammonium sulfate for being 8% with concentration

It is eluted, eluent is filtered, and filter cake is retained;

(2) filter cake adds 3 times of water dissolutions, adjusts pH5.0-6.0,12 times of polyethylene glycol are added, stir 40min, stand, sheet frame mistake

Filter, sediment freeze-drying;

(3) step (2) sediment is dissolved in water, during which 4 DEG C of dialysis are replaced 5 water, are freeze-dried to get ulinastatin crude product;

(4) ulinastatin crude product is dissolved in water, and crosses sephadex G -75, distills water elution, flow velocity 0.5ml/min, and collection is washed

De- component, is added 0.4gEDTA-Na/ liter, the ultrafiltration membrane ultrafiltration for being 30,000 with molecular weight;

(5) 732 type cation-exchange chromatography post loadings are added in step (4) sample, are eluted with 3mol/L hydrochloric acid, flow velocity 1ml/min,

Eluent concentration is collected, is freeze-dried to get ulinastatin sterling.

Wherein, dialysis uses bag filter MD40(6000-8000 in step (3)), flatten width 40mm, diameter 25.5mm.

100,000,000 unit of ulinastatin sterling is taken, 30g mannitol is weighed, adds 500ml water for injection to dissolve, is adjusted after mixing

PH6-7, then add and inject water to 2000ml, filter membrane is sterile filtered, and is sub-packed in cillin bottle, is lyophilized up to ulinastatin drug

Composition.

Obtained ulinastatin sterling is diluted to every milliliter of 100,000 unit income Physico-chemical tests:

Heavy metal analysis: it is detected according to 2015 editions the 4th 0821 the second method of heavy metal inspection technique of Chinese Pharmacopoeia.

Molecular weight HPLC method:

(1) liquid-phase condition: Bio-sil exclusion chromatography column;Detection wavelength 280nm;Adjust the guarantor that flow velocity makes bovine serum albumin(BSA)

Staying the time is about 36 minutes.

(2) prepared by mobile phase: take 16.33 grams of potassium dihydrogen phosphates and 124.15 grams of ethylene glycol to dissolve and be diluted to 1000ml,

When necessary with phosphorus acid for adjusting pH to 4.0.

(3) sample preparation: taking ulinastatin, is made in every 1ml with flowing phase dilution containing about the solution of 6500 units, as

Sample solution.

(4) referring to product solution: with the flowing suitable gamma Globulin of phased soln (molecular weight 160,000), bovine serum albumin(BSA)

(molecular weight 67,000), flesh hyperglobulinemia (molecular weight 17,000) and the mixed solution that 1mg/ml is made.

(5) it measures: respectively taking sample solution and carry out high performance liquid chromatography experiment referring to product solution 50ul, referring to product point

The logarithm of son amount is ordinate, prepares standard curve by abscissa of retention time.Sample is acquired according to referring to product standard curve

The molecular weight of product.

Remaining is referring to Chinese Pharmacopoeia 2015 editions the first enlarged editions ulinastatin of page -8213 of page 212 and ulinastatin solution item

Lower detection project is detected, as a result as follows:

Inspection item

Inspection result

Color A405

0.16

The content of human urokinase-type peptidase

0.0003PNAU

Heavy metal

1.0 μ g/ml of <

Bacterial endotoxin

< 5.0EU

SDS-PAGE molecular weight

40,706Da

HPLC molecular weight

66,439Da

The ulinastatin and preparation method thereof that comparative example 1 replaces nitrite to be prepared using chitin

500g chitin is added in (1) 1 ton of urine, stirs 50min, is eluted with the ammonium sulfate that concentration is 10%, takes out

Filter retains filter cake;

(2) filter cake adds 2 times of water dissolutions, adjusts pH5.0-6.0,8 times of polyethylene glycol are added, stir 30min, stand, plate-frame filtering,

Sediment freeze-drying;

(3) step (2) sediment is dissolved in water, during which 4 DEG C of dialysis are replaced 4 water, are freeze-dried to get ulinastatin crude product;

(4) ulinastatin crude product is dissolved in water, and crosses sephadex G -75, distills water elution, flow velocity 0.6ml/min, and collection is washed

De- component, is added 0.3gEDTA-Na/ liter, the ultrafiltration membrane ultrafiltration for being 30,000 with molecular weight;

(5) 732 type cation-exchange chromatography post loadings are added in step (4) sample, are eluted with 1.5mol/L hydrochloric acid, flow velocity 1ml/

Min collects eluent concentration, is freeze-dried to get ulinastatin sterling.

