CN114652840A - 一种犬类病毒四联活疫苗的冻干保护剂及其制备方法和应用 - Google Patents
一种犬类病毒四联活疫苗的冻干保护剂及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开一种犬类病毒四联活疫苗的冻干保护剂及其制备方法和应用,属于疫苗保护剂技术领域。为了提供一种在2‑8℃温度下,保护犬类病毒四联活疫苗的冻干保护剂。本发明提供的配方的成分按照如下质量分数组成:40%的蔗糖、2%‑4%的甘露醇、4%‑8%的右旋糖酐40、2%的甘氨酸和1%的叔丁醇。本发明的冻干保护剂在提高四联冻干疫苗稳定性以及安全性中的应用。
Description
技术领域
本发明属于疫苗保护剂技术领域,具体涉及一种犬类病毒四联活疫苗的冻干保护剂及其制备方法和应用。
背景技术
现有申请公开号为CN112370531A专利文献,专利名称为:一种犬瘟热、犬细小、犬腺病毒、犬副流感病毒四联活疫苗耐热保护剂及其制备方法,公开耐热保护剂为蔗糖、海藻糖、蛋白胨、甘氨酸和PEG等成分,因配方中含有明胶,明胶为动物源性成分,增加了疫苗的杂质含量,免疫动物易发生过敏反应。在预防性疫苗制品中,不含明胶能够减低对人体的刺激性,目前已经被推广和应用在各类疫苗制品中,但是对于动物疫苗尤其是宠物(犬、猫)疫苗的制品不含明胶的疫苗保护效果方面还没有相关报道及研究。因此本领域里急需一种安全有效的无明胶的预防性疫苗。
发明内容
本发明的目的是为了提供一种在2-8℃温度下,保护犬类病毒四联活疫苗的冻干保护剂,使得犬类病毒四联活疫苗的有效性维持16个月。
本发明提供了一种犬类病毒四联活疫苗的冻干保护剂,所述冻干保护剂的成分按照如下质量分数组成:40%的蔗糖、2%-4%的甘露醇、4%-8%的右旋糖酐40、2%的甘氨酸和1%的叔丁醇。
进一步地限定,所述冻干保护剂的成分按照如下质量分数组成:40%的蔗糖、2%的甘露醇、4%的右旋糖酐40、2%的甘氨酸和1%的叔丁醇。
进一步地限定,所述冻干保护剂的成分按照如下质量分数组成:40%的蔗糖、2%的甘露醇、8%的右旋糖酐40、2%的甘氨酸和1%的叔丁醇。
进一步地限定,所述冻干保护剂的成分按照如下质量分数组成:40%的蔗糖、4%的甘露醇、8%的右旋糖酐40、2%的甘氨酸和1%的叔丁醇。
进一步地限定,所述冻干保护剂的成分按照如下质量分数组成:40%的蔗糖、4%的甘露醇、4%的右旋糖酐40、2%的甘氨酸和1%的叔丁醇。
进一步地限定,冻干保护剂的溶剂为注射用水。
本发明还提供了上述的冻干保护剂的制备方法,所述制备方法如下:
(1)配置A液:按上述的冻干保护剂的配方称取蔗糖,加入注射用水至完全溶解,灭菌;
(2)配置B液:按上述的冻干保护剂的配方称取甘露醇、右旋糖酐40、甘氨酸和叔丁醇,加入注射用水中至完全溶解,除菌过滤;
(3)将A液和B液混合后用无菌注射用水定容,制得冻干保护剂。
本发明提供一种冻干犬类病毒四联活疫苗的制备方法,在冻干过程中利用上述的干保护剂。
本发明提供上述的冻干保护剂在提高冻干疫苗稳定性中的应用。
本发明提供上述的冻干保护剂在提高冻干疫苗安全性中的应用。
有益效果:机理:1)蔗糖:蔗糖呈无定型的结构,具有化学性质稳定的特点,对阻止蛋白质二级结构的改变、冻干处理过程中及贮藏期内蛋白质多肽链的伸展和聚集起着显著作用。
2)甘露醇:完全复性.甘露醇不仅可作为优良的骨架剂使用,而且在保护剂配方中它能够兼作蛋白质的冻干保护剂.甘露醇对蛋白质的保护作用与其浓度、形态结构有关,而浓度与结晶形态有时又有一定的关联性。
3)甘氨酸:具有酸、碱两性,因此能够在制品的低温保存和冷冻干燥过程中能抑制溶液的pH变化,从而达到保护活性组分的目的。
4)右旋糖酐40,在冻结过程中优先析出,具有一定的表面活性,在蛋白质分子之间产生位阻(steric hindrance)作用,抑制小分子赋形剂(如蔗糖)的结晶。
5)叔丁醇:冷冻干燥过程耗时长、耗能多,因此迫切需要对冻干循环进行优化,降低生产成本。在冻干配方中加入适量的叔丁醇(Tertiary Butyl Alcohol)后,冻结时会形成针状结晶;冰晶升华后,留下了管状通道,使水蒸汽流动阻力大大减小,升华速率显著提高。(1)可以降低干燥层阻力,从而加速了干燥过程,缩短了干燥时间;
(2)使产品具有高的比表面积、好的外观、并易于复水;
(3)可提高药品溶液和冻干品的稳定性;
(4)有一定的抑菌作用。
具体实施方式
实施例1.犬瘟热、犬细小、犬腺病毒、犬副流感病毒四联活疫苗的冻干保护剂配方
(1)冻干保护剂各成分配制浓度:蔗糖:40%、右旋糖酐40:4%、甘露醇2%、甘氨酸2%、叔丁醇1%,配制冻干保护剂的溶剂为注射用水。
(2)制备方法如下:
A液,按上述配方称取蔗糖,加入注射用水至完全溶解,灭菌。
B液,按上述配方称取或量取甘露醇、右旋糖酐、甘氨酸和叔丁醇,加入注射用水中至完全溶解,除菌过滤。
将A液和B液混合后用无菌注射用水定容,制得冻干保护剂。
实施例2.犬瘟热、犬细小、犬腺病毒、犬副流感病毒四联活疫苗的冻干保护剂配方
(1)冻干保护剂各成分配制浓度:蔗糖:40%、右旋糖酐40:8%、甘露醇2%、甘氨酸2%、叔丁醇1%,配制冻干保护剂的溶剂为注射用水。
(2)制备方法如下:
A液,按上述配方称取蔗糖,加入注射用水至完全溶解,灭菌。
B液,按上述配方称取或量取甘露醇、右旋糖酐、甘氨酸和叔丁醇,加入注射用水中至完全溶解,除菌过滤。
将A液和B液混合后用无菌注射用水定容,制得冻干保护剂。
实施例3.犬瘟热、犬细小、犬腺病毒、犬副流感病毒四联活疫苗的冻干保护剂配方
(1)冻干保护剂各成分配制浓度:蔗糖:40%、右旋糖酐40:8%、甘露醇4%、甘氨酸2%、叔丁醇1%,配制冻干保护剂的溶剂为注射用水。
(2)制备方法如下:
A液,按上述配方称取蔗糖,加入注射用水至完全溶解,灭菌。
B液,按上述配方称取或量取甘露醇、右旋糖酐、甘氨酸和叔丁醇,加入注射用水中至完全溶解,除菌过滤。
将A液和B液混合后用无菌注射用水定容,制得冻干保护剂。
实施例4.犬瘟热、犬细小、犬腺病毒、犬副流感病毒四联活疫苗的冻干保护剂配方
(1)冻干保护剂各成分配制浓度:蔗糖:40%、右旋糖酐40:4%、甘露醇4%、甘氨酸2%、叔丁醇1%,配制冻干保护剂的溶剂为注射用水。
(2)制备方法如下:
A液,按上述配方称取蔗糖,加入注射用水至完全溶解,灭菌。
B液,按上述配方称取或量取甘露醇、右旋糖酐、甘氨酸和叔丁醇,加入注射用水中至完全溶解,除菌过滤。
将A液和B液混合后用无菌注射用水定容,制得冻干保护剂。
利用以下实验验证实验效果:
