CN116693657A - 一种猪纤维蛋白止血剂的制备方法 - Google Patents
一种猪纤维蛋白止血剂的制备方法 Download PDFInfo
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Abstract
本发明涉及一种猪纤维蛋白止血剂的制备方法,包括如下步骤:S1,将冷冻猪血浆进行融浆后,离心去除冷沉淀得到猪血浆上清液;S2,将所述猪血浆上清液进行有机溶剂/去污剂混合物病毒灭活,然后分别提取病毒灭活后的猪血浆上清液中的功能蛋白,得到至少两种功能蛋白;S3,分别在每种所述功能蛋白中加入蛋白质保护剂溶液,经过巴氏病毒灭活后,再经过超滤透析以提纯,按照处方配制得到猪纤维蛋白止血剂。采用本发明的方法,对所提取的功能蛋白含量和活性的损害较小,功能蛋白的产率较高。
Description
技术领域
本发明涉及动物血液制品分离纯化技术领域,具体涉及一种猪纤维蛋白止血剂的制备方法。
背景技术
血浆具体包括白蛋白、免疫球蛋白和一组凝血因子等。猪纤维蛋白止血剂为从猪血浆中提取和纯化所得的凝血因子,主要组分包括猪纤维蛋白原和猪凝血酶,其止血原理是模拟人体内凝血的最后一步反应,但它并不依赖于机体自身的凝血成分,因此它对具有凝血功能障碍或正在服用抗凝药物的患者仍有理想止血效果。
但使用这种类型的制剂具有血浆供体感染性疾病传播的潜在可能问题以及蛋白稳定性差的问题。因此,能够有必要提供一种有效且无病毒感染危险的高度纯化的血浆制剂和凝血因子制剂。
目前,已经提出了多种病毒灭活和/或病毒消除的方法。中国专利一种冻干型人凝血因子Ⅷ的制备方法(申请号202011107419.9),公开了一种凝血因子的病毒灭活方法,具体包括S/D病毒灭活与干热病毒灭活,但其干热病毒灭活工艺为在98~100℃中保温30min,此工艺仅适用于冻干制剂,而且对凝血因子活性的影响较大,导致成品的效用和收率下降。
发明内容
为了解决血浆制品安全性问题以及产品收率低的问题,而提供一种猪纤维蛋白止血剂的制备方法。本发明方法能够将血浆制品中的包膜病毒和/或非包膜病毒灭活从而保障血浆来源生物制品的安全性,且蛋白质在病毒灭活的过程中被稳定保护,提高了产品的收率。
一种猪纤维蛋白止血剂的制备方法,包括如下步骤:
S1,将冷冻猪血浆进行融浆后,离心去除冷沉淀得到猪血浆上清液;
S2,将所述猪血浆上清液进行有机溶剂/去污剂混合物病毒灭活,然后分别提取病毒灭活后的猪血浆上清液中的功能蛋白,得到至少两种功能蛋白;
S3,分别在每种所述功能蛋白中加入蛋白质保护剂溶液,经过巴氏病毒灭活后,再经过超滤透析以提纯,按照处方配制得到猪纤维蛋白止血剂。
进一步地,步骤S1中所述融浆的过程是在18-23℃的水浴中进行脱袋融化;所述离心去除冷沉淀的过程是在融浆完成后于0℃进行离心。
进一步地,步骤S2所述有机溶剂/去污剂混合物病毒灭活的过程是:在所述猪血浆上清液中加入1.0±0.3w/v%聚山梨酯80和1.0±0.3w/v%磷酸三丁酯,在24±1℃下维持至少6h进行病毒灭活。有机溶剂/去污剂混合物病毒灭活能够有效灭活潜在的病毒。
进一步地,步骤S2所述功能蛋白包括猪纤维蛋白原、猪凝血酶、猪免疫球蛋白、猪白蛋白。
再进一步地,所述提取过程为:
①对所述猪血浆上清液控制温度为-2℃±0.5℃下进行乙醇沉淀,离心收集第一沉淀和第一上清液,所得第一沉淀为猪纤维蛋白原;
②对所述第一上清液按照体积比9:1加入3-5wt%的柠檬酸钠,搅拌均匀后加入化学当量的8wt%氯化钡溶液,形成的柠檬酸钡会对所述第一上清液中的凝血酶进行吸附生成第二沉淀,离心收集第二沉淀和第二上清液,所得第二沉淀为猪凝血酶。
