Removal of endotoxins from plasmid DNA : Analysis of aggregative interaction of mobile divalent metal cations with endotoxins and plasmid DNA

Endotoxin lipopolysaccharide removal from plasmid DNA ‐based vaccine remains a very challenging task for bioprocess engineers. This paper examined the potential use and advantages of divalent cation ( Z n 2+ , C a 2+ , M g 2+ ) induced aggregation as a plasmid DNA purification method for lipopolysaccharide removal. Analysis of zeta potential, hydrodynamic size, percentage of aggregation; UV ‐ V is spectroscopy and electron microscopy were performed to determine the optimal cation for preferential aggregation of lipopolysaccharide over plasmid DNA . The results from the hydrodynamic size analysis showed that the addition of Z n 2+ resulted in the maximum theoretical number of lipopolysaccharide molecules per aggregate particle. Dynamic light scattering analysis showed that plasmid DNA aggregates formed a larger maximum hydrodynamic size when it was treated with C a 2+ than the other two cations. The K m value for lipopolysaccharide‐ Z n 2+ was substantially low (0.28 M) and considerably large (>2 M) for plasmid DNA ‐ Z n 2+ . Scatchard plots for plasmid DNA cations showed positive slopes indicating that there was a minimum concentration of plasmid DNA or cations before a significant aggregation occurred. This work concluded that Z n 2+ had the most preferential aggregative interaction with lipopolysaccharide compared to M g 2+ and C a 2+ .