Wherein, dialysis uses bag filter MD40(6000-8000 in step (3)), flatten width 40mm, diameter 25.5mm.

100,000,000 unit of ulinastatin sterling is taken, 30g mannitol is weighed, adds 500ml water for injection to dissolve, is adjusted after mixing

PH6-7, then add and inject water to 2000ml, filter membrane is sterile filtered, and is sub-packed in cillin bottle, is lyophilized up to ulinastatin drug

Composition.

Obtained ulinastatin sterling is diluted to every milliliter of 100,000 unit income Physico-chemical tests:

Heavy metal analysis: it is detected according to 2015 editions the 4th 0821 the second method of heavy metal inspection technique of Chinese Pharmacopoeia.

Molecular weight HPLC method:

(1) liquid-phase condition: Bio-sil exclusion chromatography column;Detection wavelength 280nm;Adjust the guarantor that flow velocity makes bovine serum albumin(BSA)

Staying the time is about 36 minutes.

(2) prepared by mobile phase: take 16.33 grams of potassium dihydrogen phosphates and 124.15 grams of ethylene glycol to dissolve and be diluted to 1000ml,

When necessary with phosphorus acid for adjusting pH to 4.0.

(3) sample preparation: taking ulinastatin, is made in every 1ml with flowing phase dilution containing about the solution of 6500 units, as

Sample solution.

(4) referring to product solution: with the flowing suitable gamma Globulin of phased soln (molecular weight 160,000), bovine serum albumin(BSA)

(molecular weight 67,000), flesh hyperglobulinemia (molecular weight 17,000) and the mixed solution that 1mg/ml is made.

(5) it measures: respectively taking sample solution and carry out high performance liquid chromatography experiment referring to product solution 50ul, referring to product point

The logarithm of son amount is ordinate, prepares standard curve by abscissa of retention time.Sample is acquired according to referring to product standard curve

The molecular weight of product.

Remaining is referring under Chinese Pharmacopoeia 2015 editions the first enlarged editions ulinastatin of page -213 of page 212 and ulinastatin solution item

Detection project is detected, as a result as follows:

Inspection item

Inspection result

Color A405

0.19

The content of human urokinase-type peptidase

0.0005PNAU

Heavy metal

2.0 μ g/ml of <

Bacterial endotoxin

< 6.25EU

SDS-PAGE molecular weight

41,141Da

HPLC molecular weight

68,413Da

Comparative example 2 is without the ulinastatin and preparation method thereof being prepared of dialysing

14.7kg ammonium nilrite is added in (1) 1 ton of urine, adjusts pH to 5.0, stirs 50min, the ammonium sulfate for being 10% with concentration

Solution is eluted, and eluent is filtered, and retains filter cake;

(2) filter cake adds 2 times of water dissolutions, adjusts pH5.0-6.0,8 times of polyethylene glycol are added, stir 30min, stand, plate-frame filtering,

Sediment freeze-drying, obtains ulinastatin crude product;

(3) ulinastatin crude product is dissolved in water, and crosses sephadex G -75, distills water elution, flow velocity 0.6ml/min, and collection is washed

De- component, is added 0.3gEDTA-Na/ liter, the ultrafiltration membrane ultrafiltration for being 30,000 with molecular weight;

(4) 732 type cation-exchange chromatography post loadings are added in step (3) sample, are eluted with 1.5mol/L hydrochloric acid, flow velocity 1ml/

Min collects eluent concentration, is freeze-dried to get ulinastatin sterling.

Obtained ulinastatin sterling is diluted to every milliliter of 100,000 unit income Physico-chemical tests:

Heavy metal analysis: it is detected according to 2015 editions the 4th 0821 the second method of heavy metal inspection technique of Chinese Pharmacopoeia.

Molecular weight HPLC method:

(1) liquid-phase condition: Bio-sil exclusion chromatography column;Detection wavelength 280nm;Adjust the guarantor that flow velocity makes bovine serum albumin(BSA)

Staying the time is about 36 minutes.

(2) prepared by mobile phase: take 16.33 grams of potassium dihydrogen phosphates and 124.15 grams of ethylene glycol to dissolve and be diluted to 1000ml,

When necessary with phosphorus acid for adjusting pH to 4.0.

(3) sample preparation: taking ulinastatin, is made in every 1ml with flowing phase dilution containing about the solution of 6500 units, as

Sample solution.

