Characterization of an affinity purification system for recombinant proteins containing a cellulose binding domain

The selective binding of the cellulose binding domain (CBD) of the exo-ß-l,4-gIycanase Cex from Cellulomonas fimi (CBDcex) to a variety of cellulosic substrates offers the possibility of a new, cost effective, highly flexible affinity chromatography system for purification of recombinant proteins. Genetic linkage of CBDCex to a target protein results in a fusion protein which binds strongly to cellulose and retains the biological activity of the fusion partner. This thesis is concerned with development, testing, and modeling of an affinity chromatography column based on a sorbent resin of Avicel PHI01, a pure semicrystalline cellulose particulate, for one-step purification of recombinant fusion proteins containing a CBDcex tag. Appropriate mobile-phase compositions for the loading, wash, and elution steps are described, as are the general requirements for column set-up. Limitations of the current column configuration and components are discussed along with suggestions for improving the system. Frontal-loading and first-moment analyses are used to characterize basic column properties, including voidage and packing uniformity. Second moment analysis is combined with breakthrough data as a function of flow rate and concentration to determine axial diffusion coefficients, film/particle mass transfer coefficients, and solute diffusivities. The pore-size distribution and effective porosity of Avicel PHI01 particles are determined by a solute exclusion technique using probe molecules of varying hydrodynamic radius. Batch adsorptionisotherm data are used to estimate binding constants for CBDcex and maximum sorbent surface capacity, which are then confirmed on column by breakthrough curve analysis. The measured column parameters are combined with the equations governing continuity and intraparticle diffusion to develop a column model which accounts for gradients both along the column axis and in the radial direction within the sorbent particles. Flow and solute concentrations are assumed to be uniform across any cross-section of the column. Breakthrough curves predicted by the model are in good agreement with experiment over the range of realistic operating conditions. Column performance and model predictions are verified by their application to the purification of a Protein A-CBDcex fusion and the comparison of predicted breakthrough curves with those observed experimentally.Applied Science, Faculty ofChemical and Biological Engineering, Department ofGraduat