Abstract
This study set out to examine the practicalities of producing silica-based (PS and CPG) affinity solid phases for purifying materials of biological origin. The silica based solid-phases produced in this study were used in order to assess the rapid affinity clean-up of materials in a manner applicable to HPLAC and FPLC systems. In addition, the feasibility of using silica based solid phases in process-scale affinity purification processes was explored. This has required the development of techniques for activating silica-based matrices, immobilizing ligands to activated matrices, and evaluating the performance of the affinity solid phases. In order to compare matrices commonly used as stationary phase supports for affinity purifying proteins, a specific non-competitive enzyme-linked immunosorbent assay (ELISA) was developed to detect and quantify protein A (SpA) levels, present as a trace contaminant in therapeutic murine monoclonal antibodies (MAb), purified on immobilized SpA solid phases. This assay was used to compare eight commercially available SpA solid phases, demonstrating a number of matrix materials, and ligand immobilization chemistries; to establish their potential for the large-scale purification of a murine monoclonal antibody (MAb, mlgG1). Solid phases were tested for the effects of ionic strength, pH, the presence of MAb, flow rate and mode of operation (stirred-tank or flow-through) on the level of ligand leakage. Solid phases have also been evaluated to determine the effect of buffer composition on their non-specific protein adsorption (NSA), and plasma protein (albumin and transferrin) contamination of purified antibodies. The porous silica Matrex™ Si (particle size, 90 - 130 μm; pore size 1000 A Amicon Ltd. ), was used as the starting material for the development of a number of silica-based solid phases. In particular, the grade of Matrex chosen had a pore accessibility suited to the model affinity chromatographic applications in this study; involving the purification of biological macromolecules from biological matrices (filtered tissue homogenates, serum, whole blood, and bile) on immobilized bioligands (SpA and Ab’s). The introduction of functional groups onto the surface of silica-based matrices by the process of silanization was assessed. This technique was used for preparing epoxide and aminopropyl derivatives of PS as the initial stages in matrix activation. The bis-oxirane 1,4-butane diol diglycidyl ether was also evaluated, to determine its merit as a bifunctional agent for immobilizing ligands. The most appropriate technique for coupling ligands to silica based matrices (PS or CPG) appeared to be by: firstly aldehyde activating the matrix surface, followed by Schiff’s base bond formation with the free amine groups of the ligand, and finally reduction of the Schiff’s base bonds. This proved the quickest of the techniques investigated and resulted in the greatest degree of ligand substitution. Activated Matrex based matrices were used to immobilise a number of proteins (BSA, MAb, PAb, SpA), charged molecules (DEAE, QAM), a hapten (Tb-CMO), and a dye (Rivanol). These solid phases were used in order to judge the success of immobilization procedures, determine the best operating conditions for silica-based matrices and to purify biological samples. The solid phases produced in this study were able to operate at high flow rates, allowing the rapid of purification biological samples. This was demonstrated by the HPLAC resolution of Ig’s from plasma, and the development of a rapid automated IAC-ELISA system for the anabolic agent clenbuterol in bovine bile samples. Strategies to limit the level of bioligand leakage from silica-based solid phases during normal purification and hydroxide sanitising operations were investigated using BSA and SpA as model bioligands (proteins). One approach to limiting the level of ligand leakage from affinity solid phases investigated was to empirically optimise their production an operating conditions. Another approach explored was to incorporate metal ions into chromatographic buffers or onto the surface of affinity solid phases. Sanitising conditions which may be used to clean up SpA-(PS or CPG) solid phases without any ill effects were also investigated as alternatives to molar NaOH. The chromatographic performance of solid phases produced using PS and CPG matrices in this study have performed in a manner comparable to many commercially available solid phases. Furthermore, silica based matrices have allowed the development of rapid AC processes without loss of purification efficiency. In the light of the findings presented within this study, many of the more common objections aimed at the use of silica-based matrices for AC applications would appear to no longer be a problem or easily overcome.