A strategic crossflow filtration methodology for the initial purification of promegapoietin from inclusion bodies.
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 10mM EDTA in 50mM 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.
Department of Chemical Engineering, School of Sustainable Development of Society and Technology, Mälardalen University, Eskilstuna, Sweden. erika@ledung.net
Erika
E
Ledung
Per-Olov
PO
Eriksson
Sven
S
Oscarsson
D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005374:Filtration / Q000379:methods*; D002479:Inclusion Bodies / Q000737:chemistry*; D007377:Interleukin-3 / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification*; D013926:Thrombopoietin / Q000302:isolation & purification*
D007377:Interleukin-3; D011993:Recombinant Fusion Proteins; C438891:promegapoietin-1a; D013926:Thrombopoietin