title: Integrated system for temperature-controlled fast protein liquid chromatography. II. Optimized adsorbents and 'single column continuous operation'.
abstract: Continued advance of a new temperature-controlled chromatography system, comprising a column filled with thermoresponsive stationary phase and a travelling cooling zone reactor (TCZR), is described. Nine copolymer grafted thermoresponsive cation exchangers (thermoCEX) with different balances of thermoresponsive (N-isopropylacrylamide), hydrophobic (N-tert-butylacrylamide) and negatively charged (acrylic acid) units were fashioned from three cross-linked agarose media differing in particle size and pore dimensions. Marked differences in grafted copolymer composition on finished supports were sourced to base matrix hydrophobicity. In batch binding tests with lactoferrin, maximum binding capacity (qmax) increased strongly as a function of charge introduced, but became increasingly independent of temperature, as the ability of the tethered copolymer networks to switch between extended and collapsed states was lost. ThermoCEX formed from Sepharose CL-6B (A2), Superose 6 Prep Grade (B2) and Superose 12 Prep Grade (C1) under identical conditions displayed the best combination of thermoresponsiveness (qmax,50°C/qmax,10°C ratios of 3.3, 2.2 and 2.8 for supports 'A2', 'B2' and 'C1' respectively) and lactoferrin binding capacity (qmax,50°C∼56, 29 and 45mg/g for supports 'A2', 'B2' and 'C1' respectively), and were selected for TCZR chromatography. With the cooling zone in its parked position, thermoCEX filled columns were saturated with lactoferrin at a binding temperature of 35°C, washed with equilibration buffer, before initiating the first of 8 or 12 consecutive movements of the cooling zone along the column at 0.1mm/s. A reduction in particle diameter (A2→B2) enhanced lactoferrin desorption, while one in pore diameter (B2→C1) had the opposite effect. In subsequent TCZR experiments conducted with thermoCEX 'B2' columns continuously fed with lactoferrin or 'lactoferrin+bovine serum albumin' whilst simultaneously moving the cooling zone, lactoferrin was intermittently concentrated at regular intervals within the exiting flow as sharp uniformly sized peaks. Halving the lactoferrin feed concentration to 0.5mg/mL, slowed acquisition of steady state, but increased the average peak concentration factor from 7.9 to 9.2. Finally, continuous TCZR mediated separation of lactoferrin from bovine serum albumin was successfully demonstrated. While the latter's presence did not affect the time to reach steady state, the average lactoferrin mass per peak and concentration factor both fell (respectively from 30.7 to 21.4mg and 7.9 to 6.3), and lactoferrin loss in the flowthrough between elution peaks increased (from 2.6 to 12.2mg). Fouling of the thermoCEX matrix by lipids conveyed into the feed by serum albumin is tentatively proposed as responsible for the observed drops in lactoferrin binding and recovery.
authors.affiliation: School of Chemical Engineering, College of Engineering and Physical Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, England, UK.
authors.forename: Ping
authors.initials: P
authors.lastname: Cao
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.affiliation: Institute for Functional Interfaces, Karlsruhe Institute of Technology, Hermann-von-Helmholtz-Platz 1, 76344 Eggenstein-Leopoldshafen, Germany.
authors.forename: Benedikt
authors.initials: B
authors.lastname: Ketterer
authors.affiliation: School of Chemical Engineering, College of Engineering and Physical Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, England, UK; Institute for Functional Interfaces, Karlsruhe Institute of Technology, Hermann-von-Helmholtz-Platz 1, 76344 Eggenstein-Leopoldshafen, Germany.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Ewert
authors.affiliation: School of Chemical Engineering, College of Engineering and Physical Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, England, UK.
authors.forename: Eirini
authors.initials: E
authors.lastname: Theodosiou
authors.affiliation: School of Chemical Engineering, College of Engineering and Physical Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, England, UK. Electronic address: o.r.t.thomas@bham.ac.uk.
authors.forename: Owen R T
authors.initials: OR
authors.lastname: Thomas
authors.affiliation: Institute for Functional Interfaces, Karlsruhe Institute of Technology, Hermann-von-Helmholtz-Platz 1, 76344 Eggenstein-Leopoldshafen, Germany. Electronic address: matthias.franzreb@kit.edu.
authors.forename: Matthias
authors.initials: M
authors.lastname: Franzreb
mesh_terms: D000178:Acrylamides / Q000737:chemistry; D002021:Buffers; D002412:Cations; D002623:Chemistry Techniques, Analytical / Q000295:instrumentation / Q000379:methods*; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation*; D007781:Lactoferrin / Q000378:metabolism; D011108:Polymers / Q000737:chemistry; D011485:Protein Binding; D012685:Sepharose / Q000031:analogs & derivatives / Q000737:chemistry; D013696:Temperature
chemical_list: D000178:Acrylamides; D002021:Buffers; D002412:Cations; D011108:Polymers; C047264:sepharose CL 6B; D012685:Sepharose; C049225:Superose 12; C067295:N-isopropylacrylamide; D007781:Lactoferrin; C115287:tert-butylacrylamide
keywords: Copolymer modified agarose adsorbents; Ion exchange adsorption; Lower critical solution temperature (LCST); N-Isopropylacrylamide; Smart polymers; Travelling cooling zone reactor