Recombinant human parvovirus B19 virus-like particles (VLPs), a candidate vaccine, were produced using the insect cell (Sf-9)-baculovirus (AcNPV) expression system. The synthesis and assembly of the particles in Sf-9 cells are directed by double infections with one recombinant virus (bacVP1) expressing the parvovirus minor viral protein VP1 and a second virus (bacVP2) expressing the major viral protein VP2. Previous animal studies demonstrated that the polypeptide composition of the VLPs strongly affects the elicitation of virus neutralizing antibodies. The key factor controlling the production of an immunologically potent product in bioreactors was identified to be the multiplicity of infection (MOI) of bacVP1 and bacVP2 used for infection. A probabilistic model, which correlates well with the experimental results, was employed to facilitate the selection of MOIs and to provide a better understanding of the baculovirus co-infection process. A novel production process based on secondary infections was developed to ensure product consistency and to simplify large-scale logistics. The effects of other critical process parameters, such as temperature, dissolved oxygen concentration, lactate concentration, cell concentration at infection, and harvest time, were also investigated.