Downstream Processing with Affinity Chromatography. A Study of a Continuous Process for Biospecific Adsorption
A polymer matrix of hydroxy ethyl methacrylate - ethylene glycol dimethacrylate was synthesised and coupled with the ligand p-aminobenzamidine. Although the adsorbent displayed sufficient affinity towards trypsin, the overall adsorption performance was not satisfactory. Subsequent studies were carried out with a p-aminobenzamidine silica adsorbent, which gave far superior results. Amounts of up to 35 mg of trypsin per g of silica could be adsorbed from a porcine pancreatic extract at buffer conditions of 0.05 M Tris, 0.5 M NaCl, pH 8. Complete elution was achieved with a buffer of 0.1 M acetic acid, pH 3. The system was characterised by fixed bed breakthrough studies of the adsorption, wash and desorption step. A method of data analysis was developed and applied to the experimental results. Axial dispersion could be identified as the predominant 'mass transfer' mechanism. A simulation model was build, based on the data analysis results. The model was verified successfully against the experimental data. Continuous separation experiments were performed with three fluidised beds, namely an adsorption, desorption and wash column, with circulation of solids between the columns. Purities of 83% and 66% were achieved in these runs with complete recovery of trypsin. A continuous process model, also based on the data analysis results and on fluidised bed characteristics, was found to agree fairly well with the experimental data. Simulation experiments were conducted to find the optimum configuration of the process. Best performances with respect to product purity were obtained for an adsorption column height of 3 cm and a wash column height of 4 cm. Step experiments revealed that the process is relatively easy to control if the solid flow rate is chosen as the control parameter.
Networked Digital Library of Theses and Dissertations