CN1209067A - 活疫苗的稳定剂 - Google Patents
活疫苗的稳定剂 Download PDFInfo
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Abstract
本发明涉及活疫苗和减毒的单或多价活疫苗的稳定剂以及含有这些稳定剂的稳定的疫苗,和用于制备这样一些疫苗的方法。用于含一种或多种减毒病毒的活疫苗的这些稳定剂包括一种或多种选自下列的组分:氨基酸、二糖、多元醇和缓冲液,其条件是选自二糖中的一个组分是蔗糖。
Description
本发明涉及用于单价或多价活疫苗的稳定剂,还涉及含这些试剂的稳定的疫苗,以及用于制备所述疫苗的方法。
含活病毒,具体地说,减毒活病毒的疫苗对其制备和贮存条件非常敏感。在冻干步骤、贮存过程,以及培养后收集病毒的步骤中,损失了大部分病毒效价。由于病毒效价对于免疫效力的重要性,所以使用使病毒效价得到最大程度保护的稳定剂是必不可少的。稳定剂是可在疫苗制备的不同阶段加入的化学的和/或生物的化合物,从而确保疫苗在使用时(有时可能在开始贮存之后数年)时有最大效力。
已经试验了许多化合物对含减毒活病毒的不同疫苗的稳定能力。现有技术中已试验的化合物(参见,例如US3783098、US3915794和EP028563),第一种是SPGA,它主要含有牛或人血清白蛋白、或多或少水解的酪蛋白。或明胶,任选地是谷氨酸的碱金属盐、糖(葡萄糖、蔗糖或葡聚糖)、以及碱金属的一金属或二金属磷酸盐,或它们的一种混合物。EP065905描述了用于抗黄热病疫苗的稳定化合物,所述化合物包括选自11种氨基酸中的一种或多种氨基酸、乳糖、山梨糖醇和磷酸盐缓冲液(PBS)。
但是,所有这些组合物都有很大的缺陷。首先,它们含有具有潜在的生物学危险并且化学组成不确定的动物或人源的蛋白质或蛋白质水解产物(白蛋白等)。此外,本技术领域现有这些组合物未能使减毒活疫苗具有足够的稳定性,这样使贮存这样一些疫苗的可能性受到限制,并且疫苗在贮存或运输时,暴露于高温下而变得很危险。因此,这些疫苗活性的损失会导致无效免疫接种,因而在实际感染时没有保护作用。另外,当涉及稳定含不同病毒的多效价疫苗的不同效价时,已知组合物的效力是比较有限的。因此,四效价疫苗MMRV(麻疹/腮腺炎/风疹/水痘)是多效价疫苗的一个实例,但目前尚未有与四价相符的稳定剂。
本发明的稳定剂没有上述缺陷。用于含一种或多种减毒活病毒的组合物,特别是疫苗的这些稳定剂含有选自下列的一种或多种组分:氨基酸、二糖、多元醇和缓冲液,其条件是选自二糖的一种组分是蔗糖。在本发明的稳定剂中,选自二糖中的组分的比例可以是可以2至6%。有利地,选自二糖的一种或多种组分的比例可以是2.5%。
作为本发明稳定剂一种组分的多元醇,具体地说,包括山梨糖醇和甘露糖醇。本发明的稳定剂可以含有山梨糖醇和甘露糖醇。当本发明的稳定剂含有山梨糖醇时,山梨糖醇的比例优选地是2至6%。当本发明的稳定剂含有山梨糖醇时,山梨糖醇的比例有利地是3至5%。最佳地,山梨糖醇的比例可以是5%。当本发明的稳定剂含有甘露糖醇时,甘露糖醇的比例是1至2.5%。当本发明的稳定剂含有甘露糖醇时,甘露糖醇的比例优选地是1.5%。
作为本发明稳定剂组分之一的缓冲液,具体地说,可以包括磷酸盐缓冲液和乙二胺四乙酸(EDTA)。优选地,本发明的稳定剂含有磷酸盐缓冲液。本发明的稳定剂还可含有葡聚糖。当本发明的稳定剂含有葡聚糖时,葡聚糖的比例优选地是1至4%。当本发明的稳定剂含有葡聚糖时,葡聚糖的比例有利地是3%。
本发明的稳定剂还可含有脲、脲衍生物或其混合物。可以使用的脲衍生物的实例是烯丙基脲、乙酰胺、氨基甲酸甲酯或氨基甲酸丁酯。当本发明的稳定剂含有脲、脲衍生物或其混合物时,后者的比例是0.125-2%。当本发明的稳定剂含有脲、脲衍生物或其混合物时,后者的比例优选地是0.5%。
优选地,可根据待稳定的病毒颗粒调整本发明组合物的pH。将pH调到7是特别有利的。
