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Combined In-Fermenter Extraction and Cross-Flow Microfiltration for Improved Inclusion Body Processing

In this study we demonstrate a new in-fermenter chemical extraction procedure that degrades the cell wall of Escherichia coli and releases inclusion bodies (IBs) into the fermentation medium. We then prove that cross-flow microfiltration can be used to remove 91% of soluble contaminants from the rel...

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Main Authors: Chew, Tin Lee, Morreale, Giacomo, Middelberg, Anton P. J.
Format: Article
Language:English
Published: Wiley-Blackwell 2004
Subjects:
TP Chemical technology
Online Access:http://eprints.utm.my/id/eprint/3085/1/chewtinlee2004_combined%20in-fermenterextraction.pdf
http://eprints.utm.my/id/eprint/3085/
http://www3.interscience.wiley.com/cgi-bin/fulltext/106567417/PDFSTART
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spelling my.utm.30852017-03-02T07:21:56Z http://eprints.utm.my/id/eprint/3085/ Combined In-Fermenter Extraction and Cross-Flow Microfiltration for Improved Inclusion Body Processing Chew, Tin Lee Morreale, Giacomo Middelberg, Anton P. J. TP Chemical technology In this study we demonstrate a new in-fermenter chemical extraction procedure that degrades the cell wall of Escherichia coli and releases inclusion bodies (IBs) into the fermentation medium. We then prove that cross-flow microfiltration can be used to remove 91% of soluble contaminants from the released IBs. The extraction protocol, based on a combination of Triton X-100, EDTA, and intracellular T7 lysozyme, effectively released most of the intracellular soluble content without solubilising the IBs. Cross-flow microfiltration using a 0.2 m ceramic membrane successfully recovered the granulocyte macrophage-colony stimulating factor (GM-CSF) IBs with removal of 91% of the soluble contaminants and virtually no loss of IBs to the permeate. The filtration efficiency, in terms of both flux and transmission, was significantly enhanced by in-fermenter Benzonase® digestion of nucleic acids following chemical extraction. Both the extraction and filtration methods exerted their efficacy directly on a crude fermentation broth, eliminating the need for cell recovery and resuspension in buffer. The processes demonstrated here can all be performed using just a fermenter and a single cross-flow filtration unit, demonstrating a high level of process intensification. Furthermore, there is considerable scope to also use the microfiltration system to subsequently solubilise the IBs, to separate the denatured protein from cell debris, and to refold the protein using diafiltration. In this way refolded protein can potentially be obtained, in a relatively pure state, using only two unit operations. Wiley-Blackwell 2004-01 Article PeerReviewed application/pdf en http://eprints.utm.my/id/eprint/3085/1/chewtinlee2004_combined%20in-fermenterextraction.pdf Chew, Tin Lee and Morreale, Giacomo and Middelberg, Anton P. J. (2004) Combined In-Fermenter Extraction and Cross-Flow Microfiltration for Improved Inclusion Body Processing. Biotechnology & Bioengineering, 85 (1). pp. 103-113. ISSN 0006-3592 http://www3.interscience.wiley.com/cgi-bin/fulltext/106567417/PDFSTART doi:10.1002/bit.10878
institution Universiti Teknologi Malaysia
building UTM Library
collection Institutional Repository
continent Asia
country Malaysia
content_provider Universiti Teknologi Malaysia
content_source UTM Institutional Repository
url_provider http://eprints.utm.my/
language English
topic TP Chemical technology
spellingShingle TP Chemical technology
Chew, Tin Lee
Morreale, Giacomo
Middelberg, Anton P. J.
Combined In-Fermenter Extraction and Cross-Flow Microfiltration for Improved Inclusion Body Processing
description In this study we demonstrate a new in-fermenter chemical extraction procedure that degrades the cell wall of Escherichia coli and releases inclusion bodies (IBs) into the fermentation medium. We then prove that cross-flow microfiltration can be used to remove 91% of soluble contaminants from the released IBs. The extraction protocol, based on a combination of Triton X-100, EDTA, and intracellular T7 lysozyme, effectively released most of the intracellular soluble content without solubilising the IBs. Cross-flow microfiltration using a 0.2 m ceramic membrane successfully recovered the granulocyte macrophage-colony stimulating factor (GM-CSF) IBs with removal of 91% of the soluble contaminants and virtually no loss of IBs to the permeate. The filtration efficiency, in terms of both flux and transmission, was significantly enhanced by in-fermenter Benzonase® digestion of nucleic acids following chemical extraction. Both the extraction and filtration methods exerted their efficacy directly on a crude fermentation broth, eliminating the need for cell recovery and resuspension in buffer. The processes demonstrated here can all be performed using just a fermenter and a single cross-flow filtration unit, demonstrating a high level of process intensification. Furthermore, there is considerable scope to also use the microfiltration system to subsequently solubilise the IBs, to separate the denatured protein from cell debris, and to refold the protein using diafiltration. In this way refolded protein can potentially be obtained, in a relatively pure state, using only two unit operations.
format Article
author Chew, Tin Lee
Morreale, Giacomo
Middelberg, Anton P. J.
author_facet Chew, Tin Lee
Morreale, Giacomo
Middelberg, Anton P. J.
author_sort Chew, Tin Lee
title Combined In-Fermenter Extraction and Cross-Flow Microfiltration for Improved Inclusion Body Processing
title_short Combined In-Fermenter Extraction and Cross-Flow Microfiltration for Improved Inclusion Body Processing
title_full Combined In-Fermenter Extraction and Cross-Flow Microfiltration for Improved Inclusion Body Processing
title_fullStr Combined In-Fermenter Extraction and Cross-Flow Microfiltration for Improved Inclusion Body Processing
title_full_unstemmed Combined In-Fermenter Extraction and Cross-Flow Microfiltration for Improved Inclusion Body Processing
title_sort combined in-fermenter extraction and cross-flow microfiltration for improved inclusion body processing
publisher Wiley-Blackwell
publishDate 2004
url http://eprints.utm.my/id/eprint/3085/1/chewtinlee2004_combined%20in-fermenterextraction.pdf
http://eprints.utm.my/id/eprint/3085/
http://www3.interscience.wiley.com/cgi-bin/fulltext/106567417/PDFSTART
_version_ 1643643731213025280
score 13.211869

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