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  • Title: Separation of protein charge variants by ultrafiltration.
    Author: Ebersold MF, Zydney AL.
    Journal: Biotechnol Prog; 2004; 20(2):543-9. PubMed ID: 15059001.
    Abstract:
    The removal of product variants that form during downstream processing remains a challenge in the purification of recombinant therapeutic proteins. We examined the feasibility of separating variants with slightly different net charge using high-performance membrane ultrafiltration. A myoglobin variant was formed by reaction of the lysine epsilon-amino group with succinic anhydride. Sieving data were obtained over a range of solution conditions using commercial polyethersulfone ultrafiltration membranes. Maximum selectivity of about 7-fold was obtained at very low conductivity due to the strong electrostatic repulsion of the more negatively charged variant. Protein separations were performed by diafiltration. A two-stage process generated solutions of the normal myoglobin (in the permeate) and the charge variant (in the retentate), both at greater than 9-fold purification and 90% yield. These results provide the first demonstration that membrane systems can be used to separate proteins that differ by only a single charged amino acid residue.
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