2 research outputs found
Integrated system for temperature-controlled fast protein liquid chromatography. II. Optimized adsorbents and 'single column continuous operation'
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 (q max) 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 (q max,50°C/q max,10°C ratios of 3.3, 2.2 and 2.8 for supports 'A2', 'B2' and 'C1' respectively) and lactoferrin binding capacity (q max,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
Dual-Gated Microparticles for Switchable Antibody Release
We
pioneer the design of dual-gated microparticles, both responsive to
changes in temperature and pH, for stimuli-responsive chromatography
targeted at the efficient separation of antibodies. Dual-gated microspheres
were synthesized by introducing RAFT-based thiol-terminal block copolymers
of polyÂ(<i>N</i>-isopropylacrylamide-<i>b</i>-4-vinylpyridine)
(PÂ(NIPAM-<i>b</i>-4VP, 4800 ≤ <i>M</i><sub>n</sub>/Da ≤ 10 000, featuring block length ratios
of 29:7, 29:15, and 29:30, respectively) by thiol-epoxy driven ligation
to the surface of polyÂ(glycidyl methacrylate) (PGMA) microparticles
(10–12 μm), whereby the 4-vinylpyridine units within
the lateral chain enable protein binding. The switchable protein release
abilities of the resulting microparticle resins are demonstrated by
adsorption of immunoglobulins at 40 °C and pH 8 and their release
at 5 °C or pH 3, respectively. We demonstrate that PÂ(NIPAM<sub>29</sub>-<i>b</i>-4VP<sub>30</sub>)-grafted PGMA particles
show a maximum adsorption capacity for immunoglobulins of 18.9 mg
mL<sup>–1</sup> settled resin at 40 °C/pH 8, whereas the
adsorption capacity decreased to 7.5 mg mL<sup>–1</sup> settled
resin at 5 °C while retaining the pH value, allowing the unloading
of the chromatographic column by a facile temperature switch. Critically,
regeneration of the dual-gated microspheres became possible by lowering
the pH to 3