The use of plasmid DNA (pDNA) for human vaccines is a novel approach against leishmaniasis, a neglected tropical disease with severe clinical manifestations. The development of feasible bioprocesses to obtain such vaccines is a public-health priority. The aim of this work was to investigate the substrate-source flexibility of an exponential-fed perfusion (EFP) system to produce the plasmid pVAX1-NH36 for use as a leishmaniasis vaccine. Batch and EFP cultures were conducted using Escherichia coli DH5a as a host and glucose or glycerol as a carbon source. The culture kinetics of the cell, substrate and plasmid concentrations were measured. Mathematical kinetics models were fitted to experimental data and used to describe the system comportment (r 2 > 0.95). Plasmid productivities of 13.3 mg/(L h) using glucose and 19.4 mg/(L h) using glycerol were obtained. These levels represent a 1-3-fold increase in performance index compared with previously reported cultures using E. coli DH5a. The novel aspect of this work is the demonstration of the flexibility of EFP cultures for production of pDNA vaccines. Our data suggest that E. coli engineering to increase pDNA production using glucose can be circumvented with an EFP culture, reducing the host strain development costs. In addition, the greater productivity of EFP cultures entails a reduction in manufacturing costs.