Bioprocess & Bioanalytical Research, Merck Research Laboratories, West Point, PA 19486, USA.
J Virol Methods. 2010 Oct;169(1):13-21. doi: 10.1016/j.jviromet.2010.06.006. Epub 2010 Jun 25.
Cell culture derived rotavirus preparations contain a mixture of double-layered particles (DLPs) and triple-layered particles (TLPs). Characterization of rotavirus vaccine products is important to demonstrate a consistent manufacturing process. A capillary zone electrophoresis (CZE) method was developed to separate and quantitate rotavirus DLPs and TLPs in cell lysate samples and CsCl-purified vaccine preparations of each of the five reassortant rotavirus vaccine strains (G1, G2, G3, G4 and P1) contained in the pentavalent rotavirus vaccine, RotaTeq. The CZE electropherograms showed that migration of DLPs and TLPs from both CsCl-purified and cell lysates resulted in a separation distance of approximately 3 min between the two rotavirus particle types. The identification of the peak(s) containing TLPs was confirmed for both CsCl-purified and cell lysate samples by treatment of the samples with 50mM EDTA, which converted TLPs to DLPs. The migration pattern of the DLPs was consistent (23-24 min) among all reassortant strains tested, whether the DLPs were CsCl-purified or from cell lysates. However, the migration pattern of the TLP electropherograms of the reassortant rotavirus strains in cell lysates differed from those of the CsCl-purified reassortant rotavirus strains. In the cell lysate samples, the TLPs of the G1 and G2 reassortant rotavirus strains migrated slower that the corresponding TLPs from the CsCl-purified samples, while the migration time of the TLPs of the G3, G4 and P1 reassortants strains from the cell lysate and CsCl-purified samples appeared similar. Also, the TLPs from the CsCl-purified samples appeared as a defined single peak, while most of the TLPs from the cell lysate samples appeared as a broad peak or as multiple peaks. All the migration patterns were reproducible and consistent. Taking into account reproducibility, objective quantitation, and minimal sample manipulation as well as volume, CZE allowed consistent and quantitative characterization of rotavirus vaccine preparations, which is required for evaluation of vaccine products, including process validation.
细胞培养衍生的轮状病毒制剂包含双层颗粒 (DLPs) 和三层颗粒 (TLPs) 的混合物。轮状病毒疫苗产品的特征分析对于证明一致的生产工艺非常重要。本研究建立了毛细管区带电泳 (CZE) 方法，用于分离和定量细胞裂解样品和 CsCl 纯化的五价轮状病毒疫苗（RotaTeq）中五种重组轮状病毒疫苗株 (G1、G2、G3、G4 和 P1) 中的轮状病毒 DLPs 和 TLPs。CZE 电泳图谱显示，从 CsCl 纯化和细胞裂解物中迁移的 DLPs 和 TLPs 导致两种轮状病毒颗粒类型之间的分离距离约为 3 分钟。通过用 50mM EDTA 处理样品，证实了 CsCl 纯化和细胞裂解物样品中 TLPs 峰的鉴定，该处理将 TLPs 转化为 DLPs。无论 DLPs 是 CsCl 纯化的还是来自细胞裂解物，所有重组株测试的 DLPs 的迁移模式都是一致的 (23-24 分钟)。然而，细胞裂解物中重组轮状病毒株 TLP 的电泳图谱迁移模式与 CsCl 纯化的重组轮状病毒株的迁移模式不同。在细胞裂解物样品中，G1 和 G2 重组轮状病毒株的 TLPs 的迁移速度比 CsCl 纯化样品中的相应 TLPs 慢，而 G3、G4 和 P1 重组株的 TLPs 的迁移时间从细胞裂解物和 CsCl 纯化样品中似乎相似。此外，CsCl 纯化样品中的 TLPs 表现为定义明确的单个峰，而大多数细胞裂解物样品中的 TLPs 表现为宽峰或多个峰。所有迁移模式均具有重现性和一致性。考虑到重现性、客观定量和最小的样品处理以及体积，CZE 允许对轮状病毒疫苗制剂进行一致和定量的特征分析，这是评估疫苗产品包括工艺验证所必需的。