1、按MOI为0.04±0.01接种犬瘟热弱毒CDV/R-20/8株生产毒种(记载在[1]步志高,夏咸柱,王喜军,等.犬瘟热病毒cdv/r-20/8疫苗株的反向遗传操作系统及其应用中),调整培养温度为33℃,每日观察病变情况,当病变率达80%以上时收获,冻融后1000rpm离心10min去除沉淀,收获上清,即为犬瘟热弱CDV/R-20/8株病毒液。
2、按MOI为0.04±0.01接种犬副流感弱毒CPIV FA株生产毒种(记载在闫喜军,博士论文,犬副流感病毒进化分析及核酸疫苗和犬腺病毒重组疫苗的研究),调整培养温度为33℃,每日观察病变情况,当病变率达80%以上时收获,冻融后1000rpm离心10min去除沉淀,收获上清,即为犬副流感弱毒CPIV FA株病毒液。
3、按MOI为0.004±0.001接种犬腺病毒弱毒Ⅱ型YCA18株生产毒种(记载在夏咸柱,范泉水,扈荣良,等.Ⅱ型犬腺病毒自然弱毒YCA18株的实验免疫研究[J].中国兽药杂志,2001,35(2):1-4.DOI:10.3969/j.issn.1002-1280.2001.02.001.),调整培养温度为33℃,每日观察病变情况,当病变率达80%以上时收获,冻融后1000rpm离心10min去除沉淀,收获上清,即为犬腺病毒弱毒Ⅱ型YCA18株病毒液。
4、按MOI为0.02±0.013接种犬细小病毒CPV自然弱毒(PV/貉/CC/1/86)(记载在高丽,硕士论文,犬细小病毒单克隆抗体的制备及初步应用)生产毒种,调整培养温度为33℃,每日观察病变情况,当病变率达80%以上时收获,冻融后1000rpm离心10min去除沉淀,收获上清,即为犬细小病毒弱毒CR86106株病毒液。
5、检验
5.1犬瘟热弱毒CDV/R-20/8株,用待分种的Vero细胞的E-MEM细胞培养液将毒种作10倍系列稀释,接种微量细胞培养板,每个稀释度接种4孔,每孔0.1ml。观察6天细胞融合性病变,计算TCID50,每0.1ml应含病毒≥105.0TCID50。
5.2犬副流感弱毒CPIV FA株,用待分种的MDCK细胞的E-MEM细胞培养液将毒种作10倍系列稀释,接种微量细胞培养板,每个稀释度接种4孔,每孔0.1ml。观察6天细胞融合性病变,计算TCID50,每0.1ml应含病毒≥104.5TCID50。
5.3犬腺病毒弱毒Ⅱ型YCA18株,用待分种的MDCK细胞的E-MEM细胞培养液将毒种作10倍系列稀释,每个稀释度接种已长成单层并倾弃培养液的MDCK微量细胞培养板4孔,每孔0.1ml。观察6天细胞葡萄串状病变,计算TCID50,每0.1ml应含病毒≥105.0TCID50。
5.4犬细小病毒弱毒CR86106株,用待分种的F81细胞的E-MEM细胞培养液将毒种作10倍系列稀释,接种微量细胞培养板,每个稀释度接种4孔,每孔0.1ml。观察6天细胞拉网样病变,计算TCID50,每0.1ml应含病毒≥104.5TCID50。
5.5按每头份1.3ml病毒液中含CDV/R-20/8毒液0.5ml,CPIV FA毒液0.5ml,YCA18毒液0.1ml,CR86106毒液0.2ml的比例置于同一灭菌容器内混合均匀。
5.6加冻干保护剂按每7份病毒液加冻干保护剂1份,充分摇匀。
5.7分装:以每瓶1.5ml(1头份)定量分装。