再进一步地,所述提取过程根据需要对所述第二上清液按照等体积比加入pH=7.2、0.2mol/L的PBS缓冲溶液,所述PBS缓冲溶液中含0.15mol/L的氯化钠,搅拌均匀后加入饱和硫酸铵溶液,使硫酸铵的质量分数为50%形成第三沉淀,离心收集第三沉淀和第三上清液,所得第三沉淀为猪免疫球蛋白;对第三上清液进行透析、浓缩可得猪白蛋白。
进一步地,步骤S3中所述巴氏病毒灭活是在60±1℃的水浴中保持水浴加热10h。
进一步地,步骤S3中所述功能蛋白在所述蛋白质保护剂溶液中的浓度为30-50g/L;所述蛋白质保护剂溶液由如下组分组成:400-600g/L蔗糖、50-70g/L盐酸精氨酸、20-50g/L枸橼酸钠、1-5g/L氯化钙、以及水。
优选地,所述蛋白质保护剂溶液由如下组分组成:500g/L蔗糖、60g/L盐酸精氨酸、40g/L枸橼酸钠、1-5g/L氯化钙、以及水。
有益技术效果:
目前血液制品主要采用S/D病毒灭活工艺和干热灭活工艺组合的方式,但是这种病毒灭活方法仅适用于冻干制剂,而且干热灭活对凝血因子活性的影响较大,导致成品的收率下降。本发明的方法包含两个灭活病毒的工艺步骤,即有机溶剂/去污剂混合物病毒灭活和巴氏病毒灭活工艺。两种病毒灭活工艺在病毒灭活过程中都具有较可靠和完善的灭活条件,而且两种灭活工艺的组合对包膜病毒和非包膜病毒均能够起到有效的灭活效果。因此,针对猪纤维蛋白止血剂和其它血浆蛋白生物制品,本发明的病毒灭活工艺都是可靠的、稳定的病毒灭活方法。本发明所使用的巴氏病毒灭活工艺不仅适用于冻干制剂,还适用于液体制剂,并且对凝血因子的活性影响较小,收率高。此外,通过向蛋白溶液中添加蛋白质保护剂,能够进一步减少巴氏灭活工艺对凝血因子活性的影响,提高成品的收率。
具体实施方式
下面将结合本发明的实施例,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。以下对至少一个示例性实施例的描述实际上仅仅是说明性的,决不作为对本发明及其应用或使用的任何限制。基于本发明中的实施例,本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
除非另外具体说明,否则在这些实施例中阐述的数值不限制本发明的范围。对于相关领域普通技术人员已知的技术、方法可能不作详细讨论,但在适当情况下,所述技术、方法应当被视为说明书的一部分。在这里示出和讨论的所有示例中,任何具体值应被解释为仅仅是示例性的,而不是作为限制。因此,示例性实施例的其它示例可以具有不同的值。
此外,需要说明的是,使用“第一”、“第二”等词语来限定离心后得到的沉淀和上清液,仅仅是为了便于对各步骤的产物进行区别,如没有另行声明,上述词语并没有特殊含义,因此不能理解为对本发明保护范围的限制。
以下实施例中未注明具体条件的实验方法,通常按照国家标准测定;若没有相应的国家标准,则按照通用的国际标准、或相关企业提出的标准要求进行。除非另有说明,否则所有的份数为重量份,所有的百分比为重量百分比。
实施例1
一种猪纤维蛋白止血剂的制备方法,包括如下步骤:
S1,将冷冻猪血浆在20℃的水浴中进行脱袋融化,在融浆完成后于0℃进行离心以去除冷沉淀,得到猪血浆上清液。
S2,按照质量-体积百分含量计算,在每毫升所述猪血浆上清液中加入0.01g聚山梨酯80和0.007g磷酸三丁酯,在24±1℃下维持6h进行病毒灭活,以对潜在的病毒进行有效灭活,然后分别提取病毒灭活后的猪血浆上清液中的功能蛋白。
所述功能蛋白包括猪纤维蛋白原、猪凝血酶、猪免疫球蛋白、猪白蛋白,猪纤维蛋白止血剂中主要用到的功能蛋白为猪纤维蛋白原、猪凝血酶,功能蛋白提取过程如下:
