Fourier transform infrared spectroscopy, a powerful tool to monitor biopharmaceuticals production

• Autor(a) principal: Sales, Kevin Costa
• Data de Publicação: 2014
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10400.14/15655
• topic: Escherichia coli; MIR spectroscopy; NIR spectroscopy; Metabolic profiling; Bioprocess monitoring; Espectroscopia MIR; Espectroscopia NIR; Caraterização metabólica; Monitorização de bioprocessos; Domínio/Área Científica::Engenharia e Tecnologia::Outras Engenharias e Tecnologias

Escherichia coli is the most used microorganism as host for the production of recombinant products, such as plasmids used for gene therapy and DNA vaccination. Therefore, it is important to understand the complex metabolic relationships and the plasmid bioproduction process occurring in dynamic culture environments, in order to control and optimize the performance of the recombinant expression system. The main goal of this work is to evaluate the potential of Fourier Transform Infrared (FT-IR) spectroscopy to monitor and characterize recombinant E. coli cultures producing the plasmid model pVAX-LacZ, namely to extract information concerning the critical variables (biomass, plasmid, carbon sources and the by-product acetate) and metabolic information regarding the host E. coli. To achieve that cultures of E. coli conducted with different mixture of glucose and glycerol and different cultivation strategies (batch and fed-batch) were monitored in-situ by a fiber optic probe in near- infrared (NIR) and of the cell pellets in at-line in high-throughput mode by mid-infrared (MIR) spectroscopy. Both NIR and MIR spectroscopy setup enabled to extract information regarding the critical variables of the bioprocess by the implementation of partial least square regression models that result in high regression coefficients and low prediction errors. The NIR setup presents the advantage of acquiring in real time the knowledge of the bioprocess variables, where the at-line measurements with the MIR setup presents more advantageous in cases of micro-bioreactors used in optimization protocols, enabling the simultaneously information acquisition of hundreds samples by using multi-microplates. Furthermore, as the MIR spectra presents more information than the NIR spectra, since it represents the fundamental vibration modes of biomolecules while the NIR spectra represents overtones and combinations of vibrations, the MIR data also enabled to acquire biochemical information along the E. coli cultures as pointed out in an principal component analysis and by the estimation of biochemical features as glycogen reserves and apparent transcriptional levels. Therefore, FT-IR spectroscopy presents relevant features towards the understanding and monitoring of the production process of recombinant cultures for control and optimization purposes, in according to the Quality-by-Design and the Process Analytical Technology.