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Title: Polypeptide extraction using reverse micelles
Author: Gunther, Selina Lavinia
ISNI:       0000 0004 2682 0787
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2010
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The reverse micelle (RM) extraction of human IgG4 monoclonal antibodies (MAbs), humanized IgG4 MAb B72.3 Fab fragments and horse heart cytochrome-c was investigated. The effects of system parameters on forward (FE) and backward (BE) extraction was examined, and optimal extraction conditions were determined. Sodium bis(2-ethylhexyl)sulfosuccinate (AOT), bis(2-ethylhexyl)phosphate (HDEHP), isooctane and corn oil were the model surfactants and solvents. Precipitate formation was investigated, and non-ionic (polyoxyethylene(4) lauryl ether i.e. Brij 30) and counterionic (trioctylmethylammonium chloride i.e. TOMAC) surfactants were assessed to reduce precipitate formation and increase extraction yields. Protein in the precipitate was measured using acetone precipitation. Conventional RM extraction of IgG4 for AOT- and HDEHP-isooctane resulted in yields of up to 99% FE, 58% BE for AOT-isooctane, and 92% BE for HDEHP-isooctane. It was found for AOT-isooctane with TOMAC for cytochrome-c 81% FE and 98% BE; and 41% FE and 85% BE for AOT-isooctane with Brij 30 and TOMAC, 56% FE and 71% BE for AOT-corn oil, 62% FE and 89% BE for HDEHP-isooctane, and 57% FE and 59% BE for HDEHP-corn oil with TOMAC for Fab fragments. Hollow fibre membrane (HFM) extraction of cytochrome-c resulted in complete FE and 40% BE with TOMAC; and with Fab fragments resulted in 99% FE and 79% BE for AOT, and in 88% FE and 46% BE for HDEHP with Brij 30. HFM FE using two aqueous phases showed that the quantity of Fab fragments adsorbed to the membrane surface and/or stuck in the membrane pores were negligible. Structural analysis revealed that precipitate formation during HFM module extraction was not an issue compared to conventional FE, where it structurally damaged and prevented the Fab fragments and MAbs from successfully transferring into the RM phase. RM phase water content analysis from both extraction methods confirmed that RM extraction was successful.
Supervisor: Stuckey, David Sponsor: Engineering and Physical Sciences Research Council ; Lonza Biologics
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral