Membrane-based separations for solid/liquid clarification and protein purification

Abstract

<p>Recombinant protein technology has become increasingly important in recent years. Recombinant proteins can be found in everything from pharmaceuticals to detergents. The increasing demand and the high cost associated with the production and purification of recombinant proteins highlights the need to develop efficient and inexpensive methods for the solid/liquid clarification and purification of these proteins. Membrane technologies, which offer the advantage of high throughput and ever-improving selectivity, have the potential to meet the increased demands placed on the downstream processing of recombinant proteins. The overall objective of this work was to develop methods for the solid/liquid clarification and purification of recombinant proteins using membrane based separations. First, we explored the use of polyelectrolyte flocculation to improve the microfiltration of an industrial fermentation broth. We evaluated the effectiveness of several cationic polyelectrolytes of differing repeating unit, molecular weight, and charge density for the flocculation of an industrial Bacillus subtilis fermentation and the subsequent effect on the permeate flux and product rejection in both dead-end and tangential flow microfiltration. We also investigated the effect of crossflow rate on the permeate flux, product rejection, and the rate of floc breakup. Finally, we used flocculation to examine particle deposition in the highly variable shear environment that occurs in flow through a spacer filled channel. Then, we investigated microfiltration for the solids/liquid clarification of transgenic corn extracts. Within this work we characterized the resistance and compressibility of filter cakes formed in dead-end microfiltration of endosperm- and germ-rich extracts. In tangential flow microfiltration the effects of crossflow rate and transmembrane pressure (TMP) on the permeate flux and rejection of corn host cell proteins (HCP), recombinant Type 1 human collagen (rCollagen), and GFP were investigated. We also examined both a ceramic and a poly (vinylidene fluoride) (PVDF) membrane for the extent of membrane fouling and the subsequent effect on the rejection of HCP, GFP, and rCollagen. Finally, we examined ultrafiltration for the purification of recombinant proteins from transgenic corn extracts. We investigated the effects of membrane pore size, crossflow rate, TMP, filtration pH, and corn tissue (i.e. endosperm or germ) on the permeate flux and protein sieving in ultrafiltration. We also developed a purification strategy to purify rCollagen from transgenic corn extracts using only acid salt precipitation and ultrafiltration.</p>