Research Article Received: 31 March 2014 Revised: 12 May 2014 Accepted article published: 16 May 2014 Published online in Wiley Online Library: (wileyonlinelibrary.com) DOI 10.1002/jctb.4431 In situ NIR spectroscopy monitoring of plasmid production processes: effect of producing strain, medium composition and the cultivation strategy Marta B. Lopes, a§ Geisa A. L. Gonçalves, b,c,d§ Daniel Felício-Silva, a Kristala L. J. Prather, b,e Gabriel A. Monteiro, b,c,d Duarte M. F. Prazeres b,c,d and Cecília R. C. Calado a* Abstract BACKGROUND: While the pharmaceutical industry keeps an eye on plasmid DNA production for new generation gene therapies, real-time monitoring techniques for plasmid bioproduction are as yet unavailable. This work shows the possibility of in situ monitoring of plasmid production in Escherichia coli cultures using a near infrared (NIR) fiber optic probe. RESULTS: Partial least squares (PLS) regression models based on the NIR spectra were developed for predicting bioprocess critical variables such as the concentrations of biomass, plasmid, carbon sources (glucose and glycerol) and acetate. In order to achieve robust models able to predict the performance of plasmid production processes, independently of the composition of the cultivation medium, cultivation strategy (batch versus fed-batch) and E. coli strain used, three strategies were adopted, using: (i) E. coli DH5 cultures conducted under different media compositions and culture strategies (batch and fed-batch); (ii) engineered E. coli strains, MG1655endArecApgi and MG1655endArecA, grown on the same medium and culture strategy; (iii) diverse E. coli strains, over batch and fed-batch cultivations and using different media compositions. PLS models showed high accuracy for predicting all variables in the three groups of cultures. CONCLUSION: NIR spectroscopy combined with PLS modeling provides a fast, inexpensive and contamination-free technique to accurately monitoring plasmid bioprocesses in real time, independently of the medium composition, cultivation strategy and the E. coli strain used. © 2014 Society of Chemical Industry Keywords: plasmid bioprocess; Escherichia coli; near infrared spectroscopy; in situ monitoring INTRODUCTION There is currently a high demand for pharmaceutical plasmid DNA for non-viral human gene therapy and DNA vaccination, as regulatory approval is being granted for products in clinical trials. 1 The development of adequate methods for monitoring plasmid amplification in Escherichia coli in real time is of the utmost importance, in order to ensure high-quality plasmid yields and bioprocess reproducibility according to regulatory compliance. However, in situ bioprocess monitoring remains a challenge, espe- cially due to contamination issues, aeration, agitation, and the fact that matrices are very complex and drastically change during the process. A number of publications describe the use of NIR fiber optic probes to monitor in situ biomass growth of bacteria and mam- malian cells, carbon source consumption, by-product formation and consumption, and their corresponding products, which include amino acids and protein complexes. 2 – 10 NIR spectroscopy provides less informative spectra compared with mid-infrared (MIR) spectra, as the former represents combinations and overtones of molecular vibrations, and not fundamental vibrations modes provided by MIR spectroscopy, which makes the extraction of quantitative relevant information possible only by the use ∗ Correspondence to: Cecília Calado, Faculdade de Engenharia, Universidade Católica Portuguesa, Estrada Octávio Pato, 2635-631 Rio de Mouro, Portugal. E-mail: c.calado@fe.lisboa.ucp.pt § Both authors contributed equally to this work a Engineering Faculty, Catholic University of Portugal, Lisbon, Portugal b MIT-Portugal Program c Department of Bioengineering, Instituto Superior Técnico (IST), Lisbon, Portugal d IBB-Institute for Biotechnology and Bioengineering, Center for Biological and Chemical Engineering, IST, Lisbon, Portugal e Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA J Chem Technol Biotechnol (2014) www.soci.org © 2014 Society of Chemical Industry