(4) referring to product solution: with the flowing suitable gamma Globulin of phased soln (molecular weight 160,000), bovine serum albumin(BSA)

(molecular weight 67,000), flesh hyperglobulinemia (molecular weight 17,000) and the mixed solution that 1mg/ml is made.

(5) it measures: respectively taking sample solution and carry out high performance liquid chromatography experiment referring to product solution 50ul, referring to product point

The logarithm of son amount is ordinate, prepares standard curve by abscissa of retention time.Sample is acquired according to referring to product standard curve

The molecular weight of product.

Remaining is referring under Chinese Pharmacopoeia 2015 editions the first enlarged editions ulinastatin of page -213 of page 212 and ulinastatin solution item

Detection project is detected, as a result as follows:

Inspection item

Inspection result

Color A405

0.19

The content of human urokinase-type peptidase

0.0005PNAU

Heavy metal

2.0 μ g/ml of <

Bacterial endotoxin

< 6.25EU

SDS-PAGE molecular weight

41,884Da

HPLC molecular weight

68,435Da

Ulinastatin and preparation method thereof is prepared using anion-exchange column in comparative example 3

14.7kg ammonium nilrite is added in (1) 1 ton of urine, adjusts pH to 5.0, stirs 50min, the ammonium sulfate for being 10% with concentration

Solution is eluted, and eluent is filtered, and retains filter cake;

(2) filter cake adds 2 times of water dissolutions, adjusts pH5.0-6.0,8 times of polyethylene glycol are added, stir 30min, stand, plate-frame filtering,

Sediment freeze-drying;

(3) step (2) sediment is dissolved in water, during which 4 DEG C of dialysis are replaced 4 water, are freeze-dried to get ulinastatin crude product;

(4) ulinastatin crude product is dissolved in water, and crosses sephadex G -75, distills water elution, flow velocity 0.6ml/min, and collection is washed

De- component, is added 0.3gEDTA-Na/ liter, the ultrafiltration membrane ultrafiltration for being 30,000 with molecular weight;

(5) anion exchange chromatography QAE Sephadex A-25 loading is added in step (4) sample, with containing 1mol/LNaCl and

The phosphate buffer of 0.3mol/L elutes, flow velocity 1ml/min, collects eluent concentration, is freeze-dried pure to get ulinastatin

Product.

Wherein, dialysis uses bag filter MD40(6000-8000 in step (3)), flatten width 40mm, diameter 25.5mm.

100,000,000 unit of ulinastatin sterling is taken, 30g mannitol is weighed, adds 500ml water for injection to dissolve, is adjusted after mixing

PH6-7, then add and inject water to 2000ml, filter membrane is sterile filtered, and is sub-packed in cillin bottle, is lyophilized up to ulinastatin drug

Composition.

Obtained ulinastatin sterling is diluted to every milliliter of 100,000 unit income Physico-chemical tests:

Heavy metal analysis: it is detected according to 2015 editions the 4th 0821 the second method of heavy metal inspection technique of Chinese Pharmacopoeia.

Molecular weight HPLC method:

(1) liquid-phase condition: Bio-sil exclusion chromatography column;Detection wavelength 280nm;Adjust the guarantor that flow velocity makes bovine serum albumin(BSA)

Staying the time is about 36 minutes.

(2) prepared by mobile phase: take 16.33 grams of potassium dihydrogen phosphates and 124.15 grams of ethylene glycol to dissolve and be diluted to 1000ml,

When necessary with phosphorus acid for adjusting pH to 4.0.

(3) sample preparation: taking ulinastatin, is made in every 1ml with flowing phase dilution containing about the solution of 6500 units, as

Sample solution.

(4) referring to product solution: with the flowing suitable gamma Globulin of phased soln (molecular weight 160,000), bovine serum albumin(BSA)

(molecular weight 67,000), flesh hyperglobulinemia (molecular weight 17,000) and the mixed solution that 1mg/ml is made.

(5) it measures: respectively taking sample solution and carry out high performance liquid chromatography experiment referring to product solution 50ul, referring to product point

The logarithm of son amount is ordinate, prepares standard curve by abscissa of retention time.Sample is acquired according to referring to product standard curve

The molecular weight of product.

