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- Gonzalez-Ortega, Omar, et al.
(författare)
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Adsorption of peptides and small proteins with control access polymer permeation to affinity binding sites. Part I : Polymer permeation-immobilized metal ion affinity chromatography separation adsorbents with polyethylene glycol and immobilized metal ions
- 2012
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Ingår i: Journal of Chromatography A. - : Elsevier BV. - 0021-9673 .- 1873-3778. ; 1227, s. 115-125
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Tidskriftsartikel (refereegranskat)abstract
- Despite the many efforts to develop efficient protein purification techniques, the isolation of peptides and small proteins on a larger than analytical scale remains a significant challenge. Recovery of small biomolecules from diluted complex biological mixtures, such as human serum, employing porous adsorbents is a difficult task mainly due to the presence of concentrated large biomolecules that can add undesired effects in the system such as blocking of adsorbent pores, impairing diffusion of small molecules, or competition for adsorption sites. Adsorption and size exclusion chromatography (AdSEC) controlled access media, using polyethylene glycol (PEG) as a semi-permeable barrier on a polysaccharide matrix, have been developed and explored in this work to overcome such effects and to preferentially adsorb small molecules while rejecting large ones. In the first part of this work, adsorption studies were performed with small peptides and proteins from synthetic mixtures using controlled access polymer permeation adsorption (CAPPA) media created by effectively grafting PEG on an immobilized metal affinity chromatography (IMAC) agarose resin, where chelating agents and immobilized metal ions were used as the primary affinity binding sites. Synthetic mixtures consisted of bovine serum albumin (BSA) with small proteins, peptides, amino acids (such as histidine or Val(4)-Angiotensin III), and small molecules-spiked human serum. The synthesized hybrid adsorbent consisted of agarose beads modified with iminodiacetic (IDA) groups, loaded with immobilized Cu(II) ions, and PEG. These CAPPA media with grafted PEG on the interior and exterior surfaces of the agarose matrix were effective in rejecting high molecular weight proteins. Different PEG grafting densities and PEG of different molecular weight were tested to determine their effect in rejecting and controlling adsorbent permeation properties. Low grafting density of high molecular weight PEG was found to be as effective as high grafting density of low molecular weight PEG in the rejecting properties of the semi-permeable synthesized media. (C) 2012 Elsevier B.V. All rights reserved.
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| 2. |
- Gonzalez-Ortega, Omar, et al.
(författare)
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Adsorption of peptides and small proteins with control access polymer permeation to affinity binding sites. Part II : Polymer permeation-ion exchange separation adsorbents with polyethylene glycol and strong anion exchange groups
- 2012
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Ingår i: Journal of Chromatography A. - : Elsevier BV. - 0021-9673 .- 1873-3778. ; 1227, s. 126-137
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Tidskriftsartikel (refereegranskat)abstract
- In chromatographic separations, the most general problem in small biomolecule isolation and purification is that such biomolecules are usually found in extremely low concentrations together with high concentrations of large molecular weight proteins. In the first part of this work, adsorption and size exclusion chromatography (AdSEC) controlled access media, using polyethylene glycol (PEG) as a semi-permeable barrier on a polysaccharide Immobilized Metal Affinity Chromatography (IMAC) matrix was synthesized and used to develop chromatographic adsorbents that preferentially adsorb and separate low molecular weight biomolecules while rejecting large molecular weight proteins. In this second part, we expand the concept of controlled access polymer permeation adsorption (CAPPA) media by grafting polyethylene glycol (PEG) on a high capacity polysaccharide ion exchange (IEX) chromatographic resin where PEG acts as a semi-permeable barrier that preferentially allows the permeation of small molecules while rejecting large ones. The IEX resin bearing quaternary ammonium groups binds permeated biomolecules according to their ion exchange affinity while excluding large biomolecules by the PEG barrier and thus cannot compete for the binding sites. This new AdSEC media was used to study the retention of peptides and proteins covering a wide range of molecular weights from to 150 kDa. The effect of protein molecular weight towards retention by ion exchange was performed using pure protein solutions. Recovery of insulin from insulin-spiked human serum and insulin-spiked human urine was evaluated under polymer controlled permeation conditions. The CAPPA media consisted of agarose beads modified with amino-PEG-methoxy and with trimethyl ammonium groups, having chloride capacities between 20 and 40 mu eq/mL and were effective in rejecting high molecular weight proteins while allowing the preferential adsorption of small proteins and peptides.
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