Integrated system for temperature-controlled fast protein liquid chromatography comprising imprved copolymer modified beaded agarose adsorebnts and a travelling cooling zone reactor arrangement.pdf (915.48 kB)

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

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posted on 25.03.2013, 16:19 by Tobias K.H. Muller, Ping Cao, Stephanie Ewert, Jonas Wohlgemuth, Matthias Franzreb, Haiyang Liu, Thomas C. Willett, Eirini Theodosiou, Owen R.T. Thomas
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.

History

School

  • Aeronautical, Automotive, Chemical and Materials Engineering

Department

  • Chemical Engineering

Citation

MULLER, T.K.H. ... et al, 2013. Integrated system for temperature-controlled fast protein liquid chromatography comprising improved copolymer modified beaded agarose adsorbents and a travelling cooling zone reactor arrangement. Journal of Chromatography A, 1285, pp.97–109

Publisher

© Elsevier

Version

AM (Accepted Manuscript)

Publication date

2013

Notes

NOTICE: this is the author’s version of a work that was accepted for publication in Journal of Chromatography A. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published at: http://dx.doi.org/10.1016/j.chroma.2013.02.025

ISSN

0021-9673

eISSN

1873-3778

Language

en

Exports