Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
Department of Pharmaceutical Chemistry, Vaccine Analytics and Formulation Center, University of Kansas, Lawrence, Kanas, USA.
Biotechnol Bioeng. 2021 Apr;118(4):1750-1756. doi: 10.1002/bit.27699. Epub 2021 Feb 19.
Nonreplicating rotavirus vaccine (NRRV) candidates are being developed with the aim of serving the needs of developing countries. A significant proportion of the cost of manufacturing such vaccines is the purification in multiple chromatography steps. Crystallization has the potential to reduce purification costs and provide new product storage modality, improved operational flexibility, and reduced facility footprints. This communication describes a systematic approach for the design of the crystallization of an NRRV candidate, VP8 subunit proteins fused to the P2 epitope of tetanus toxin, using first-principles models and preliminary experimental data. The first-principles models are applied to literature data to obtain feasible crystallization conditions and lower bounds for nucleation and growth rates. Crystallization is then performed in a hanging-drop vapor diffusion system, resulting in the nucleation and growth of NRRV crystals. The crystals obtained in a scaled-up evaporative crystallization contain proteins truncated in the P2 region, but have no significant differences with the original samples in terms of antibody binding and overall conformational stability. These results demonstrate the promise of evaporative crystallization of the NRRV.
正在开发非复制型轮状病毒疫苗 (NRRV) 候选物，以满足发展中国家的需求。制造此类疫苗的成本中，相当大的一部分是经过多次色谱步骤的纯化。结晶有可能降低纯化成本，并提供新的产品储存方式、提高操作灵活性和减少设施占地面积。本通讯描述了一种使用第一性原理模型和初步实验数据设计与破伤风毒素 P2 表位融合的 NRRV 候选物 VP8 亚单位蛋白结晶的系统方法。第一性原理模型应用于文献数据，以获得可行的结晶条件和成核与生长速率的下限。然后在悬滴蒸汽扩散系统中进行结晶，导致 NRRV 晶体的成核和生长。在规模化蒸发结晶中获得的晶体在 P2 区域截断，但在抗体结合和整体构象稳定性方面与原始样品没有显著差异。这些结果表明蒸发结晶 NRRV 具有前景。