可用本发明的稳定剂稳定许多组合物,特别是基本上含一种病毒株或种的单价疫苗,或含数种病毒株或种的多价疫苗。本发明还涉及用上述稳定剂稳定的组合物,特别是疫苗。
根据本发明稳定的这样一些疫苗例如可含有疱疹病毒科的至少一种病毒。疱疹病毒科具体地说包括水痘病毒、巨细胞病毒和单纯性疱疹病毒。与其它病毒相比,水痘病毒是特别脆弱并不耐热的。因此,当涉及稳定象水痘病毒这样不稳定的病毒时,本发明的稳定剂是特别适宜的。
可将本发明的稳定剂用于其它病毒,具体地说,这些病毒具体地选自副粘病毒科(麻疹病毒,包括麻疹、腮腺炎病毒、副流感病毒1、2、3和4型以及肺病毒)、披盖病毒科(风疹)和流感(A、B和C)病毒。
可用本发明的稳定剂稳定从上述病毒或其它病毒得到的、并构成多价疫苗的混合物。可以列举的并可想到的多价疫苗是麻疹-腮腺炎-风疹疫苗。在效价组合情况下,本发明的稳定剂有除上述优点外,还有有利的特性,特性在于稳定每种效价的能力使得可将这些效价组合起来。事实上,不同疾病的组合感染剂代表了一种经济实用的进步,达到了通过一种预防处理即可抵御数种疾病的程度,这与单价疫苗的情况相反。但是,不同感染剂的共存提出了有关这些试剂相容性的实质性问题,这些问题使得很难同时完成有效的多种免疫接种。本发明的稳定剂提供了解决这些问题的办法。由于三价组合有热不稳定性,因此,用四价麻疹/腮腺炎/风疹/水痘疫苗的制备说明了疫苗组合的这些问题。使用本发明的稳定剂时,在四种效价的每一种效价的情况下,均稳定并保持了足够的感染潜能。
再进一步改善最终疫苗的稳定性,如果可能的话,这些疫苗优选地是冻干的。在制备冻干的活疫苗时,优选地将所述疫苗与适当量的本发明稳定剂接触。在收集所述病毒之前、之中或之后,将病毒与本发明的稳定剂接触。有利地在收集所述病毒前,将病毒与本发明的稳定剂接触。
在本说明书的以下部分将更详细地描述本发明。我们选择解释如何制备稳定的单价水痘疫苗和制备稳定的多价麻疹/腮腺炎/风疹/水痘疫苗作为实施本发明的实施例,因为在这些情况下,稳定性和组合的问题特别突出。
实施例
实施例1:根据本发明制备的稳定的水痘疫苗
用于制备本发明稳定的疫苗的减毒水痘株是OKA株。
用下列组分,以下列比例在注射用水中混合制备稳定剂:
-山梨糖醇,5%
-甘露糖醇,2.5%
-蔗糖,5%
-葡聚糖,3%
-氨基酸混合物,
-脲,0.5%
-谷氨酸钠,0.05%
通过在该稳定剂中将细胞分裂(声处理、剪切、高压等),或在稳定剂中自发破裂来从感染的细胞中收集或释放病毒。用这种方法,在澄清后就可得到稳定的病毒悬浮液。
在通过稀释调整效价后,将检测样品按等份放到小瓶中,然后按照适当的循环冻干。
所得疫苗的稳定性试验
将冻干的待测制品再水化并滴定。对于不同的试验稀释度,通过计数25 cm2平皿中产生的致细胞病变效应,用噬菌斑形成单位(pfu)数来确定活性。
所用的对照稳定剂是已知类型的传统稳定剂,这些稳定剂相应于例如美国专利4147772描述的那种稳定剂。
在制备疫苗的不同阶段稳定性比较
检测条件和阶段 | 效价(pfu/ml) | |
对照(如:SPGA) | 本发明的稳定剂 | |
收集过程中 | X | X+1 log |
冻干后 | X | X+0.2 log |
在37℃贮存7天 | X | X+1.1 log |
在上述表中,X是标准效价。
在不同贮存条件下,用本发明稳定剂达到的稳定性
贮存期 | 在22.5°的效价损失(Δpfu/剂量) | 贮存期 | 在5°的效价损失(Δpfu/剂量) | 贮存期 | 在-70°的效价损失(Δpfu/剂量) |
1个月 | 0.3 | 6个月 | 0 | 9个月 | 0.1 |
1.5个月 | 0.3 | 12个月 | 0.1 | 18个月 | 0.3 |
2个月 | 0.46 | 18个月 | 0.15 | 30个月 | 0.1 |
3个月 | 0.54 | 24个月 | 0.28 | 36个月 | 0.06 |
30个月 | 0.35 | ||||
36个月 | 0.4 |
上表中所示的值是三次试验进行平均得到的。
