Batch binding studies with thermo-responsive polymer grafted sepharose 6 fast flow sorbents under different temperature and protein loading conditions.

This study has examined the batch binding behaviour of different thermo-responsive co-polymer grafted chromatographic materials under different temperature and protein loading conditions. The effect of molecular composition of poly(N-isopropylacrylamide) (PNIPAAm)-based co-polymers on the phase transition properties has been documented. Sixteen co-polymers of different compositions were synthesized by free radical polymerization methods. Most underwent relatively sharp phase transitions upon application of increasing temperature. However, the value of the lower critical solution temperature (LCST) varied due to differences in co-polymer compositions. In general, it was found that the LCST increased for co-polymers containing more hydrophilic moieties, but decreased for co-polymers with more hydrophobic moieties. Moreover, the LCST increased, together with increased width of the transition temperature, when highly branched monomeric moieties (i.e. N­tert­octyl groups) were present. When bulky side chains (octadecyl or triphenylmethyl groups) were located in the polymer structures the LCST transition was absent. Based on these findings, 6 thermo-responsive co-polymers of different compositions were individually immobilised onto cross-linked Sepharose 6 Fast Flow by a "grafting-from" method. Bovine holo-lactoferrin and bovine holo-transferrin at different concentrations in the range 1-100 mg/mL were then employed as target proteins to evaluate the adsorption behaviour under batch binding conditions with these different polymer grafted Sepharose 6 Fast Flow sorbents at two different temperatures. In general, all sorbents exhibited greater affinity and adsorption capacity for bovine holo-lactoferrin at 50 °C compared to 20 °C. In addition, the affinity and adsorption capacity of bovine holo-lactoferrin with positively charged copolymer grafted Sepharose 6 Fast Flow chromatographic sorbents at 20 °C and 50 °C were much lower than that found for negatively charged copolymer grafted Sepharose 6 Fast Flow sorbents, whilst the opposite trend was found with bovine holo-transferrin due to differences in the surface charge properties of these two proteins, indicative of different separation selectivity. Furthermore, the structure of the side chains present in the grafted copolymer structure was found to affect the adsorption performance of both proteins at 20 °C and 50 °C.

Studies on the binding sites of IgG2 monoclonal antibodies recognized by terpyridine-based affinity ligands.

This investigation has examined the origin of the molecular recognition associated with the interaction of monoclonal IgG2's with terpyridine-based ligands immobilized onto agarose-derived chromatographic adsorbents. Isothermal titration calorimetric (ITC) methods have been employed to acquire thermodynamic data associated with the IgG2-ligand binding. These ITC investigations have documented that different enthalpic and entropic processes are involved depending on the nature of the chemical substituents in the core structure of the terpyridinyl moiety. In addition, molecular docking studies have been carried out with IgG2 structures with the objective to identify possible ligand binding sites and key interacting amino acid residues. These molecular docking experiments with the different terpyridine-based ligands have shown that all of the examined ligands can potentially undergo favorable interactions with a site located within the Fab region of the IgG2. However, another favorable binding site was also identified from the docking poses to exist within the Fc region of the IgG2 for some, but not all, of the ligands studied. These investigations have provided a basis to elucidate the unique binding properties and chromatographic behaviors shown by several substituted terpyridine ligands in their interaction with IgGs of different isotype. Copyright © 2016 John Wiley & Sons, Ltd.

Studies on the application of temperature-responsive ion exchange polymers with whey proteins.

Several new types of temperature-responsive ion exchange resins of different polymer composition have been prepared by grafting the products from the co-polymerisation of N-phenylacrylamide, N-iso-propylacrylamide and acrylic acid derivatives onto cross-linked agarose. Analysis of the binding isotherms for these different resins obtained under batch adsorption conditions indicated that the resin based on N-iso-propylacrylamide containing 5% (w/w) N-phenylacrylamide and 5% (w/w) acrylic acid resulted in the highest adsorption capacity, Bmax, for the whey protein, bovine lactoferrin, e.g. 14 mg bovine lactoferrin/mL resin at 4 °C and 62 mg bovine lactoferrin/mL resin at 40 °C, respectively. Under dynamic loading conditions at 40 °C, 94% of the loaded bovine lactoferrin on a normalised mg protein per mL resin basis was adsorbed by this new temperature-responsive ion-exchanger, and 76% was eluted by a single cycle temperature shift to 4 °C without varying the composition of the 10mM sodium dihydrogen phosphate buffer, pH 6.5, or the flow rate. The binding characteristics of these different ion exchange resins with bovine lactoferrin were also compared to results obtained using other resins based on N-isopropylacrylamide but contained N-tert-butylacrylamide rather than N-phenylacrylamide, where the corresponding dynamic capture and release properties for bovine lactoferrin required different temperature conditions of 20 °C and 50 °C, respectively for optimal desorption/adsorption. The cationic protein, bovine lactoperoxidase, was also adsorbed and desorbed with these temperature-responsive resins under similar conditions of changing temperature, whereas the anionic protein, bovine ß-lactoglobulin, was not adsorbed under this regime of temperature conditions but instead eluted in the flow-through.

Application of 4'-terpyridinylsulfanylethylamine resins for the purification of monoclonal antibodies by mixed-mode chromatography.

In this study, a pyridine-based compound, 4'-terpyridinylsulfanylethylamine (4'-TerPSEA), has been employed as a ligand to purify via mixed-mode chromatographic procedures a humanised monoclonal antibody of the IgG1 sub-class directly from crude supernatants derived from cultured CHO cells. The antibody binding capacity, selectivity and reusability of the adsorbent, derived from the immobilisation of this ligand onto Sepharose FF™, were compared to a Protein A affinity resin. The chromatographic performance of this mixed mode adsorbent was similar to that shown by the Protein A-based adsorbent with this IgG1 mAb. In addition, the IgG1 mAb was able to bind to the immobilised 4'-TerPSEA under reducing conditions. Through the use of papain-digested IgG1 mAb, fractionated with both the 4'-TerPSEA and Protein A adsorbents, it was found that this IgG1 mAb preferentially bound to the immobilised 4'-TerPSEA Sepharose FF™ resin through its Fc region.

N-Heterocyclic-based adsorbents for antibody purification-effect of ligand structure.

A new set of ligands based on substituted pyridine and other N-heterocyclic structures, possessing an aliphatic primary amino group tether and an exocyclic sulphur atom, has been prepared and immobilized onto epoxy-activated matrices such as Sepharose 6 Fast Flow®. The derived adsorbents have been evaluated for their utility to capture and purify humanized monoclonal antibodies. Favourable binding properties were assessed from screening assays to determine optimal conditions for the capture and elution of the monoclonal antibodies. Static and dynamic binding experiments were employed to derive the equilibrium dissociation constants KD 's and binding capacities Qmax 's. Typically, the KD values were in the range of 2-5 µM and the Qmax values between 20 and 75 mg mAb/ml resin, depending on the stereo-electronic properties of the substituent in the N-heterocyclic ring structure. The effect of ligand structure on the selectivity of these adsorbents was also investigated, and criteria for their use in the purification of monoclonal antibodies from cell culture supernatants established.

A highly enantioselective synthesis of cyclic alpha-amino acids involving a one-pot, single catalyst, tandem hydrogenation-hydroformylation sequence.

Tandem enantioselective hydrogenation followed by a hydroformylation-cyclisation sequence leading to cyclic alpha-amino acids with ee's > 95% can be achieved in a single pot, one catalyst system by successive reactions of prochiral dienamide esters with H2 followed by H2/CO using Rh(I)-DuPHOS.