GSK, Analytical Research and Development (ARD), Via Fiorentina 1, 53100 Siena, Italy; Department of Chemistry "U. Schiff", University of Florence, Via U. Schiff 6, 50019 Sesto Fiorentino, Florence, Italy.
Department of Chemistry "U. Schiff", University of Florence, Via U. Schiff 6, 50019 Sesto Fiorentino, Florence, Italy.
Talanta. 2018 Feb 1;178:552-562. doi: 10.1016/j.talanta.2017.09.077. Epub 2017 Sep 29.
Bexsero is the first approved vaccine for active immunization of individuals from 2 months of age and older to prevent invasive disease caused by Neisseria meningitidis serogroup B. The active components of the vaccine are Neisseria Heparin Binding Antigen, factor H binding protein, Neisseria adhesin A, produced in Escherichia coli cells by recombinant DNA technology, and Outer Membrane Vesicles (expressing Porin A and Porin B), produced by fermentation of Neisseria meningitidis strain NZ98/254. All the Bexsero active components are adsorbed on aluminum hydroxide and the unadsorbed antigens content is a product critical quality attribute. In this paper the development of a fast, selective and sensitive ultra-high-performance liquid chromatography (UHPLC) method for the determination of the Bexsero antigens in the vaccine supernatant is presented. For the first time in the literature, the Quality by Design (QbD) principles were applied to the development of an analytical method aimed to the quality control of a vaccine product. The UHPLC method was fully developed within the QbD framework, the new paradigm of quality outlined in International Conference on Harmonisation guidelines. Critical method attributes (CMAs) were identified with the capacity factor of Neisseria Heparin Binding Antigen, antigens resolution and peak areas. After a scouting phase, aimed at selecting a suitable and fast UHPLC operative mode for the vaccine antigens separation, risk assessment tools were employed to define the critical method parameters to be considered in the screening phase. Screening designs were applied for investigating at first the effects of vial type and sample concentration, and then the effects of injection volume, column type, organic phase starting concentration, ramp time and temperature. Response Surface Methodology pointed out the presence of several significant interaction effects, and with the support of Monte-Carlo simulations led to map out the design space, at a selected probability level, for the desired CMAs. The selected working conditions gave a complete separation of the antigens in about 5min. Robustness testing was carried out by a multivariate approach and a control strategy was implemented by defining system suitability tests. The method was qualified for the analysis of the Bexsero vaccine.
Bexsero 是首个获批准的疫苗，用于对 2 个月及以上的个体进行主动免疫接种，以预防由脑膜炎奈瑟氏菌 B 群引起的侵袭性疾病。疫苗的活性成分包括：通过重组 DNA 技术在大肠杆菌细胞中产生的奈瑟菌肝素结合抗原、因子 H 结合蛋白、奈瑟菌黏附素 A；以及通过脑膜炎奈瑟氏菌菌株 NZ98/254 发酵产生的外膜囊泡（表达 Porin A 和 Porin B）。Bexsero 的所有活性成分都吸附在氢氧化铝上，未吸附的抗原含量是产品关键质量属性。本文介绍了一种快速、选择性和灵敏的超高效液相色谱（UHPLC）方法，用于测定疫苗上清液中的 Bexsero 抗原。本文首次在文献中应用质量源于设计（QbD）原则，开发一种旨在控制疫苗产品质量的分析方法。该 UHPLC 方法完全在 QbD 框架内开发，该新方法是国际协调会议指南中概述的质量新范例。使用奈瑟菌肝素结合抗原的容量因子、抗原分辨率和峰面积等关键方法属性（CMA）进行识别。在选择适合且快速的 UHPLC 操作模式分离疫苗抗原的探索阶段之后，采用风险评估工具确定筛选阶段需要考虑的关键方法参数。筛选设计首先用于研究小瓶类型和样品浓度的影响，然后研究进样体积、柱类型、有机相起始浓度、梯度时间和温度的影响。响应面方法指出存在多个显著的相互作用效应，并且通过蒙特卡罗模拟的支持，在选定的概率水平下，映射出设计空间，以满足所需的 CMA。选择的工作条件可在大约 5 分钟内实现抗原的完全分离。通过多元方法进行稳健性测试，并通过定义系统适用性测试实施控制策略。该方法已通过验证可用于 Bexsero 疫苗的分析。
