title: Integrated system for temperature-controlled fast protein liquid chromatography comprising improved copolymer modified beaded agarose adsorbents and a travelling cooling zone reactor arrangement.
abstract: An integrated approach to temperature-controlled chromatography, involving copolymer modified agarose adsorbents and a novel travelling cooling zone reactor (TCZR) arrangement, is described. Sepharose CL6B was transformed into a thermoresponsive cation exchange adsorbent (thermoCEX) in four synthetic steps: (i) epichlorohydrin activation; (ii) amine capping; (iii) 4,4'-azobis(4-cyanovaleric acid) immobilization; and 'graft from' polymerization of poly(N-isopropylacrylamide-co-N-tert-butylacrylamide-co-acrylic acid-co-N,N'-methylenebisacrylamide). FT-IR, (1)H NMR, gravimetry and chemical assays allowed precise determination of the adsorbent's copolymer composition and loading, and identified the initial epoxy activation step as a critical determinant of 'on-support' copolymer loading, and in turn, protein binding performance. In batch binding studies with lactoferrin, thermoCEX's binding affinity and maximum adsorption capacity rose smoothly with temperature increase from 20 to 50 °C. In temperature shifting chromatography experiments employing thermoCEX in thermally jacketed columns, 44-51% of the lactoferrin adsorbed at 42 °C could be desorbed under binding conditions by cooling the column to 22 °C, but the elution peaks exhibited strong tailing. To more fully exploit the potential of thermoresponsive chromatography adsorbents, a new column arrangement, the TCZR, was developed. In TCZR chromatography, a narrow discrete cooling zone (special assembly of copper blocks and Peltier elements) is moved along a bespoke fixed-bed separation columnfilled with stationary phase. In tests with thermoCEX, it was possible to recover 65% of the lactoferrin bound at 35 °C using 8 successive movements of the cooling zone at a velocity of 0.1mm/s; over half of the recovered protein was eluted in the first peak in more concentrated form than in the feed. Intra-particle diffusion of desorbed protein out of the support pores, and the ratio between the velocities of the cooling zone and mobile phase were identified as the main parameters affecting TCZR performance. In contrast to conventional systems, which rely on cooling the whole column to effect elution and permit only batch-wise operation, TCZR chromatography generates sharp concentrated elution peaks without tailing effects and appears ideally suited for continuous operation.
authors.affiliation: Institute for Functional Interfaces, Karlsruhe Institute of Technology, Hermann-von-Helmholtz-Platz 1, 76344 Eggenstein-Leopoldshafen, Germany.
authors.forename: Tobias K H
authors.initials: TK
authors.lastname: Müller
authors.forename: Ping
authors.initials: P
authors.lastname: Cao
authors.forename: Stephanie
authors.initials: S
authors.lastname: Ewert
authors.forename: Jonas
authors.initials: J
authors.lastname: Wohlgemuth
authors.forename: Haiyang
authors.initials: H
authors.lastname: Liu
authors.forename: Thomas C
authors.initials: TC
authors.lastname: Willett
authors.forename: Eirini
authors.initials: E
authors.lastname: Theodosiou
authors.forename: Owen R T
authors.initials: OR
authors.lastname: Thomas
authors.forename: Matthias
authors.initials: M
authors.lastname: Franzreb
mesh_terms: D000178:Acrylamides / Q000737:chemistry; D000327:Adsorption; D000818:Animals; D002417:Cattle; D002852:Chromatography, Ion Exchange / Q000295:instrumentation* / Q000379:methods*; D007781:Lactoferrin / Q000032:analysis* / Q000737:chemistry; D019906:Nuclear Magnetic Resonance, Biomolecular; D011108:Polymers / Q000737:chemistry; D012685:Sepharose / Q000031:analogs & derivatives* / Q000737:chemistry; D017550:Spectroscopy, Fourier Transform Infrared; D013696:Temperature
chemical_list: D000178:Acrylamides; D011108:Polymers; C047264:sepharose CL 6B; D012685:Sepharose; C067295:N-isopropylacrylamide; D007781:Lactoferrin