①在所述猪血浆上清液中滴加-25℃的乙醇溶液使乙醇浓度为8%,控制整体温度为-2℃±0.5℃,滴加完毕后再继续搅拌30min进行乙醇沉淀,然后在0℃、5000rpm的条件下离心收集第一沉淀和第一上清液,所得第一沉淀即为猪纤维蛋白原;
②,第一上清液用于提取凝血酶:对第一上清液按照体积比9:1加入4wt%的柠檬酸钠,搅拌均匀后加入化学当量的8wt%氯化钡溶液,形成的柠檬酸钡会对第一上清液中的凝血酶进行吸附生成第二沉淀,在0℃、5000rpm的条件下离心收集第二沉淀和第二上清液,所得第二沉淀即为猪凝血酶。
(根据需要,第二上清液用于提取免疫球蛋白和白蛋白:对第二上清液按照等体积比加入pH=7.2、0.2mol/L的PBS缓冲溶液,PBS缓冲溶液中还含0.15mol/L的氯化钠,搅拌均匀后加入饱和硫酸铵溶液,使硫酸铵的质量分数为50%形成第三沉淀,在0℃、5000rpm的条件下离心收集第三沉淀和第三上清液,第三沉淀即为猪免疫球蛋白;对第三上清液进行透析、浓缩可得猪白蛋白。)
S3,猪纤维蛋白止血剂的处方中包括猪纤维蛋白原、猪凝血酶以及其他辅料,对步骤S2中得到的猪纤维蛋白原分别采用蛋白质保护剂溶液进行保护,蛋白质保护剂溶液由蔗糖、盐酸精氨酸、枸橼酸钠、氯化钙以及注射用水组成,使得每升注射用水中包含40g猪纤维蛋白原、500g蔗糖、60g盐酸精氨酸、40g枸橼酸钠、0.5g氯化钙,分别得到猪纤维蛋白原溶液和猪凝血酶溶液;
分别将猪纤维蛋白原溶液和猪凝血酶溶液置于60±1℃的水浴中保持水浴加热10h进行巴氏病毒灭活;
将经巴氏病毒灭活后的猪纤维蛋白原溶液和猪凝血酶溶液分别进行超滤透析,猪纤维蛋白原保护溶液使用100kd的超滤膜包进行透析,透析倍数为溶液体积的8~10倍,猪凝血酶保护溶液使用10kd的超滤膜包进行透析,透析倍数为蛋白溶液体积的8~10倍,超滤透析可去除杂质、蛋白质保护剂等使功能蛋白得到提纯,可省略纤维蛋白原乙醇沉淀等功能蛋白的溶解再沉淀提纯的步骤;
经过巴氏病毒灭活后,再经过超滤透析,按照猪纤维蛋白止血剂的标准处方配制得到猪纤维蛋白止血剂。
实施例2
本实施例的猪纤维蛋白止血剂与实施例1的制备方法相同,不同之处在于,步骤S3中每升注射用水中包含40g猪纤维蛋白原或猪凝血酶、500g蔗糖、60g盐酸精氨酸、40g枸橼酸钠、1.0g氯化钙。
实施例3
本实施例的猪纤维蛋白止血剂与实施例1的制备方法相同,不同之处在于,步骤S3中每升注射用水中包含40g猪纤维蛋白原或猪凝血酶、500g蔗糖、60g盐酸精氨酸、40g枸橼酸钠、1.5g氯化钙。
对比例1
本实施例的猪纤维蛋白止血剂与实施例1的制备方法相同,不同之处在于,蛋白质保护剂溶液不含有氯化钙。
对比例2
本实施例的猪纤维蛋白止血剂与实施例1的制备方法相同,不同之处在于,未添加蛋白质保护剂。
对比例3
本实施例的猪纤维蛋白止血剂与实施例1的制备方法相同,不同之处在于,步骤S3中获得功能蛋白溶液后进行常规冻干操作,然后将冻干品在隔水式恒温箱内于63℃下保温139h进行干热病毒灭活。
测定以上实施例及对比例中所获得的功能蛋白的纯度和产率,以及测定功能蛋白溶液进行巴氏病毒灭活前后其中所含功能蛋白含量,具体数据见表1。
表1功能蛋白的纯度和产率以及巴氏病毒灭活前后功能蛋白含量
(注:表中功能蛋白为猪纤维蛋白原)
由表1可知,采用本发明的方法,对功能蛋白(猪纤维蛋白原)进行病毒灭活后,功能蛋白含量虽然有所损失,但是损失率能够控制在10%以内,提高了功能蛋白的收率,且功能蛋白在病毒灭活后蛋白质活性仍然保持的较好,本发明方法对功能蛋白的活性损害非常小。本发明方法对猪凝血酶功能蛋白而言同样能够达到80%以上的纯度,以及控制巴氏病毒灭活后损失率在10%以内。
