Zika virus-like particles' production lab protocol in chemically defined medium using quality by design.

Zika virus remains a global health emergency without approved vaccines or drugs. Virus-like particles (VLP) provide a safe vaccine-development platform, but optimizing the baculovirus/insect-cell expression system to obtain these nanostructures is difficult because multiple critical parameters interact. This study used statistical experimental designs with low control requirements and operating costs to optimize multiplicity of infection (MOI), time of infection (TOI), and time of harvest (TOH) for Zika VLP production. VLP were immunochemically characterized. Dot-blot chemiluminescence provided volumetric total signal intensity as a measure of relative protein expression, while negative-stain transmission electron microscopy and immunolabeling determined morphology, mean particle size, and heterogeneity through the unbiased standard error of mean particle size. Mathematical models related MOI, TOI, TOH, and their interactions to recombinant-protein quality and relative quantity. A desirability-function optimization identified MOI 0.2 pfu cell-1, TOI 46 h, and TOH 48 h as optimal. These conditions predicted VLP with a mean diameter of 58.52 nm, a standard error of 3.52 nm, and signal intensity equal to 64.57% of the study maximum. Thus, subsequent process-development stages involving equipment-dependent parameters relevant to commercial scale can be completed faster and at lower cost, supporting efficient scale-up of a promising Zika vaccine candidate platform.