注射用A型肉毒毒素的生物学特性及质量分析

目的对注射用A型肉毒毒素(Botulinum toxin type A for injection,商品名衡力,出口商品名BTXA)制品中的活性药用成分(Active pharmaceutical ingredient,API)的性质及2009～2011年生产的注射用A型肉毒毒素原液的比活性进行分析。方法对BTXA收获物样品进行阴离子交换层析,分离毒素复合体各组分;采用血凝试验、HPLC法、SDS-PAGE、等点聚焦电泳及N-末端氨基酸测序等方法分析A型肉毒毒素复合体及其各组分的性质;统计2009～2011年连续生产的注射用A型肉毒毒素原液的比活性数据,分析连续生产的工艺稳定性和质量重现性。结果 BTXA收获物样品在碱性条件下可被解离,解离出的A型肉毒神经毒素相对分子质量约为150 000,无血凝效价;BTXA的API为完整的单体,纯度均在99.5%以上;复合物和神经毒素的等电点分别为4.97、4.91,N-末端氨基酸测序结果与GenBank基本一致;在连续3年的生产过程中,原液比活性平均为3.0×107LD50/mg蛋白,BTXA成品中API载量约为5 ng/瓶。结论注射用A型肉毒毒素的活性成分是特异的复合体结构,可在碱性条件下解离出A型肉毒神经毒素;BTXA原液的比活性在连续3年的生产中持续稳定,具备很好的连续性,为临床使用的安全性提供依据。

Biological characteristics and quality of botulinum toxin type A for injection

Objective To analyze the property of active pharmaceutical ingredient(API) of botulinum toxin type A(BTXA) for injection,as well as the specific activities of the preparations manufactured in 2009 ~ 2011.Methods Various components were separated from the harvested BTXA by anion exchange chromatography.The properties of BTXA complex and its components were analyzed by hemagglutination test,HPLC,SDS-PAGE,isoelectric focusing electrophoresis and amino acid sequencing at N-terminus.The data on specific activity of bulks of BTXA manufactured in 2009 ~ 2011 were analyzed,based on which the stability of production procedure and the reproducibility of quality were evaluated.Results The harvested BTXA was dissociated in basic environment.The obtained neurotoxin showed no hemagglutination titer,of which the relative molecular mass was about 150 000.The API of BTXA was an intact monomer,of which the purity was more than 99.5%.The isoelectric points of BTXA complex and neurotoxin were 4.97 and 4.91 respectively,of which the amino acid sequences at N-terminus were basically consistent with those reported in GenBank.The mean specific activity of bulks of BTXA manufactured in 2009 ~ 2011 was 3.0 × 107 LD50 / mg protein.The load of API in final product of BTXA was about 5 ng per container.Conclusion The API of BTXA was a specific complex which was dissociated in basic environment to obtain neurotoxin.The specific activities of bulk BTXA manufactured in 2009 ~ 2011 were stable,indicating good persistency of quality,which provided a basis for safety of BTXA in clinic.