无细胞百日咳疫苗纯化新工艺的建立

目的建立适合大规模生产的无细胞百日咳疫苗(Acellular pertussis vaccine,APV)纯化新工艺,使疫苗主要成分的比例稳定可控。方法复苏百日咳菌种并传代培养,在大罐发酵培养收获前,调整菌液的pH值至弱酸性,再通过离心获得上清液,经超滤浓缩和脱盐处理,使用阳离子交换层析进行目的组分的分离纯化,纯化产物经SDS-PAGE分离并经Western blot鉴定,确定最佳纯化条件。用建立的层析纯化新工艺连续纯化3批APV,检测各项指标,验证该工艺的重复性。结果收获前菌液pH值调至5.8～6.0,可使疫苗主要保护抗原在上清液中的含量明显提高;采用强阳离子交换层析的方法,从目的蛋白的捕获到多组分的分离提纯可一步完成,各目的蛋白组分的纯度均可达85%以上,回收率可达90%以上;建立的纯化新工艺具有良好的重复性。结论初步建立了可线性放大、适合APV大规模生产的层析纯化新工艺,为疫苗质量标准的提高奠定了基础。

Development of a novel procedure for purification of acellular pertussis vaccine

Objective To develop a novel procedure suitable for large-scale production of acellular pertussis vaccine(APV).Methods Bordetella pertussis strain was resuscitated and subcultured.The pH value of fermentation liquid of B.pertussis was adjusted to weak acidity before harvest.Supernatant was collected after centrifugation,concentrated by ultrafiltration and subjected to desalting,from which the target components were purified by cation exchange chromatography.The purified protein was identified by SDS-PAGE and Western blot,based on which the condition for purification was optimized.Three successive batches of APV were purified by the developed procedure and tested for various quality indexes to verify the reproducibility of the procedure.Results Adjusting the pH value of fermentation liquid to 5.8 ～ 6.0 before harvest increased the content of major protective antigen in supernatant significantly.By strong cation exchange chromatography,the capture of target protein and purification of various components were completed in a single step.All the purities of various target protein components were more than 85%,while the recovery rates were more than more than 90%.The developed procedure showed high reproducibility.Conclusion A novel purification procedure which might be scaled up linearly and were suitable for large scale production of APV was developed.It laid a foundation of improvement of quality standard of vaccine.