Journal of Biotechnology 186 (2014) 38–48 Contents lists available at ScienceDirect Journal of Biotechnology j ourna l ho me pa ge: www.elsevier.com/locate/jbiotec Kinetic modeling of plasmid bioproduction in Escherichia coli DH5˛ cultures over different carbon-source compositions Marta B. Lopes a,b , Gabriel Martins a , Cecília R.C. Calado a,∗ a Engineering Faculty, Catholic University of Portugal, Estrada Octávio Pato, 2635-631 Rio de Mouro, Portugal b Institute of Telecommunications, Instituto Superior Técnico, Technical University of Lisbon, Avenida Rovisco Pais, 1049-001 Lisboa, Portugal a r t i c l e i n f o Article history: Received 3 February 2014 Received in revised form 18 June 2014 Accepted 23 June 2014 Available online 3 July 2014 Keywords: Escherichia coli Kinetic model Multiple substrate Plasmid Acetate Fermentation a b s t r a c t The need for the development of economic high plasmid production in Escherichia coli cultures is emerg- ing, as a result of the latest advances in DNA vaccination and gene therapy. In order to contribute to achieve that, a model describing the kinetics involved in the bioproduction of plasmid by recombinant E. coli DH5˛ is presented, as an attempt to understand the complex and non-linear metabolic relationships and the plasmid production occurring in dynamic batch culture environments, run under different media compositions of glucose and glycerol, that result in distinct maximum biomass growths (between 8.2 and 12.8 g DCW/L) and specific plasmid productions (between 1.1 and 7.4 mg/g DCW). The model based on mass balance equations for biomass, glucose, glycerol, acetate and plasmid accurately described differ- ent culture behaviors, using either glucose or glycerol as carbon source, or mixtures of both. From the 17 parameters obtained after model simplification, the following 10 parameters were found to be inde- pendent of the carbon source composition: the substrate affinity constants, the inhibitory constants of biomass growth on glycerol by glucose, of biomass growth on acetate by glycerol and the global biomass growth by acetate, and the yields of biomass on acetate, acetate on glucose and glycerol, and plasmid on glucose. The parameters that depend on the culture composition, and that might explain the differ- ences found between cultures, were: maximum specific growth rates on glucose, glycerol and acetate; biomass yield on glucose and glycerol; and plasmid yield on glycerol and acetate. Moreover, a crucial role of acetate in the plasmid production was revealed by the model, with most of plasmid production being associated to the acetate consumption. The model provides meaningful insight on the E. coli dynamic cell behavior concerning the plasmid bioproduction, which might lead to important guidelines for culture optimization and process scale-up and control. © 2014 Elsevier B.V. All rights reserved. 1. Introduction The use of plasmids as vectors for gene therapy has gained considerable attention as they are easily modified by genetic engi- neering, they are stable and easily stored and distributed, as do not need refrigeration, they can be safely manufactured on a large scale and, most importantly, they present much less human security con- cerns in relation to viral vectors. The number of “naked” plasmids protocols used in a total of 1996 ongoing gene therapy clinical tri- als reached 17.8% (www.wiley.co.uk/genmed/clinical, May 2014). Despite this, “naked” plasmid DNA usually leads to a weak immune response, unless a relatively large amount, on the order of sev- eral milligrams per dose in humans, is administered (Leitner et al., 2000). New adjuvants (Lurescia et al., 2014; Capitani et al., 2014) ∗ Corresponding author. Tel.: +351 214269770; fax: +351 214269800. E-mail address: c.calado@fe.lisboa.ucp.pt (C.R.C. Calado). and plasmid delivery techniques such as nano- and microparticles can result in an induced immunogenicity (Cadete et al., 2012; Chadwick et al., 2010; Donnelly et al., 2005). However, these new techniques have yet to translate into a significant lowering of the dose size, resulting in the continuous need for the development of cost-effective plasmid production processes on E. coli cultures (Silva et al., 2012; Bower and Prather, 2009; Carnes, 2005; Prather et al., 2003). When considering plasmid optimization protocols it is very relevant to maximize the plasmid yields while minimizing the manufacturing costs. High plasmid concentrations and plasmid productions per biomass, i.e., plasmid specific yields, will capital- ize the use of bioreactors and will maximize the final downstream purification yield (Bower and Prather, 2009). Several studies across distinct research areas have approached this topic with the goal of obtaining E. coli cultures producing plasmid high-yields. Strategies for achieving this include the optimization of the medium compo- sition and the cultivation strategy (Lopes et al., 2014; Silva et al., http://dx.doi.org/10.1016/j.jbiotec.2014.06.022 0168-1656/© 2014 Elsevier B.V. All rights reserved.