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A strategic crossflow filtration methodology for the initial purification of promegapoietin from inclusion bodies

Paper ID Volume ID Publish Year Pages File Format Full-Text
24151 43501 2009 9 PDF Available
Title
A strategic crossflow filtration methodology for the initial purification of promegapoietin from inclusion bodies
Abstract

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 IB solvent. Preliminary results from circular dichroism spectroscopy measurements indicate that the protein secondary structure was restored when arginine was used in the second step.

Keywords
Inclusion body solubilization; Crossflow filtration; Lysate clarification; Cell debris separation; Initial protein purification
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A strategic crossflow filtration methodology for the initial purification of promegapoietin from inclusion bodies
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 141, Issues 1–2, 20 April 2009, Pages 64–72
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us