Isothermal modelling of protein adsorption to thermo-responsive polymer grafted Sepharose Fast Flow sorbents.

In this study, five adsorption isotherm models, that is, the Langmuir, Freundlich, Langmuir-Freundlich, Temkin and Brunauer-Emmett-Teller isotherms, were utilized for the analysis of the experimental adsorption data for six classes of poly(N-isopropylacrylamide)-based thermo-responsive copolymer-grafted Sepharose Fast Flow sorbents of different copolymer compositions with two structurally related proteins, namely bovine holo-lactoferrin and bovine holo-transferrin at 20 and 50°C. The experimental data for bovine holo-lactoferrin could be mathematically fitted to the Freundlich and Temkin isotherms when the protein feed concentrations were in the range of 1-40 mg/mL at both 20 and 50°C. Similar analysis of the binding of the homologous protein, bovine holo-transferrin, to the same thermo-responsive copolymer-grafted sorbents revealed that the experimental data could be fitted to the Langmuir, Freundlich and Temkin isotherms with coefficients of determination value over 0.90.

Adsorption of a Humanized Monoclonal Antibody onto Thermoresponsive Copolymer-Grafted Sepharose Fast Flow Sorbents.

The batch adsorption behavior of a humanized monoclonal antibody (hIgG2 mAb) with thermoresponsive polymer (TRP)-modified Sepharose Fast Flow sorbents with different compositions of grafted copolymers is described. At high protein loadings, the adsorption with negatively charged copolymer-modified sorbents exhibited S-shaped isotherms in most cases, indicative of unrestricted multilayer adsorption. The adsorption capacity of the negatively charged copolymer-modified sorbents increased with an increase in the applied environmental temperature due to increased protein-sorbent surface hydrophobic and electrostatic interactions. The affinity of the hIgG2 mAb for a positively charged copolymer-grafted sorbent was much lower than that found for the negatively charged copolymer-grafted sorbents at both 20 and 50 °C due to electrostatic repulsive effects. This study has documented that the molecular functionalities of the grafted copolymer can significantly affect the adsorption behavior of this humanized mAb at both 20 and 50 °C with the isothermal dependencies revealing subtle effects due to copolymer composition.

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.

Stimuli-responsive polymeric materials for separation of biomolecules.

Stimuli-responsive polymeric (SRP) materials undergo changes in their physical morphologies and chemical properties in response to small changes in their external environment, such as temperature, pH or light. When immobilised, SRP materials, fabricated in various formats and compositions, provide new opportunities for the separation of products generated by the biotechnology industry. This Current Opinion highlights the potential of these functional materials for the capture, purification and analysis of these products via batch capture methods, column chromatography or electrophoresis, drawing on break-through developments achieved particularly over the last five years.

Ultrasonic synthesis of stable oil filled microcapsules using thiolated chitosan and their characterization by AFM and numerical simulations.

An experimental protocol has been developed for synthesizing stable core-shell microcapsules using a biopolymer, chitosan, lacking cross-linkable thiol functional groups. In the first step, thiol moieties were introduced into the backbone of chitosan using dl-N-acetylhomocysteine thiolactone (AHT). In the second step, AHT-modified chitosan shelled microcapsules, encapsulating an oil core, were successfully prepared using high intensity 20 kHz ultrasound. The size of chitosan and AHT modified chitosan microcapsules was found to be in the range of 1-15 µm. The thickness of the microcapsule shell increased with an increase in thiol content. The mechanical properties of microcapsules were evaluated by subjecting the microcapsules to compressive forces by colloidal probe AFM. The stiffness and the Young's modulus of the shell of microcapsules were determined by analyzing the force versus indentation data using Reissner's theory for indentation of thin elastic shells. The stiffness of AHT modified chitosan microcapsules was found to be higher than unmodified chitosan microcapsules. The viability of microcapsules to be embedded into processed food, pharmaceutical and cosmetic products was tested via numerical simulations. The confined capsule in the micro-channel was subjected to linear shear and uniform flows. We used finite element numerical simulations to determine the deformation of microcapsules in flow as a function of shear rate and thickness of the shell. The deformation of capsule was found to be linear with an increase in the shear rate. The deformation decreased with an increase in the thickness of the shell. Based on the simulations, we predict that the microcapsules would survive processing conditions and shear rates used in industrial applications.