本发明的稳定剂在制备疫苗的不同阶段以及疫苗的贮存过程(在4种不同的温度下试验)中都能够保持病毒效价。实施例2:根据本发明制备稳定的麻疹/腮腺炎/风疹/水痘多效价疫苗
将已经在低温冷冻贮存的浓缩的麻疹、腮腺炎、风疹和水痘病毒产物融解,然后立即混合。然后将该混合物分配到不同的试验样品,所述试验样品补充了上述实施例1说明的本发明稳定产物。用该方法达到下列最终溶液比例:麻疹1.5个体积;腮腺炎0.01个体积;风疹1.0个体积和水痘2.5个体积。这些实施例列出的,这些不同的比例可随浓缩的病毒产物的效价而变化,并且不受限制。
将试验样品按等份放到小瓶中,然后按照标准循环冻干。通过将小瓶在37℃放置7天或在22℃放置21天,或在+4℃放置30个月来完成稳定性试验。
在不同贮存条件下用本发明稳定剂得到的稳定性
损失的效价(log) | |||
试验样品 | 37℃放置7天 | 22℃放置14天 | 22℃放置21天 |
麻疹 | 0.5 | #0 | #0 |
腮腺炎 | 1 | 0.3 | 0.4 |
风疹 | 0.09 | #0 | #0 |
水痘 | 1 | 0.15 | 0.16 |
在4℃试验样品 | 损失(log) | |||
麻疹 | 腮腺炎 | 风疹 | 水痘 | |
3个月 | 0.37 | 0.33 | 0.23 | 0.03 |
6个月 | #0 | 0.03 | 0.16 | 0.06 |
9个月 | 0.3 | 0.27 | 0.2 | 0.1 |
12个月 | 0.1 | 0.36 | #0 | #0 |
18个月 | 0.23 | 0.6 | 0.07 | 0.1 |
24个月 | 0.33 | 0.34 | 0.07 | 0.3 |
30个月 | 0.63 | 0.43 | 0.4 | 0.25 |
未观察到损失,这明显不同于相应的单价疫苗的情况。
Claims (29)
1.用于组合物,具体地含有一种或多种减毒病毒的疫苗的稳定剂,其特征在于所述稳定剂含有一种或多种选自下列各组中的组分:
-氨基酸,
-二糖,
-多元醇,
和缓冲液;
其条件是选择二糖的一种组分是蔗糖。
2.根据权利要求1所述的稳定剂,其特征在于选自二糖的一种或多种组分的比例是以2至6%。
3.根据权利要求2所述的稳定剂,其特征在于选自二糖的一种或多种组分的比例是2.5%。
4.根据权利要求1-3中任一权利要求所述的稳定剂,其特征在于所述多元醇包括山梨糖醇和甘露糖醇。
5.根据权利要求4所述的稳定剂,其特征在于它含有山梨糖醇和甘露糖醇。
6.根据权利要求4和5中的任一权利要求所述的稳定剂,其中山梨糖醇的比例是2至6%。
7.根据权利要求6所述的稳定剂,其中山梨糖醇的比例是5%。
8.根据权利要求4和5中任一权利要求所述的稳定剂,其中甘露糖醇的比例是1至2.5%。
9.根据权利要求8所述的稳定剂,其中甘露糖醇的比例是2.5%。
10.根据权利要求1至9中任一权利要求所述稳定剂,其特征在于缓冲液包括磷酸盐缓冲液和乙二胺四乙酸。
11.根据权利要求10所述的稳定剂,其特征在于它含有磷酸盐缓冲液。
12.根据权利要求1至11中的任一权利要求所述的稳定剂含有葡聚糖。
13.根据权利要求12所述的稳定剂,其中葡聚糖的比例是1至4%。
14.根据权利要求13所述的稳定剂,其中葡聚糖的比例是3%。
15.根据权利要求1至14中任一权利要求所述的稳定剂含有脲、一种脲衍生物或其混合物。
16.根据权利要求15所述稳定剂,其中脲、一种脲衍生物或其混合物的比例是0.125至2%。
17.根据权利要求16所述的稳定剂,其中脲、一种脲衍生物或其混合物的比例是0.5%。
18.根据权利要求1至17中任一权利要求所述的稳定剂,其中将pH调至7。
19.含有权利要求1-18中任一权利要求所述的稳定剂的稳定的、减毒的活病毒疫苗。
20.根据权利要求19所述的疫苗,它含有疱疹病毒科种的至少一种。
21.根据权利要求20所述的疫苗,其中疱疹病毒科含有水痘病毒、巨细胞病毒和单纯性疱疹病毒。