5.8冻干分装后,迅速进行冷冻真空干燥,轧盖。
冻干处理时对以下参数进行控制
5.8.1预冻阶段,温度-45℃,时间:3小时。
升华阶段:温度-30℃~-25℃,时间:12小时。
解析干燥阶段:温度-10℃~20℃,时间:10小时。
从升华阶段开始抽真空一直持续。
5.9检验
5.9.1性状本品呈微黄白色海绵状疏松团块,易与瓶壁脱离。加稀释液后,迅速溶解成粉红色澄清液体。
5.9.2无菌检验按现行《中国兽药典》附录进行检验,应无菌生长。
5.9.3支原体检验按现行《中国兽药典》附录进行检验,应无支原体生长。
5.9.4鉴别检验每批样品随机抽取样品5瓶,每瓶用1.5ml注射用水溶解,各加入等量经56℃,灭活30分钟的4种病毒的特异性血清混合液,37℃中和1小时,每瓶分别接种微量细胞培养板培养的Vero、MDCK和F81细胞各4孔,每孔0.1ml,观察6天,应不致细胞病变。
5.9.5外源病毒检验用3.6项安全检验犬,观察至21天时,分别采取血清检测伪狂犬病毒、牛病毒性腹泻病毒的中和抗体(按现行《中国兽药典》附录进行检验),应均小于1:2。
5.9.6安全检验将疫苗用注射用水稀释,肌肉注射2~3月龄易感犬(血清对4种制苗病毒中和抗体均≤1:2)5只,各注射10头份疫苗,逐日观察至21天,精神、食欲、体温与粪便性状均应正常,疫苗判为合格。
5.9.7效力检验用2~3月龄易感犬5只,每只肌肉注射疫苗1头份,21天后分别采血,按下列方法测定血清对4种制苗病毒中和抗体效价。
5.9.7.1犬瘟热病毒组份分别用100TCID50的犬瘟热病毒弱毒CDV/R-20/8株培养物与各试验犬血清于37℃作用1小时,然后按现行《中国兽药典》附录,用Vero细胞测定各血清的犬瘟热中和抗体效价,5/5犬抗犬瘟热SN抗体≥1:50合格。
5.9.7.2犬副流感病毒组份分别用100TCID50的犬副流感病毒弱毒CPIV FA株培养物与各试验犬血清于37℃作用1小时,然后按现行《中国兽药典》附录,用MDCK细胞测定各血清的犬副流感中和抗体效价,5/5犬抗犬副流感SN抗体≥1:4合格。
5.9.7.3犬腺病毒组份分别用100TCID50的犬腺病毒弱毒YCA18株培养物与各试验犬血清于37℃作用1小时,然后按现行《中国兽药典》附录,用MDCK细胞测定各血清的犬腺病毒中和抗体效价,5/5犬抗犬腺病毒SN抗体≥1:10合格。
5.9.7.4犬细小病毒组份分别用100TCID50的犬细小病毒弱毒CR86106株培养物与各试验犬血清于37℃作用1小时,然后按现行《中国兽药典》附录,用F81细胞测定各血清的犬细小病毒中和抗体效价,5/5犬抗犬细小病毒SN抗体≥1:16合格。
5.9.8剩余水分测定按现行《中国兽药典》附录进行测定,应符合规定。
5.9.9真空度测定按现行《中国兽药典》附录进行测定,应符合规定。
5.10结果:表1说明冻干保护剂配方在犬四联活疫苗的应用中,冻干保护剂可以有效减轻犬四联活疫苗在干燥过程中受到水分子的损伤,从而保证制品的外观质量。无菌、支原体及外源病毒检验合格,证明冻干制品在冻干过程中,制品密封性良好。
表1犬瘟热、犬副流感、犬腺病毒与犬细小病毒病四联活疫苗成品检验结果
表2说明实施例1-实施例4的冻干保护剂是安全的。
