Title:
Virus-like particle formulation optimization by miniaturized high-throughput screening.

Abstract:
Virus-like particles (VLPs) are non-infectious and immunogenic virus-mimicking protein assemblies that are increasingly researched as vaccine candidates. Stability against aggregation is an important determinant dictating the viability of a pipeline VLP product, making multivariable stability data highly desirable especially in early product development stages. However, comprehensive formulation studies are challenging due to low sample availability early in developability assessment. This issue is exacerbated by industry-standard analytical techniques which are low-throughput and/or sample-consuming. This study presents a miniaturized high-throughput screening (MHTS) methodology for VLP formulation by integrating dynamic light scattering (DLS) and asymmetrical flow field-flow fractionation (AF4) in a formulation funnel analysis. Using only 2 μg of sample and 100 s per measurement, a DLS plate reader was deployed to effectively pre-screen a large experimental space, allowing a smaller set of superior formulation conditions to be interrogated at high-resolution with AF4. The stabilizing effects of polysorbate 20, sucrose, trehalose, mannitol and sorbitol were investigated. MHTS data showed that addition of 0.5% w/v polysorbate 20 together with either 40% w/v sucrose or 40% w/v sorbitol could stabilize VLPs at elevated temperatures up to 58 °C. AF4 data further confirmed that the formulation containing 40% w/v sorbitol and 0.5% w/v polysorbate 20 effectively protected VLPs during freeze-thawing and freeze-drying, increasing recoveries from these processes by 80 and 50 percentage points, respectively. The MHTS strategy presented here could be used to rapidly explore a large formulation development space using reduced amounts of sample, without sacrificing the analytical resolution needed for quality control. Such a method paves the way for rapid formulation development and could potentially hasten the commercialization of new VLP vaccines.