Remaining is referring under Chinese Pharmacopoeia 2015 editions the first enlarged editions ulinastatin of page -213 of page 212 and ulinastatin solution item

Detection project is detected, as a result as follows:

Inspection item

Inspection result

Color A405

0.20

The content of human urokinase-type peptidase

0.0005PNAU

Heavy metal

2.0 μ g/ml of <

Bacterial endotoxin

< 6.25EU

SDS-PAGE molecular weight

42,351Da

HPLC molecular weight

68,759 Da

Ulinastatin and preparation method thereof is prepared without using gel filtration chromatography in comparative example 4

14.7kg ammonium nilrite is added in (1) 1 ton of urine, adjusts pH to 5.0, stirs 50min, the ammonium sulfate for being 10% with concentration

Solution is eluted, and eluent is filtered, and retains filter cake;

(2) filter cake adds 2 times of water dissolutions, adjusts pH5.0-6.0,8 times of polyethylene glycol are added, stir 30min, stand, plate-frame filtering,

Sediment freeze-drying;

(3) step (2) sediment is dissolved in water, during which 4 DEG C of dialysis are replaced 4 water, are freeze-dried to get ulinastatin crude product;

(4) ulinastatin crude product is dissolved in water, and 0.3gEDTA-Na/ liter is added, the ultrafiltration membrane ultrafiltration for being 30,000 with molecular weight;

(5) 732 type cation-exchange chromatography post loadings are added in step (4) sample, are eluted with 2mol/L hydrochloric acid, flow velocity 1ml/min,

Collect eluent;

Wherein, dialysis uses bag filter MD40(6000-8000 in step (3)), flatten width 40mm, diameter 25.5mm.

100,000,000 unit of ulinastatin sterling is taken, 30g mannitol is weighed, adds 500ml water for injection to dissolve, is adjusted after mixing

PH6-7, then add and inject water to 2000ml, filter membrane is sterile filtered, and is sub-packed in cillin bottle, is lyophilized up to ulinastatin drug

Composition.

Obtained ulinastatin sterling is diluted to every milliliter of 100,000 unit income Physico-chemical tests:

Heavy metal analysis: it is detected according to 2015 editions the 4th 0821 the second method of heavy metal inspection technique of Chinese Pharmacopoeia.

Molecular weight HPLC method:

(1) liquid-phase condition: Bio-sil exclusion chromatography column;Detection wavelength 280nm;Adjust the guarantor that flow velocity makes bovine serum albumin(BSA)

Staying the time is about 36 minutes.

(2) prepared by mobile phase: take 16.33 grams of potassium dihydrogen phosphates and 124.15 grams of ethylene glycol to dissolve and be diluted to 1000ml,

When necessary with phosphorus acid for adjusting pH to 4.0.

(3) sample preparation: taking ulinastatin, is made in every 1ml with flowing phase dilution containing about the solution of 6500 units, as

Sample solution.

(4) referring to product solution: with the flowing suitable gamma Globulin of phased soln (molecular weight 160,000), bovine serum albumin(BSA)

(molecular weight 67,000), flesh hyperglobulinemia (molecular weight 17,000) and the mixed solution that 1mg/ml is made.

(5) it measures: respectively taking sample solution and carry out high performance liquid chromatography experiment referring to product solution 50ul, referring to product point

The logarithm of son amount is ordinate, prepares standard curve by abscissa of retention time.Sample is acquired according to referring to product standard curve

The molecular weight of product.

Remaining is referring under Chinese Pharmacopoeia 2015 editions the first enlarged editions ulinastatin of page -213 of page 212 and ulinastatin solution item

Detection project is detected, as a result as follows:

Inspection item

Inspection result

Color A405

0.19

The content of human urokinase-type peptidase

0.0005PNAU

Heavy metal

2.0 μ g/ml of <

Bacterial endotoxin

< 6.25EU

SDS-PAGE molecular weight

37,764Da

HPLC molecular weight

63,673Da

The different ulinastatin of embodiment 4 compare

According to official method measurement ulinastatin than living, while comparing the yield and its purity of ulinastatin preparation method.

Above-mentioned detailed description is illustrating for one of them possible embodiments of the present invention, the embodiment not to

The scope of the patents of the invention is limited, all equivalence enforcements or change without departing from carried out by the present invention are intended to be limited solely by the technology of the present invention

In the range of scheme.

图1
©2018 IPPH.cn   PatViewer·专利搜索
主办单位:知识产权出版社有限责任公司  咨询热线:01082000860-8588
浏览器:IE9及以上、火狐等  京ICP备09007110号 京公网安备 11010802026659号