表2犬瘟热、犬副流感、犬腺病毒与犬细小病毒病四联活疫苗成品检验结果
表3说明冻干效果良好,制品免疫实验动物后,实验动物能产生较高的效价。
表3犬瘟热、犬副流感、犬腺病毒与犬细小病毒病四联活疫苗成品检验结果
表4.说明冻干保护剂配方在犬四联活疫苗的应用中,制品的剩余水分≤3%,冻干效果好,真空度良好。
表4犬瘟热、犬副流感、犬腺病毒与犬细小病毒病四联活疫苗成品检验结果
实施例1-实施例4涉及的犬瘟热、犬细小、犬腺病毒、犬副流感病毒四联活疫苗冻干产品,稳定性良好,在2-8℃条件下,保存16个月后,检测结果符合规定,结果如下。
表5犬瘟热、犬副流感、犬腺病毒与犬细小病毒病四联活疫苗成品检验结果
表6犬瘟热、犬副流感、犬腺病毒与犬细小病毒病四联活疫苗成品检验结果
批次 | 批号 | 安全检验 |
1 | 实施例1 | 5/5接种犬精神、食欲、体温与粪便均正常 |
2 | 实施例2 | 5/5接种犬精神、食欲、体温与粪便均正常 |
3 | 实施例3 | 5/5接种犬精神、食欲、体温与粪便均正常 |
4 | 实施例4 | 5/5接种犬精神、食欲、体温与粪便均正常 |
表7犬瘟热、犬副流感、犬腺病毒与犬细小病毒病四联活疫苗成品检验结果
表8犬瘟热、犬副流感、犬腺病毒与犬细小病毒病四联活疫苗成品检验结果
Claims (10)
1.一种犬类病毒四联活疫苗的冻干保护剂,其特征在于,所述冻干保护剂的成分按照如下质量分数组成:40%的蔗糖、2%-4%的甘露醇、4%-8%的右旋糖酐40、2%的甘氨酸和1%的叔丁醇。
2.根据权利要求1所述的冻干保护剂,其特征在于,所述冻干保护剂的成分按照如下质量分数组成:40%的蔗糖、2%的甘露醇、4%的右旋糖酐40、2%的甘氨酸和1%的叔丁醇。
3.根据权利要求1所述的冻干保护剂,其特征在于,所述冻干保护剂的成分按照如下质量分数组成:40%的蔗糖、2%的甘露醇、8%的右旋糖酐40、2%的甘氨酸和1%的叔丁醇。
4.根据权利要求1所述的冻干保护剂,其特征在于,所述冻干保护剂的成分按照如下质量分数组成:40%的蔗糖、4%的甘露醇、8%的右旋糖酐40、2%的甘氨酸和1%的叔丁醇。
5.根据权利要求1所述的冻干保护剂,其特征在于,所述冻干保护剂的成分按照如下质量分数组成:40%的蔗糖、4%的甘露醇、4%的右旋糖酐40、2%的甘氨酸和1%的叔丁醇。
6.根据权利要求1-5任一项所述的冻干保护剂,其特征在于,冻干保护剂的溶剂为注射用水。
7.权利要求1-5任一项所述的冻干保护剂的制备方法,其特征在于,所述制备方法如下:
(1)配置A液:按权利要求1-5任一项所述的冻干保护剂的配方称取蔗糖,加入注射用水至完全溶解,灭菌;
(2)配置B液:按权利要求1-5任一项所述的冻干保护剂的配方称取甘露醇、右旋糖酐40、甘氨酸和叔丁醇,加入注射用水中至完全溶解,除菌过滤;
(3)将A液和B液混合后用无菌注射用水定容,制得冻干保护剂。
8.一种冻干犬类病毒四联活疫苗的制备方法,其特征在于,在冻干过程中利用权利要求1-5任一项所述的干保护剂。
9.权利要求1-5任一项所述的冻干保护剂在提高冻干疫苗稳定性中的应用。
10.权利要求1-5任一项所述的冻干保护剂在提高冻干疫苗安全性中的应用。
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