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。 Objective To establish a new purification technology of acellular pertussis vaccine (APV) which is suitable for large-scale production and make the ratio of the main components of the vaccine stable and controllable. Methods Resuscitation of B. pertussis strains and subculture were carried out. Before the fermentation of large tank, the pH value of the broth was adjusted to weak acidity, and the supernatant was obtained by centrifugation. The supernatant was concentrated and desalted by ultrafiltration and was subjected to cation exchange chromatography The components were isolated and purified. The purified products were separated by SDS-PAGE and identified by Western blot to determine the best purification conditions. Three batches of APV were continuously purified by a new chromatographic purification process and the indexes were tested to verify the repeatability of the process. Results Before harvest, the pH value of the broth was adjusted to 5.8-6.0, which significantly increased the content of the main protective antigen in the supernatant. The strong cation exchange chromatography was used to separate and purify the target protein from multi-component Can be completed in one step, the purity of each objective protein component can reach more than 85%, the recovery rate can reach more than 90%; the established purification process has good repeatability. Conclusion The chromatographic purification process that can be linearly amplified and suitable for large-scale production of APV was initially established, laying a foundation for the improvement of vaccine quality standards.