A thiol functionalized cryogel as a solid phase for selective reduction of a cysteine residue in a recombinant human growth hormone variant.

Site selective chemical modification is a preferred method, employed to prolong the circulation half-life of biopharmaceuticals. Cysteines have been used as attachment point for such modification, however, to be susceptible for chemical modification the involved thiol must be in its reduced form. Proteins often contain disulfides, which aid to maintain their tertiary structure and therefore must remain intact. Thus, methods for selectively reducing cysteine residues, introduced through site-directed mutagenesis, are of interest. In this study a macroporous, polymeric monolith was designed for selectively reducing a single cysteine residue inserted in recombinant human growth hormone (hGH). Advantages of such a material are the circumvention of the need to remove the reducing agent after reaction, as well as milder reduction conditions and a concomitant lower risk of reducing the native disulfides. The designed monolith showed very high capacity towards the selective reduction of an unpaired cysteine residue in a recombinant hGH variant. Factors influencing the selectivity and rate of reaction were investigated and it was found that monolith thiol loading, and buffer pH had an effect on the rate of reduction, whereas hGH variant concentration and buffer conductivity influenced both rate of reduction and selectivity. The developed system constitutes the basis for the development of a scalable platform for selective reduction of a capped cysteine residue in hGH.

An improved capillary model for describing the microstructure characteristics, fluid hydrodynamics and breakthrough performance of proteins in cryogel beds.

A capillary-based model modified for characterization of monolithic cryogels is presented with key parameters like the pore size distribution, the tortuosity and the skeleton thickness employed for describing the porous structure characteristics of a cryogel matrix. Laminar flow, liquid dispersion and mass transfer in each capillary are considered and the model is solved numerically by the finite difference method. As examples, two poly(hydroxyethyl methacrylate) (pHEMA) based cryogel beds have been prepared by radical cryo-copolymerization of monomers and used to test the model. The axial dispersion behaviors, the pressure drop vs. flow rate performance as well as the non-adsorption breakthrough curves of different proteins, i.e., lysozyme, bovine serum albumin (BSA) and concanavalin A (Con A), at various flow velocities in the cryogel beds are measured experimentally. The lumped parameters in the model are determined by matching the model prediction with the experimental data. The results showed that for a given cryogel column, by using the model based on the physical properties of the cryogel (i.e., diameter, length, porosity, and permeability) together with the protein breakthrough curves one can obtain a reasonable estimate and detailed characterization of the porous structure properties of cryogel matrix, particularly regarding the number of capillaries, the capillary tortuousness, the pore size distribution and the skeleton thickness. The model is also effective with regards to predicting the flow performance and the non-adsorption breakthrough profiles of proteins at different flow velocities. It is thus expected to be applicable for characterizing the properties of cryogels and predicting the chromatographic performance under a given set of operating conditions.