Synthesis of adsorbents with dendronic structures for protein hydrophobic interaction chromatography

Abstract

a b s t r a c tHere, we introduced a new technology based on the incorporation of dendrons-branched chemical structures-onto supports for synthesis of HIC adsorbents. In doing so we studied the synthesis and performance of these novel HIC dendron-based adsorbents. The adsorbents were synthesized in a facile two-step reaction. First, Sepharose 4FF (R) was chemically modified with polyester dendrons of different branching degrees i.e. third (G3) or fifth (G5) generations. Then, butyl-end valeric acid ligands were coupled to dendrons via ester bond formation. UV-vis spectrophotometry and FTIR analyses of the modified resins confirmed the presence of the dendrons and their ligands on them. Inclusion of dendrons allowed the increment of ligand density, 82.5 ± 11 and 175.6 ± 5.7 mol ligand/mL resin for RG3 and RG5, respectively. Static adsorption capacity of modified resins was found to be ∼60 mg BSA/mL resin. Interestingly, dynamic binding capacity was higher at high flow rates, 62.5 ± 0.8 and 58.0 ± 0.5 mg/mL for RG3 and RG5, respectively. RG3 was able to separate lipase, ␤-lactoglobulin and ␣-chymotrypsin selectively as well as fractionating of a whole proteome from yeast. This innovative technology will improve the existing HIC resin synthesis methods. It will also allow the reduction of the amount of adsorbent used in a chromatographic procedure and thus permit the use of smaller columns resulting in faster processes. Furthermore, this method could potentially be considered as a green technology since both, dendrons and ligands, are formed by ester bonds that are more biodegradable allowing the disposal of used resin waste in a more ecofriendly manner when compared to other exiting resins.

The architecture of responsive polymeric ligands on protein binding and recovery

“…20,21 Recently, dendritic butyl HIC ligands immobilized on resins were shown to improve graing density and capacity. 22 However, only two branching degrees of the dendritic ligands were investigated in their study. In order to improve protein binding capacity and recovery for HIC applications, comb-like PVCL ligands were graed on a primary hydrophilic polymer backbone graed on the regenerated cellulose membrane substrate using ATRP as shown schematically in Fig.…”

Magnetic hydrophobic‐charge induction adsorbents for the recovery of immunoglobulins from antiserum feedstocks by high‐gradient magnetic fishing

“…To cope with higher Ig titres found in animal sera, human plasma and highly expressing CHO cultures, more selective adsorption is required to make full use of the available surface area for target binding (200–250 mg g ‐1 with present magnetic support design) thereby reducing the amounts of magnetic support required to treat a given feed. This could be achieved either through the prior and/or post addition of sodium caprylate to reduce serum albumin adsorption (the potential downsides here are reduced Ig yield and compromised isolation of other targets), boosting the immobilised ligand density – perhaps through grafting of ligand‐bearing polymers or dendrons, and/or use of more potent ‘capture’ ligands …”

Development and Characterization of PEGylated Chromatographic Monoliths as a Novel Platform for the Separation of PEGylated RNase a Isomers

“…Figure 2 shows the separation pro les of PEGylation reactions of RNase A. An important point to mention is that in previous reports, the PEGylation reaction is diluted in AS bu er before column injection [4,6,17] since proteins bind to HIC ligands at high salt concentrations and are eluted at low ones [25]. In this work, the reaction mixtures were not diluted in AS bu er before injection.…”