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

Integrated system for temperature-controlled fast protein liquid chromatography. II. Optimized adsorbents and ‘single column continuous operation’

“…For detailed descriptions of the procedures involved in the four step conversion of underivatized beaded agarose chromatography supports into thermoCEX media, the reader is referred to our previous study [23]. Exactly the same methods were applied here to three different beaded agarose starting materials (i.e.…”

“…Most of this has involved modification of small pored inorganic or hydrophobic (polystyrene based) chromatography supports for use in analytical separations of small biomolecules (especially steroids). In stark contrast, very little has been done on the modification of softer macroporous media for preparative separations of much larger macromolecules, such as proteins [22][23][24]. Maharjan et al [22] and subsequently we [23] grafted lightly cross-linked networks of poly(N-isopropylacrylamide-co-N-tertbutylacrylamide-co-acrylic acid) onto the surfaces of cross-linked agarose supports to produce thermoresponsive cation exchangers (hereafter abbreviated to thermoCEX).…”

“…In stark contrast, very little has been done on the modification of softer macroporous media for preparative separations of much larger macromolecules, such as proteins [22][23][24]. Maharjan et al [22] and subsequently we [23] grafted lightly cross-linked networks of poly(N-isopropylacrylamide-co-N-tertbutylacrylamide-co-acrylic acid) onto the surfaces of cross-linked agarose supports to produce thermoresponsive cation exchangers (hereafter abbreviated to thermoCEX). In tests with the thermally robust protein lactoferrin (LF) and jacketed columns of thermoCEX media, LF previously adsorbed at a higher temperature could be desorbed by lowering the mobile phase and column temperature.…”

“…To exploit thermoresponsive chromatography media more effectively, we invented a bespoke column arrangement [23], the so-called Travelling Cooling Zone Reactor (TCZR). TCZR chromatography employs a vertically held stainless steel walled column filled with thermoresponsive stationary phase and a Fig.…”

“…A large body of work on endowing chromatographic packing materials with temperature switchable behavior, through their modification with e.g. pNIPAAm or pNIPAAm copolymers, has appeared since the 1990s [19][20][21][22][23][24]. Most of this has involved modification of small pored inorganic or hydrophobic (polystyrene based) chromatography supports for use in analytical separations of small biomolecules (especially steroids).…”