Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Morelos, México.
Electrophoresis. 2010 Apr;31(8):1376-81. doi: 10.1002/elps.200900558.
Virus-like particles have been successfully used as safe vaccines, as their structure is identical to their native counterparts but devoid of the viral genetic material. However, production of these complex structures is not easy, as recombinant proteins must assemble into virus-like particles. Techniques to differentiate assembled and soluble proteins, as well as assembly intermediaries often present in a sample, are required. An example of complex virus-like particles mixture occurs when rotavirus proteins are recombinantly expressed. Rotavirus-like particles (RLP) can be single (sl), double (dl), or triple layered (tl). The use of RLP preparations as vaccines requires their complete characterization, including separation and quantification of each RLP in a sample. In this work, CZE was evaluated for the separation and quantification of dl and triple-layered rotavirus-like particles (tlRLP). A fused-silica capillary with a deoxycholate running buffer efficiently separated dl and tlRLP in RLP preparations, as they migrated in two discrete peaks with electrophoretic mobilities of 1.24+/-0.04 and 2.95+/-0.03 Ti, respectively. Standard curves for dl and tlRLP were generated, and the response was linearly proportional to analyte concentration. The methodology developed was quantitative, specific, accurate, precise, and reproducible. CZE allowed the quantitative characterization of RLP preparations, which is required for evaluation of immunogens, for process development, and for quality control protocols.
病毒样颗粒已成功用作安全疫苗，因为它们的结构与其天然对应物相同，但不含病毒遗传物质。然而，这些复杂结构的生产并不容易，因为重组蛋白必须组装成病毒样颗粒。需要区分组装和可溶性蛋白的技术，以及样品中通常存在的组装中间体。当轮状病毒蛋白被重组表达时，就会出现复杂的病毒样颗粒混合物的例子。轮状病毒样颗粒 (RLP) 可以是单层 (sl)、双层 (dl) 或三层 (tl)。RLP 制剂用作疫苗需要对其进行完全表征，包括分离和定量样品中的每个 RLP。在这项工作中，CZE 用于分离和定量 dl 和三层轮状病毒样颗粒 (tlRLP)。带有脱氧胆酸钠运行缓冲液的熔融石英毛细管能够有效地分离 RLP 制剂中的 dl 和 tlRLP，因为它们以两个离散的峰迁移，电泳迁移率分别为 1.24+/-0.04 和 2.95+/-0.03 Ti。生成了 dl 和 tlRLP 的标准曲线，响应与分析物浓度呈线性比例。开发的方法是定量的、特异性的、准确的、精密的和可重复的。CZE 允许定量表征 RLP 制剂，这是评估免疫原、进行工艺开发和质量控制协议所必需的。
