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Overall Key Performance Indicator to Optimizing Operation of High-Pressure Homogenizers for a Reliable Quantification of Intracellular Components in Pichia pastoris

The most commonly used cell disruption procedures may present lack of reproducibility, which introduces significant errors in the quantification of intracellular components. In this work, an approach consisting in the definition of an overall key performance indicator (KPI) was implemented for a lab...

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Autores principales: Garcia-Ortega, Xavier, Reyes, Cecilia, Montesinos, José Luis, Valero, Francisco
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2015
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4522904/
https://www.ncbi.nlm.nih.gov/pubmed/26284241
http://dx.doi.org/10.3389/fbioe.2015.00107
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author Garcia-Ortega, Xavier
Reyes, Cecilia
Montesinos, José Luis
Valero, Francisco
author_facet Garcia-Ortega, Xavier
Reyes, Cecilia
Montesinos, José Luis
Valero, Francisco
author_sort Garcia-Ortega, Xavier
collection PubMed
description The most commonly used cell disruption procedures may present lack of reproducibility, which introduces significant errors in the quantification of intracellular components. In this work, an approach consisting in the definition of an overall key performance indicator (KPI) was implemented for a lab scale high-pressure homogenizer (HPH) in order to determine the disruption settings that allow the reliable quantification of a wide sort of intracellular components. This innovative KPI was based on the combination of three independent reporting indicators: decrease of absorbance, release of total protein, and release of alkaline phosphatase activity. The yeast Pichia pastoris growing on methanol was selected as model microorganism due to it presents an important widening of the cell wall needing more severe methods and operating conditions than Escherichia coli and Saccharomyces cerevisiae. From the outcome of the reporting indicators, the cell disruption efficiency achieved using HPH was about fourfold higher than other lab standard cell disruption methodologies, such bead milling cell permeabilization. This approach was also applied to a pilot plant scale HPH validating the methodology in a scale-up of the disruption process. This innovative non-complex approach developed to evaluate the efficacy of a disruption procedure or equipment can be easily applied to optimize the most common disruption processes, in order to reach not only reliable quantification but also recovery of intracellular components from cell factories of interest.
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spelling pubmed-45229042015-08-17 Overall Key Performance Indicator to Optimizing Operation of High-Pressure Homogenizers for a Reliable Quantification of Intracellular Components in Pichia pastoris Garcia-Ortega, Xavier Reyes, Cecilia Montesinos, José Luis Valero, Francisco Front Bioeng Biotechnol Bioengineering and Biotechnology The most commonly used cell disruption procedures may present lack of reproducibility, which introduces significant errors in the quantification of intracellular components. In this work, an approach consisting in the definition of an overall key performance indicator (KPI) was implemented for a lab scale high-pressure homogenizer (HPH) in order to determine the disruption settings that allow the reliable quantification of a wide sort of intracellular components. This innovative KPI was based on the combination of three independent reporting indicators: decrease of absorbance, release of total protein, and release of alkaline phosphatase activity. The yeast Pichia pastoris growing on methanol was selected as model microorganism due to it presents an important widening of the cell wall needing more severe methods and operating conditions than Escherichia coli and Saccharomyces cerevisiae. From the outcome of the reporting indicators, the cell disruption efficiency achieved using HPH was about fourfold higher than other lab standard cell disruption methodologies, such bead milling cell permeabilization. This approach was also applied to a pilot plant scale HPH validating the methodology in a scale-up of the disruption process. This innovative non-complex approach developed to evaluate the efficacy of a disruption procedure or equipment can be easily applied to optimize the most common disruption processes, in order to reach not only reliable quantification but also recovery of intracellular components from cell factories of interest. Frontiers Media S.A. 2015-08-03 /pmc/articles/PMC4522904/ /pubmed/26284241 http://dx.doi.org/10.3389/fbioe.2015.00107 Text en Copyright © 2015 Garcia-Ortega, Reyes, Montesinos and Valero. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Bioengineering and Biotechnology
Garcia-Ortega, Xavier
Reyes, Cecilia
Montesinos, José Luis
Valero, Francisco
Overall Key Performance Indicator to Optimizing Operation of High-Pressure Homogenizers for a Reliable Quantification of Intracellular Components in Pichia pastoris
title Overall Key Performance Indicator to Optimizing Operation of High-Pressure Homogenizers for a Reliable Quantification of Intracellular Components in Pichia pastoris
title_full Overall Key Performance Indicator to Optimizing Operation of High-Pressure Homogenizers for a Reliable Quantification of Intracellular Components in Pichia pastoris
title_fullStr Overall Key Performance Indicator to Optimizing Operation of High-Pressure Homogenizers for a Reliable Quantification of Intracellular Components in Pichia pastoris
title_full_unstemmed Overall Key Performance Indicator to Optimizing Operation of High-Pressure Homogenizers for a Reliable Quantification of Intracellular Components in Pichia pastoris
title_short Overall Key Performance Indicator to Optimizing Operation of High-Pressure Homogenizers for a Reliable Quantification of Intracellular Components in Pichia pastoris
title_sort overall key performance indicator to optimizing operation of high-pressure homogenizers for a reliable quantification of intracellular components in pichia pastoris
topic Bioengineering and Biotechnology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4522904/
https://www.ncbi.nlm.nih.gov/pubmed/26284241
http://dx.doi.org/10.3389/fbioe.2015.00107
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