摘要 据统计,5%以上的人类癌症由人乳头瘤病毒(HPV)导致。HPV疫苗的使用,尤其是多价HPV疫苗的使用,可有效预防HPV感染和肿瘤的发生。例如,9价HPV疫苗可有效预防90%以上HPV相关癌前病变。人乳头瘤病毒样颗粒(VLP)是HPV疫苗的唯一抗原。VLP由360份衣壳蛋白L1组成。VLP的含量测定对HPV原液和HPV疫苗的质量评价至关重要。该文发展了一种以体积排阻色谱(SEC)为基础的9种型别人乳头病毒样颗粒的定量方法。实验优化了包括色谱柱类型、色谱柱孔径、流动相离子强度和流动相pH值在内的色谱条件。经过考察,以SHIMSEN Ankylo SEC-300色谱柱(300 mm×7.8 mm,3μm)为固定相,以含有300 mmol/L NaCl和50 mmol/L磷酸盐(pH 7.0)的缓冲溶液为流动相时,VLP的色谱峰更窄,从而可获得更高的响应和更好的灵敏度,因此选择该色谱条件用于VLP与基质的分离。优化所得的方法具有较宽的线性范围,良好的重复性(峰面积的相对标准偏差不大于5.0%)和灵敏度(定量限为4.58~15.24μg/mL)。将方法用于HPV原液中VLP的含量测定,监测VLP的稳定性。结果显示,HPV原液中VLP颗粒不稳定,于4℃放置一周后,VLP含量与生产后立即测得的含量相比存在一定程度的降解。此外,方法还可用于疫苗上清液中游离蛋白质的分析,监测铝佐剂对VLP的吸附情况。被测厂家的铝佐剂可较好的吸附VLP,无明显残余蛋白质检出。与传统的蛋白质定量方法相比,如Folin-酚法(Lowry法),该法具有操作简单、自动化程度高、分析通量高等优点,可实现VLP含量的批量化分析。 Cervical cancer is the fourth most common cancer among women.Human papilloma virus(HPV)is the most common cause of cervical cancer which accounts for 5%of all human cancers and results in about 528000 cases and 266000 deaths every year.HPV vaccines are considered the most effective strategy for the prevention of HPV infection and cervical carcinoma.Since 2006,three prophylactic vaccines against HPV have been available on the market,including bivalent vaccines,quadrivalent vaccines,and nine-valent vaccines.Among them,nine-valent vaccines have been reported to be the most effective.They can prevent 97%of the high-grade pre-cancer lesions.Virus-like particles(VLPs),which are arranged as 360 copies of capsid proteins L1,are the only antigens of the HPV vaccine.Nine-valent HPV vaccines are prepared by mixing nine types of VLPs with adjuvants.Thus,the quality of the VLPs,including their stability and content in the HPV bulk,is very important for developing HPV vaccines.In this study,a method was developed for the determination of the nine types of VLPs(HPV6/11/16/18/31/33/45/52/58)in HPV bulk by size exclusion chromatography(SEC).The parameters of this method were optimized in terms of column brand,pore size of stationary phase particles,buffer concentration,and pH value.SHIMSEN Ankylo SEC-300 column(300 mm×7.8 mm,3μm)combined with a buffer aqueous solution containing 300 mmol/L NaCl and 50 mmol/L phosphate(pH 7.0)was utilized to separate the VLPs from the matrix since a narrow peak shape and good repeatability for VLPs could be obtained with this column and mobile phase.The optimized method had a wide linear range,good repeatability(RSDs of peak area were not more than 5.0%),and a satisfactory sensitivity(LOQs in the range of 4.58-15.24μg/mL).The optimized method was used to determine the VLPs in the HPV bulk.The LOQs of the current method were much lower than the content of the nine types of VLPs in the HPV bulk,indicating that this method was sensitive enough for the determination of the nine types of VLPs in the
出处 《色谱》 CAS CSCD 北大核心 2021年第4期424-429,共6页 Chinese Journal of Chromatography
基金 国家科技重大药物专项(2018ZX09101001) 中国食品药品检定研究院学科带头人培养基金(2019X1).
关键词 体积排阻色谱 定量 人乳头瘤病毒 病毒样颗粒 疫苗 size exclusion chromatography(SEC) quantitative analysis human papilloma virus(HPV) virus-like particle(VLP) vaccine
分类号 O658 [理学—分析化学]
