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A strategic crossflow filtration methodology for the initial purification of promegapoietin from inclusion bodies
Mälardalen University, Department of Biology and Chemical Engineering.
Mälardalen University, Department of Biology and Chemical Engineering.
Mälardalen University, Department of Biology and Chemical Engineering.
2009 (English)In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 141, no 1-2, p. 64-72Article in journal (Refereed) Published
Abstract [en]

A novel crossflow filtration methodology is demonstrated for the initial purification of the therapeutic protein, promegapoietin-1a (PMP), produced as inclusion bodies (IBs) in a recombinant Escherichia coli bioprocess. Two strategic separation steps were performed by utilizing a filtration unit with a 1000 kDa polyethersulphone membrane. The first step, aiming for separation of soluble contaminants, resulted in a 50% reduction of the host cell proteins, quantified by total amino acid analysis and a 70% reduction of all DNA, quantified by fluorometry, when washing the particulate material with a 10 mM EDTA in 50 mM phosphate buffer, pH 8. The second step, aiming for separation of particulate contaminants from solubilized IBs, resulted in a 97-99.5% reduction of endotoxin, used as a marker for cell debris, and was quantified by the kinetic turbidimetric LAL endotoxin assay. The overall PMP yield was 58% and 33% respectively for the two solubilizations investigated, guanidine hydrochloride and arginine, as measured by RP-HPLC. The scope was also to investigate the physical characteristics of the intermediate product/s with regard to the choice of 113 solvent. Preliminary results from circular dichroism spectroscopy measurements indicate that the protein secondary structure was restored when arginine was used in the second step.

Place, publisher, year, edition, pages
2009. Vol. 141, no 1-2, p. 64-72
National Category
Natural Sciences
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URN: urn:nbn:se:mdh:diva-19900DOI: 10.1016/j.jbiotec.2009.02.016ISI: 000266174600009PubMedID: 19428732OAI: oai:DiVA.org:mdh-19900DiVA, id: diva2:635699
Available from: 2013-07-05 Created: 2013-06-26 Last updated: 2018-10-16Bibliographically approved

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