Browsing by Author "Sales, Kevin"
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- Biopharmaceuticals process monitoring based on infrared spectroscopy according to quality by designPublication . Sales, Kevin; Ribeiro Da Cunha, Bernardo; Calado, CecíliaEscherichia coli, the most common host microorganism to produce high value biopharmaceuticals, presents, however a natural variability due to high sensitivity towards small fluctuations of the cultivation environment. Therefore, to speed up the development process and to control this critical manufacturing step it is relevant to develop monitoring techniques allowing simultaneous characterization of the heterologous product synthesis and of the physiologic cell behavior under a variety of culture conditions. In the present work high-throughput mid-infrared (MIR) spectral analysis of dehydrated E. coli cell pellets and in situ near infrared (NIR) spectral analysis of the whole culture broth were compared to monitor the bioproduction of a model plasmid along recombinant Escherichia coli cultures. Good partial least square regression models were built, either based on MIR or on NIR spectral data. Besides being conducted in situ, NIR spectroscopy allowed building regression models as accurate as those obtained from the MIR setup, and even better models for estimating biomass and glycerol. However, MIR spectroscopy allows retrieving valuable biochemical and metabolic information along the cell culture, e.g. lipids, RNA, protein synthesis and turnover metabolism. This information contributed to better understand the complex interrelationships between the recombinant cell metabolism and the bioprocess environment. In resume, since NIR spectroscopy enables the in situ and real time monitoring without the risk of culture contamination, thus represents an appealing tool for control purposes specially at industrial (i.e. large-scale) productions. On the other hand, MIR spectroscopy is better suited for optimization processes, as it enables the analysis of hundreds of samples at ounce, and to acquire off line historical information along industrial production processes. Both monitoring techniques are therefore complementary, empowering optimization and control procedures more efficiently and quicker, according to the new regulatory framework based on Quality by Design.
- Molecular fingerprint of human gastric cell line infected by Helicobacter pyloriPublication . Sampaio, Pedro; Ribeiro Da Cunha, Bernardo; Rosa, Filipa; Sales, Kevin; Lopes, Marta; Calado, CecíliaHelicobacter pylori infection represents a serious health problem, given its association with serious gastric diseases as gastric ulcers, cancer and MALT lymphoma. Currently no vaccine exists and antibiotic-based eradication therapy is already failing in more than 20% of cases. To increase the knowledge on the infection process diverse gastric cell lines, e.g. the adenocarcinona gastric (AGS) cell line, are routinely used has in vitro models of gastric epithelia. In the present work the molecular fingerprint of infected and non-infected AGS cell lines, by diverse H. pylori strains, was acquired using vibrational infrared spectroscopy. These molecular fingerprints enabled to discriminate infected from non-infected AGS cells, and infection due to different strains, by performing Principal Component Analysis. It was also possible to estimate, from the AGS cells molecular fingerprint, the effect of the infection on diverse biochemical and metabolic cellular status. In resume infra-red spectroscopy enabled the acquisition of infected AGS cells molecular fingerprint with minimal sample preparation, in a rapid, high-throughput, economic process yielding highly sensitive and informative data, most useful for promoting critical knowledge on the H. pylori infection process. © 2015 IEEE.