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

Integrated system for temperature-controlled fast protein liquid chromatography comprising improved copolymer modified beaded agarose adsorbents and a travelling cooling zone reactor arrangement

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, 1H 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 ºC. In temperature shifting chromatography experiments employing thermoCEX in thermally-jacketed columns, 44 – 51% of the lactoferrin adsorbed at 42 ºC could be desorbed under binding conditions by cooling the column to 22 ºC, 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 ºC using 8 successive movements of the cooling zone at a velocity of 0.1 mm/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 3 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