Janssen Vaccines & Prevention B.V., Pharmaceutical and Analytical Development, Newtonweg 1, 2333 CP Leiden, The Netherlands.
Janssen Vaccines & Prevention B.V., Pharmaceutical and Analytical Development, Newtonweg 1, 2333 CP Leiden, The Netherlands.
Talanta. 2017 May 1;166:8-14. doi: 10.1016/j.talanta.2017.01.013. Epub 2017 Jan 12.
During development of adenovirus-based vaccines, samples have to be analyzed in order to either monitor the production process or control the quality and safety of the product. An important quality attribute is the total concentration of intact adenoviruses, which currently is determined by quantitative polymerase chain reaction (qPCR) or anion exchange-HPLC. Capillary Electrophoresis (CE) was evaluated as alternative to the current methods with the aim to have one single method that allows reliable and fast quantification of adenovirus particles throughout the full process. Intact adenoviruses samples from downstream processing and upstream processing were analyzed directly by CE with UV-detection at 214nm. Only the samples with high amounts of DNA required a simple sample pretreatment by benzonase. Adenovirus particles were separated from matrix components such as cell debris, residual cell DNA, and/or proteins on a PVA-coated capillary using a BGE consisting of 125mM Tris, 338mM tricine and 0.2% v/v polysorbate-20 at pH 7.7. Full factorial design of experiments was used for method optimization as part of the analytical quality by design (AQbD) method development approach. The method was validated for the quantification of adenoviruses on five representative samples from the manufacturing process in the range of 0.5×10-1.5×10 adenovirus particles per ml (~80 to 250pmol/l). The CE method showed intermediate precision of 7.8% RSD on concentration and an accuracy (spiked recovery) of 95-110%. CE proved highly useful for process development support and is being implemented for in-process control testing for adenovirus vaccine manufacturing.
在腺病毒疫苗的开发过程中，必须对样品进行分析，以监测生产过程或控制产品的质量和安全性。总浓度的完整腺病毒是一个重要的质量属性，目前通过定量聚合酶链反应（qPCR）或阴离子交换-HPLC 来确定。毛细管电泳（CE）被评估为替代当前方法，目的是拥有一种单一的方法，可以在整个过程中可靠和快速地定量腺病毒颗粒。直接通过 CE 用 214nm 的紫外检测分析下游和上游处理的完整腺病毒样品。只有那些 DNA 含量高的样品需要简单的预处理，即用苯甲酸钠核酸酶处理。腺病毒颗粒在 PVA 涂层毛细管上，使用包含 125mM Tris、338mM 三羟甲基氨基甲烷和 0.2%v/v 吐温-20 的 BGE，与细胞碎片、残留的细胞 DNA 和/或蛋白质等基质成分分离。作为分析质量设计（AQbD）方法开发方法的一部分，实验的完全因子设计用于方法优化。该方法在五个具有代表性的生产过程样品中的浓度范围为 0.5×10-1.5×10 腺病毒颗粒/ml（~80 至 250pmol/l）进行了验证。CE 方法在浓度上显示出 7.8%RSD 的中间精密度，准确度（加标回收率）为 95-110%。CE 对工艺开发支持非常有用，正在实施腺病毒疫苗生产的过程控制测试。
