2 research outputs found
In situ modification of chromatography adsorbents using cold atmospheric pressure plasmas
Efficient manufacturing of increasingly sophisticated biopharmaceuticals requires the development of new breeds of chromatographic materials featuring two or more layers, with each layer affording different functions. This letter reports the in situ modification of a commercial beaded anion exchange adsorbent using atmospheric pressure plasma generated within gas bubbles. The results show that exposure to He-O2 plasma in this way yields significant reductions in the surface binding of plasmid DNA to the adsorbent exterior, with minimal loss of core protein binding capacity; thus, a bi-layered chromatography material exhibiting both size excluding and anion exchange functionalities within the same bead is produced
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
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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