对比例2中在病毒灭活前未对功能蛋白采用蛋白质保护剂进行保护其最终得到的功能蛋白纯度非常低,灭活后功能蛋白损失率达到了70%以上。对比例1中对功能蛋白采用蔗糖、盐酸精氨酸和枸橼酸钠组成的蛋白质保护剂后,在灭活后功能蛋白的损失率由对比例2的70.24%减少到33.14%,说明添加蛋白质保护剂能够提高产品收率。为了进一步减少巴氏灭活工艺对功能蛋白活性的以下爱过你,在对比例1的基础上于蛋白质保护剂中加入了氯化钙,在灭活后功能蛋白的损失率由对比例1的33.14%减少到实施例1的3.85%,进一步提高了功能蛋白的成品收率。
以上所述,仅为本发明较佳的具体实施方式,但本发明的保护范围并不局限于此,任何熟悉本技术领域的技术人员在本发明揭露的技术范围内,根据本发明的技术方案及其发明构思加以等同替换或改变,都应涵盖在本发明的保护范围之内。
Claims (9)
1.一种猪纤维蛋白止血剂的制备方法,其特征在于,包括如下步骤:
S1,将冷冻猪血浆进行融浆后,离心去除冷沉淀得到猪血浆上清液;
S2,将所述猪血浆上清液进行有机溶剂/去污剂混合物病毒灭活,然后分别提取病毒灭活后的猪血浆上清液中的功能蛋白,得到至少两种功能蛋白;
S3,分别在每种所述功能蛋白中加入蛋白质保护剂溶液,经过巴氏病毒灭活后,再经过超滤透析以提纯,按照处方配制得到猪纤维蛋白止血剂。
2.根据权利要求1所述的一种猪纤维蛋白止血剂的制备方法,其特征在于,步骤S1中所述融浆的过程是在18-23℃的水浴中进行脱袋融化;所述离心去除冷沉淀的过程是在融浆完成后于0℃进行离心。
3.根据权利要求1所述的一种猪纤维蛋白止血剂的制备方法,其特征在于,步骤S2所述有机溶剂/去污剂混合物病毒灭活的过程是:在所述猪血浆上清液中加入1.0±0.3w/v%聚山梨酯80和1.0±0.3w/v%磷酸三丁酯,在24±1℃下维持至少6h进行病毒灭活。
4.根据权利要求1所述的一种猪纤维蛋白止血剂的制备方法,其特征在于,步骤S2所述功能蛋白包括猪纤维蛋白原、猪凝血酶、猪免疫球蛋白、猪白蛋白。
5.根据权利要求4所述的一种猪纤维蛋白止血剂的制备方法,其特征在于,所述提取过程为:
①对所述猪血浆上清液控制温度为-2℃±0.5℃下进行乙醇沉淀,离心收集第一沉淀和第一上清液,所得第一沉淀为猪纤维蛋白原;
②对所述第一上清液按照体积比9:1加入3-5wt%的柠檬酸钠,搅拌均匀后加入化学当量的8wt%氯化钡溶液,形成的柠檬酸钡会对所述第一上清液中的凝血酶进行吸附生成第二沉淀,离心收集第二沉淀和第二上清液,所得第二沉淀为猪凝血酶。
6.根据权利要求5所述的一种猪纤维蛋白止血剂的制备方法,其特征在于,所述提取过程根据需要对所述第二上清液按照等体积比加入pH=7.2、0.2mol/L的PBS缓冲溶液,所述PBS缓冲溶液中含0.15mol/L的氯化钠,搅拌均匀后加入饱和硫酸铵溶液,使硫酸铵的质量分数为50%形成第三沉淀,离心收集第三沉淀和第三上清液,所得第三沉淀为猪免疫球蛋白;对第三上清液进行透析、浓缩可得猪白蛋白。
7.根据权利要求1所述的一种猪纤维蛋白止血剂的制备方法,其特征在于,步骤S3中所述巴氏病毒灭活是在60±1℃的水浴中保持水浴加热10h。
8.根据权利要求1所述的一种猪纤维蛋白止血剂的制备方法,其特征在于,步骤S3中所述功能蛋白在所述蛋白质保护剂溶液中的浓度为30-80g/L;所述蛋白质保护剂溶液由如下组分组成:400-600g/L蔗糖、50-70g/L盐酸精氨酸、20-50g/L枸橼酸钠、1-5g/L氯化钙、以及水。
9.根据权利要求8所述的一种猪纤维蛋白止血剂的制备方法,其特征在于,所述蛋白质保护剂溶液由如下组分组成:500g/L蔗糖、60g/L盐酸精氨酸、40g/L枸橼酸钠、1-5g/L氯化钙、以及水。
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