Browsing by Author "Lopes, Vítor V."
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- High‐throughput analysis of the plasmid bioproduction process in Escherichia coli by FTIR spectroscopyPublication . Scholz, Teresa; Lopes, Vítor V.; Calado, CecíliaMonitoring plasmid production systems is a lab intensive task. This article proposes a methodology based on FTIR spectroscopy and the use of chemometrics for the high‐throughput analysis of the plasmid bioproduction process in E. coli. For this study, five batch cultures with different initial medium compositions are designed to represent different biomass and plasmid production behavior, with the maximum plasmid and biomass concentrations varying from 11 to 95 mg L−1 and 6.8 to 12.8 g L−1, respectively, and the plasmid production per biomass varying from 0.4 to 5.1 mg g−1. After a short sample processing consisting of centrifugation and dehydration, the FTIR spectra are recorded from the collected cellular biomass using microtiter plates with 96 wells. After spectral pre‐processing, the predictive FTIR spectra models are derived by using partial least squares (PLS) regression with the wavenumber selection performed by a Monte‐Carlo strategy. Results show that it is possible to improve the PLS models by selecting specific spectral ranges. For the plasmid model, the spectral regions between 590–1,130, 1,670–2,025, and 2,565–3,280 cm−1 are found to be highly relevant. Whereas for the biomass, the best wavenumber selections are between 900–1,200, 1,500–1,800, and 2,850–3,200 cm−1. The optimized PLS models show a high coefficient of determination of 0.91 and 0.89 for the plasmid and biomass concentration, respectively. Additional PLS models for the prediction of the carbon sources glucose and glycerol and the by‐product acetic acid, based on metabolism‐induced correlations between the nutrients and the cellular biomass are also established.
- Monitoring bacterial processes by Fourier transform infrared spectroscopy: Helicobacter pylori drug inactivation and plasmid bioproduction in recombinant Escherichia coli culturesPublication . Scholz, Teresa; Lopes, Vítor V.; Calado, CecíliaFourier transform infrared (FTIR) spectroscopy is evaluated as a tool to monitor two bacterial processes: strain discrimination and drug inactivation studies with the gastric pathogen Helicobacter pylori and the plasmid production process based on high-density cultures of recombinant Escherichia coli. Results show, that after evaluation of different incubation conditions of H.pylori with the drug model, the application of principal component analysis to the FTIR spectra assembles the samples into clusters which can be related with the minimal inhibitory concentration. Morever, the same methodology applied to FTIR spectra from 12 different strains can be used to distinguish them. For the E.coli cultures it is possible to estimate the concentration of relevant bioprocess monitoring variables, such as plasmid, biomass, and carbon sources (glucose, glycerol, acetate) by using partial least squares (PLS) models based on FTIR spectra.
- Real-time plasmid monitoring of batch and fed-batch Escherichia coli cultures by NIR spectroscopyPublication . Lopes, Marta B.; Sales, Kevin C.; Lopes, Vítor V.; Calado, CecíliaThe development of in-situ monitoring techniques enabling the real-time acquisition of information concerning the key variables over different Escherichia coli cultivation conditions and strategies is a crucial step towards the optimization of a plasmid bioproduction process. This work shows the use of a Near-InfraRed (NIR) fiber optic probe immersed in the culture broth for the real-time acquisition of NIR spectra along different E. coli cultures conducted with mixtures of the carbon sources glucose and glycerol, and performed in both batch and fed-batch modes. Accurate partial least squares models based on the acquired spectral data over such different cultivation conditions were built, yielding a R 2 = 0.97 for biomass and plasmid productions and a RMSEP of 0.34 and 7.52, respectively; a R 2 of 0.93 and a RMSEP of 0.46 and 0.33 was obtained for glucose and glycerol, respectively; the acetate model produced a R 2 of 0.96 and a RMSEP of 0.32.