22.根据权利要求21所述的疫苗,含有水痘病毒。
23.根据权利要求19所述的疫苗,它含有选自下列的至少一种病毒:副粘病毒科(麻疹病毒,包括麻疹、腮腺炎病毒、副流感病毒1、2、3和4型以及肺病毒)、披盖病毒科(风疹)和流感(A、B和C)病毒。
24.根据权利要求23所述的疫苗,它含有麻疹、风疹和腮腺炎病毒。
25.含减毒活病毒的稳定疫苗的制备方法,其特征在于优选地在冻干阶段前,将所述疫苗组合物所用的病毒与适量的权利要求1至19中任一权利要求所述的稳定剂接触。
26.根据权利要求25的方法,其特征在于在收集所述病毒前,将所述病毒与稳定剂接触。
27.根据权利要求19至24中任一权利要求所述疫苗的制备方法,其特征在于优选地在冻干阶段前,将所述疫苗组合物所用的病毒与适量的权利要求1至19中任一权利要求所述的稳定剂接触。
28.根据权利要求27所述的方法,其特征在于在收集所述病毒前,将所述病毒与稳定剂接触。
29.含权利要求1至18中任一权利要求所述稳定剂的稳定组合物。
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- 1996-12-20 AT AT96943170T patent/ATE299712T1/de active
- 1996-12-20 DK DK96943170T patent/DK0869814T3/da active
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Also Published As
Publication number | Publication date |
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NZ324742A (en) | 1999-09-29 |
DE69634960D1 (de) | 2005-08-25 |
NO320958B1 (no) | 2006-02-20 |
NO982861D0 (no) | 1998-06-19 |
DE69634960T2 (de) | 2006-03-30 |
FR2742756A1 (fr) | 1997-06-27 |
JP2000502672A (ja) | 2000-03-07 |
AU1198697A (en) | 1997-07-17 |
WO1997023238A1 (fr) | 1997-07-03 |
CA2240541C (en) | 2005-03-29 |
FR2742756B1 (fr) | 1998-04-03 |
ES2242201T3 (es) | 2005-11-01 |
MX9804965A (es) | 1998-09-30 |
EP0869814A1 (fr) | 1998-10-14 |
CN1263508C (zh) | 2006-07-12 |
HU227946B1 (en) | 2012-06-28 |
JP4020431B2 (ja) | 2007-12-12 |
US6231860B1 (en) | 2001-05-15 |
NO982861L (no) | 1998-06-19 |
AU726630B2 (en) | 2000-11-16 |
DK0869814T3 (da) | 2005-10-17 |
HUP9903713A3 (en) | 2001-10-29 |
PT869814E (pt) | 2005-09-30 |
EP0869814B1 (fr) | 2005-07-20 |
HUP9903713A2 (hu) | 2000-03-28 |
ATE299712T1 (de) | 2005-08-15 |
CA2240541A1 (en) | 1997-07-03 |
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