目的: 开发生物制剂卡介菌多糖核酸注射液质量评价方法。方法: 基于核酸物质是卡介菌多糖核酸注射液(简称注射液)的主要活性成分之一,其免疫活性表现可反映注射液质量。以免疫核酸CpG1826为标准参比核酸,非CpG核酸No-CpG为空白参比核酸,注射液或所含核酸组份为测试核酸,采用酶联免疫(ELISA试剂盒)法测定核酸刺激巨噬细胞释放细胞因子TNF-α的浓度([TNF-α]),再采用公式IFC=([TNF-α]测-[TNF-α]空)/([TNF-α]标-[TNF-α]空)计算功能指数(IFC),根据IFC大小来评价生物制剂质量。对15批次标准样品(合格产品)进行多次平行测定,训练确定合格产品的IFC范围,再用11批次实际样品(6批次合格产品,5批次超期产品)进行方法验证。结果: 从注射液提取核酸组分可显著刺激巨噬细胞释放细胞因子TNF-α,刺激现象与免疫CpG核酸一致,说明注射液中核酸物质具有类似免疫活性;15批次标准样品训练确定IFC范围为0.3~0.9;11批次实际样品中,6批次合格产品IFC在范围之内,5批次超期产品IFC偏高。结论: 基于核酸免疫活性分析技术建立的功能指数IFC较好反应了卡介菌多糖核酸注射液质量,可望用于其质量评价,也为类似生物制剂质量控制提供活性评价参考。
Objective: To develop a quality evaluation method of biologics bacillus calmette guerin (BCG) polysaccharide nucleic acid injection (abbreviated as injection). Methods: The method was constructed based on the fact that nucleic acid was one of the main active components of BCG polysaccharide nucleic acid injection and nucleic acid immunoreactivity related with the quality of the injection. The immune nucleic acid CpG1826 was used as the standard reference nucleic acid, the nucleic acid without CpG domain No-CpG was used as the blank reference nucleic acid, and the injection or the nucleic acid component contained was used as the test nucleic acid. Then enzyme linked immunosorbent assay (ELISA) Kit was used to determine the concentration of cytokine TNF-α released from macrophages which stimulated by each group of nucleic acid. The formula IFC=([TNF-α]test-[TNF-α]blank)/([TNF-α]standard-[TNF-α]blank) was used to calculate the functional index (IFC) for the evaluation of immune activity. Multiple parallel determinations were performed on 15 batches of standard samples (qualified products) to train and certify the scope of IFC, and 11 batches of actual samples (6 qualified products and 5 overdue products) were uesd for method validation. Results: The nucleic acid component extracted from the injection significantly stimulated macrophages to release cytokine TNF-α. The stimulation ability was consistent with the effect of immuned CpG nucleic acid, indicating that the nucleic acid had similar immune activity. Determination results of 15 batches of standard samples certified that the function index IFCs ranged from 0.3 to 0.9. Determination results of 11 batches of actual samples showed that the IFCs of 6 batches of qualified products were in the range, while those of 5 batches of overdue products were on the high side. Conclusion: The functional index IFC established based on the nucleic acid immunoreactivity analysis better reflects the quality of BCG polysaccharide nucleic acid injection, which is expected to be used for its quality evaluation, and also provides activity evaluation index for the quality control of similar biological products.
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