title: Preparative crystallization of a single chain antibody using an aqueous two-phase system.
abstract: A simultaneous crystallization and aqueous two-phase extraction of a single chain antibody was developed, demonstrating process integration. The process conditions were designed to form an aqueous two-phase system, and to favor crystallization, using sodium sulfate and PEG-2000. At sufficiently high concentrations of PEG, a second phase was generated in which the protein crystallization occurred simultaneously. The single chain antibody crystals were partitioned to the top, polyethylene glycol-rich phase. The crystal nucleation took place in the sodium sulfate-rich phase and at the phase boundary, whereas crystal growth was progressing mainly in the polyethylene glycol-rich phase. The crystals in the polyethylene glycol-rich phase grew to a size of >50 µm. Additionally, polyethylene glycol acted as an anti-solvent, thus, it influenced the crystallization yield. A phase diagram with an undersaturation zone, crystallization area, and amorphous precipitation zone was established. Only small differences in polyethylene glycol concentration caused significant shifts of the crystallization yield. An increase of the polyethylene glycol content from 2% (w/v) to 4% (w/v) increased the yield from approximately 63-87%, respectively. Our results show that crystallization in aqueous two-phase systems is an opportunity to foster process integration.
authors.affiliation: Boehringer Ingelheim RCV GmbH & CoKG, Dr. Boehringer Gasse 5-11, Vienna, A-1121, Austria.
authors.forename: Hauke
authors.initials: H
authors.lastname: Huettmann
authors.forename: Matthias
authors.initials: M
authors.lastname: Berkemeyer
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Buchinger
authors.forename: Alois
authors.initials: A
authors.lastname: Jungbauer
mesh_terms: D001709:Biotechnology / Q000379:methods*; D003460:Crystallization; D011092:Polyethylene Glycols / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D057127:Single-Chain Antibodies / Q000737:chemistry* / Q000302:isolation & purification*
chemical_list: D011994:Recombinant Proteins; D057127:Single-Chain Antibodies; D011092:Polyethylene Glycols
keywords: aqueous two-phase extraction; industrial crystallization; phase diagram; recombinant protein; single chain antibody