Improved adsorption reactions, kinetics and stability for model and therapeutic proteins immobilised on affinity resins
Improved adsorption reactions, kinetics and stability for model and therapeutic proteins immobilised on affinity resins
Protein adsorption on solid state media is important for the industrial affinity chromatography of biotherapeutics and for preparing materials for self-interaction chromatography where fundamental protein solution thermodynamic properties are measured. The adsorption of three model proteins (lysozyme, catalase and BSA) and two antibodies (a monoclonal and a polyclonal antibody) have been investigated on commercial affinity chromatography media with different surface functionalities (Formyl, Tresyl and Amino). Both the extent of protein immobilised (mg protein/ml media) and the reaction kinetics are reported for a range of reaction conditions, including pH, differing buffers as well as the presence of secondary reactants (glutaraldehyde, sodium cyanoborohydride, EDC and NHS). Compared to the reaction conditions recommended by manufacturers as well as those reported in previous published work, significant increases in the extent of protein immobilisation and reaction kinetics are reported here. The addition of glutaraldehyde or sodium cyanoborohydride was found to be especially effective even when not directly needed for the adsorption to happen. For mAb and pIgG, immobilisation levels of 50 and 31 mg of protein/ml of resin respectively were achieved, which are 100% or more than previously reported. Enhanced levels were achieved for lysozyme of 120 mg/ml with very rapid reaction kinetics (< 1 h) with sodium cyanoborohydride. It can be concluded that specific chromatography resins with Tresyl activated support offered enhanced levels of protein immobilisation due to their ability to react to form amine or thio-ether linkages with proteins. Additionally, glutaraldehyde can result in higher immobilisation levels whilst it can also accelerate immobilisation reaction kinetics. [Figure not available: see fulltext.].
Affinity chromatography, Coupling kinetics, Monoclonal antibodies, Polyclonal antibodies, Protein immobilisation, Self-interaction chromatography
1-14
Hedberg, S. H.M.
72ab1f70-dd40-490c-aee3-f6e3b48c2171
Brown, L. G.
43a81958-4279-4666-87ed-d341379d6108
Meghdadi, A.
2f3003a8-1b77-4763-9657-19a72b04c728
Williams, D. R.
116ed98d-8a4a-4511-9dba-00fb6cfb7c98
Hedberg, S. H.M.
72ab1f70-dd40-490c-aee3-f6e3b48c2171
Brown, L. G.
43a81958-4279-4666-87ed-d341379d6108
Meghdadi, A.
2f3003a8-1b77-4763-9657-19a72b04c728
Williams, D. R.
116ed98d-8a4a-4511-9dba-00fb6cfb7c98
Hedberg, S. H.M., Brown, L. G., Meghdadi, A. and Williams, D. R.
(2019)
Improved adsorption reactions, kinetics and stability for model and therapeutic proteins immobilised on affinity resins.
Adsorption, .
(doi:10.1007/s10450-019-00106-5).
Abstract
Protein adsorption on solid state media is important for the industrial affinity chromatography of biotherapeutics and for preparing materials for self-interaction chromatography where fundamental protein solution thermodynamic properties are measured. The adsorption of three model proteins (lysozyme, catalase and BSA) and two antibodies (a monoclonal and a polyclonal antibody) have been investigated on commercial affinity chromatography media with different surface functionalities (Formyl, Tresyl and Amino). Both the extent of protein immobilised (mg protein/ml media) and the reaction kinetics are reported for a range of reaction conditions, including pH, differing buffers as well as the presence of secondary reactants (glutaraldehyde, sodium cyanoborohydride, EDC and NHS). Compared to the reaction conditions recommended by manufacturers as well as those reported in previous published work, significant increases in the extent of protein immobilisation and reaction kinetics are reported here. The addition of glutaraldehyde or sodium cyanoborohydride was found to be especially effective even when not directly needed for the adsorption to happen. For mAb and pIgG, immobilisation levels of 50 and 31 mg of protein/ml of resin respectively were achieved, which are 100% or more than previously reported. Enhanced levels were achieved for lysozyme of 120 mg/ml with very rapid reaction kinetics (< 1 h) with sodium cyanoborohydride. It can be concluded that specific chromatography resins with Tresyl activated support offered enhanced levels of protein immobilisation due to their ability to react to form amine or thio-ether linkages with proteins. Additionally, glutaraldehyde can result in higher immobilisation levels whilst it can also accelerate immobilisation reaction kinetics. [Figure not available: see fulltext.].
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Hedberg2019_Article_ImprovedAdsorptionReactionsKin
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Accepted/In Press date: 29 April 2019
e-pub ahead of print date: 16 May 2019
Keywords:
Affinity chromatography, Coupling kinetics, Monoclonal antibodies, Polyclonal antibodies, Protein immobilisation, Self-interaction chromatography
Identifiers
Local EPrints ID: 431677
URI: http://eprints.soton.ac.uk/id/eprint/431677
ISSN: 0929-5607
PURE UUID: 8510e13f-1818-48c0-aa59-9517b601035b
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Date deposited: 13 Jun 2019 16:30
Last modified: 16 Mar 2024 02:18
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Author:
S. H.M. Hedberg
Author:
L. G. Brown
Author:
D. R. Williams
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