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Combined in-fermenter extraction and cross-flow microfiltration for improved inclusion body processing
Chew Tin Lee1, Giacomo Morreale, Anton P J Middelberg
1Department of Chemical Engineering, University of Cambridge, Pembroke Street, Cambridge CB2 3RA, United Kingdom.
Biotechnology and Bioengineering
|January 6, 2004
Summary
A novel in-fermenter chemical extraction and cross-flow microfiltration method efficiently releases and purifies inclusion bodies (IBs) from Escherichia coli. This process intensifies production by eliminating cell recovery steps, yielding high-purity protein with minimal operations.
Area of Science:
- Biotechnology
- Bioprocess Engineering
- Protein Purification
Background:
- Inclusion bodies (IBs) are aggregates of misfolded recombinant proteins often produced in Escherichia coli.
- Traditional methods for IB isolation involve cell lysis and multiple purification steps, which are time-consuming and can lead to protein loss.
- Process intensification is crucial for improving the efficiency and cost-effectiveness of biopharmaceutical manufacturing.
Purpose of the Study:
- To develop and validate an integrated, in-fermenter process for the efficient release and purification of inclusion bodies (IBs).
- To demonstrate the effectiveness of cross-flow microfiltration in removing soluble contaminants from released IBs.
- To showcase a highly intensified bioprocess requiring minimal unit operations.
Main Methods:
- An in-fermenter chemical extraction procedure using Triton X-100, EDTA, and T7 lysozyme to degrade the Escherichia coli cell wall and release IBs.
- Cross-flow microfiltration with a 0.2 µm ceramic membrane for separating IBs from soluble contaminants.
- In-fermenter Benzonase digestion of nucleic acids to enhance filtration efficiency.
Main Results:
- The chemical extraction effectively released intracellular soluble content without solubilizing IBs.
- Cross-flow microfiltration achieved 91% removal of soluble contaminants from granulocyte macrophage-colony stimulating factor (GM-CSF) IBs with minimal IB loss.
- Filtration flux and transmission were significantly improved by Benzonase digestion.
- The entire process, including extraction and filtration, was performed directly on crude fermentation broth, eliminating cell recovery steps.
Conclusions:
- An integrated in-fermenter extraction and cross-flow microfiltration process offers a highly intensified method for IB purification.
- This approach significantly reduces the number of unit operations required for protein purification.
- The microfiltration system has potential for subsequent protein solubilization, debris separation, and refolding via diafiltration, enabling high-purity protein recovery in just two steps.