Crystallization of a nonreplicating rotavirus vaccine candidate.

Moo Sun Hong,Nishant Sawant,Richard D Braatz,Sangeeta B Joshi,David B Volkin,Kawaljit Kaur

doi:10.1002/bit.27699

Abstract

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.

Highlights

Rotaviruses are one of the main causes of severe gastroenteritis among children, causing ~200,000 annual mortalities in less than 5 years of age worldwide with more than 90% occurring in low‐ income and low‐middle‐income countries (Tate et al, 2016)
The most advanced of the candidates are truncated VP8 subunit proteins fused to the P2 epitope of tetanus toxin (Kirkwood et al, 2019)
Water evaporates, which increases the concentration of protein and precipitant, until the droplet reaches equilibrium with the reservoir

Summary

Introduction

Rotaviruses are one of the main causes of severe gastroenteritis among children, causing ~200,000 annual mortalities in less than 5 years of age worldwide with more than 90% occurring in low‐ income and low‐middle‐income countries (Tate et al, 2016). Literature‐ reported results for truncated VP8 subunit proteins of rotaviruses (Dormitzer et al, 2002; Kraschnefski et al, 2008, 2005; Scott et al, 2005; Yu et al, 2008; Zhang et al, 2007) are analyzed to obtain feasible crystallization conditions and lower bounds on the crystal nucleation and growth rates. Water evaporates, which increases the concentration of protein and precipitant, until the droplet reaches equilibrium with the reservoir.

Results

Conclusion