Bioproduction of Neospora caninum: a proof-of- concept towards a scalable serum-free process

Source abstract

Abstract Neospora caninum is emerging as a promising oncolytic protozoan for cancer immunotherapy. However, large-scale production of tachyzoites remains constrained by a culture system dependent on animal-derived serum and primary-like host cells, limiting standardisation, regulatory compliance, and scalability. Moreover, bioproduction processes for obligate intracellular protozoa are scarce and largely restricted to Plasmodium and Leishmania . Developing scalable manufacturing strategies for N. caninum is therefore a critical bottleneck for clinical translation. Building on a prior regulatory analysis, which emphasised early integration of classification, quality, and manufacturing constraints, we evaluated industrially relevant mammalian cell lines and serum-reduced or serum-free conditions for protozoa production. BHK-21 and CHO-K1 cells were selected to balance physiological relevance and industrial scalability. Cells were cultured in defined media and infected with N. caninum at varying multiplicities of infection (MOI). Protozoa yield, host cell growth, and glucose/lactate dynamics were monitored to assess production performance and host–protozoa metabolic interactions. Both cell lines supported protozoa multiplication under serum-free conditions, with a better yield with CHO-K1 than BHK-21 in our experimental conditions. In CHO-K1, production was strongly influenced by medium composition and MOI, with Advanced DMEM/F-12 yielding the highest protozoa output and stable metabolic profiles, while with other media, infection increased glucose consumption. These findings demonstrate the feasibility of serum-free N. caninum production using industrial cell lines (CHO-K1) and identify Advanced DMEM/F-12 as a promising medium for scalable, standardised, and GMP-compatible manufacturing.