Characterizing and Minimizing Aggregation and Particle Formation of Three Recombinant Fusion-Protein Bulk Antigens for Use in a Candidate Trivalent Rotavirus Vaccine.

Sanjeev Agarwal,Stanley Cryz,Sangeeta B Joshi,John M Hickey,George A Robertson,David B Volkin,Robert Sitrin,Neha Sahni

doi:10.1016/j.xphs.2019.08.001

Abstract

In a companion paper, the structural integrity, conformational stability, and degradation mechanisms of 3 recombinant fusion-protein antigens comprising a non-replicating rotavirus (NRRV) vaccine candidate (currently being evaluated in early-stage clinical trials) are described. In this work, we focus on the aggregation propensity of the 3 NRRV antigens coupled to formulation development studies to identify common frozen bulk candidate formulations. The P2-VP8-P[8] antigen was most susceptible to shaking and freeze-thaw–induced aggregation and particle formation. Each NRRV antigen formed aggregates with structurally altered protein (with exposed apolar regions and intermolecular β-sheet) and dimers containing a non-native disulfide bond. From excipient screening studies with P2-VP8-P[8], sugars or polyols (e.g., sucrose, trehalose, mannitol, sorbitol) and various detergents (e.g., Pluronic F-68, polysorbate 20 and 80, PEG-3350) were identified as stabilizers against aggregation. By combining promising additives, candidate bulk formulations were optimized to not only minimize agitation-induced aggregation, but also particle formation due to freeze-thaw stress of P2-VP8-P[8] antigen. Owing to limited material availability, stabilization of the P2-VP8-P[4] and P2-VP8-P[6] was confirmed with the lead candidate P2-VP8-P[8] formulations. The optimization of these bulk NRRV candidate formulations is discussed in the context of subsequent drug product formulations in the presence of aluminum adjuvants.

Highlights

Every child before reaching 5 years of age gets infected by Rotavirus (RV), which can cause gastroenteritis and diarrhea.1 There are currently 4 WHO prequalified RV vaccines (RotaTeq®, Rotarix®, Rotavac®, and Rotasil®) which combined cover over 100 countries to reduce the burden of this viralCurrent address for Dr Agarwal: Amgen, Thousand Oaks, California, 91320
The P[4] and P[6] used for colloidal stressed stability studies were produced and purified from E. coli at Walter Reed Army Institute of Research, MD, and formulated in 0.5 mM sodium phosphate, 150 mM NaCl, pH 7.2
The P[4] and P[6] used for the bulk formulation studies, and P[8] used for all the studies in this work, were produced and purified from E. coli by Blue Sky BioServices, MA, and provided in 600 mM ammonium sulfate (AS), 50 mM Tris buffer at pH 7.5

Summary

Introduction

Every child before reaching 5 years of age gets infected by Rotavirus (RV), which can cause gastroenteritis and diarrhea.. There are currently 4 WHO prequalified RV vaccines (RotaTeq®, Rotarix®, Rotavac®, and Rotasil®) which combined cover over 100 countries to reduce the burden of this viral. Current address for Dr Agarwal: Amgen, Thousand Oaks, California, 91320. Current address for Dr Sahni: GSK Vaccines, Rockville, Maryland, 20850. Current address for Dr Robertson: Cambra Consulting, 4895 Prince William Parkway, Suite 101 Woodbridge, Virginia, 22039. S. Agarwal et al / Journal of Pharmaceutical Sciences 109 (2020) 394-406 hexavalent DTaP-IPV-HepB-Hib or the pentavalent DTwcP-HepBHib vaccines to improve compliance with the immunization schedule and encourage wider vaccination coverage.

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