Optimization of rabies virus production in neuroblastoma cells using quality by design principles for vaccine and diagnostic applications.

Efficient production of high-titer rabies virus (RABV) stocks is critical for vaccine development, diagnostic assay design, and virological research. This study employed a Quality by Design (QbD) framework to optimize RABV propagation in Neuro-2a (N2a) cells, targeting infectious titers ≥ 8.0 log₁₀ FFU/mL and cell viability ≥ 85 %. Three genetically distinct RABV strains were evaluated: the laboratory-adapted CVS-11 strain for vaccine production and two field isolates, MN-18 and V-M, relevant to diagnostics and research. A 2 ³ full factorial Design of Experiments (DOE) was implemented to assess the impact of multiplicity of infection (MOI), incubation temperature, and culture medium composition on critical quality attributes (CQAs). Distinct strain-specific responses were observed: CVS-11 and V-M were significantly influenced by MOI (p < 0.05), whereas MN-18 demonstrated a more stable replication profile. Enriched medium significantly enhanced viral titers and cell viability across all strains (p < 0.001). Monte Carlo simulations (n = 10,000) delineated an optimal operational space-MOI 0.1-1.0, temperature 34-35°C, enriched medium-ensuring process robustness with 95 % confidence. This QbD-guided strategy provides a scalable, reproducible platform for generating high-quality RABV stocks, facilitating the manufacture of CVS-11-based vaccines and supporting the diagnostic use of field isolates as biologically standardized reference reagents for diagnostics.