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J Immunol Methods. 1993 Mar 15;160(1):97-105.

Assessment of the suitability of commercially available SpA affinity solid phases for the purification of murine monoclonal antibodies at process scale.

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  • 1School of Biological Sciences, University of Surrey, Guildford, UK.


Eight commercially available staphylococcal protein A (SpA) affinity chromatography solid phases were evaluated in order to establish their potential for the large-scale purification of a murine monoclonal antibody (MAb, mIgG1). The antibody was produced in-house, serum-free, in a hollow fibre bioreactor. Solid phases were tested for the effects of salt concentration, pH, and the presence of MAb on ligand leakage and flow rate. These effects were compared using the solid phases in stirred-tank (roller-mixing) and flow-through (packed-bed) modes of operation. Ligand leakage in the absence of MAb was generally at its lowest when the solid phases were used in a flow-through mode. In this mode of operation increasing the inorganic salt concentration and pH of the washing/adsorption buffer from 150 mM at pH 8.6, to 3 M at pH 8.9, typically produced a 10% increase in MAb capacity of the solid phases (20% for Sepharose CL-4B). However, contamination of the purified antibodies was also greatly increased due to an elevated level of background ligand leakage from the matrices. Residual contaminating levels of SpA in affinity purified MAbs were lowest with a low salt (NaCl, 150 mM) glycine (1 M) adsorption/washing buffer. Maximal antibody capacity was achieved for all matrices on frontal analysis (breakthrough curves), as opposed to a pulse mode of use. The largest capacity was found for Prosep A 'high capacity' (12-15 mg/ml column volume), where capacity approached its experimentally determined theoretical capacity (C/Co = 0.5) regardless of its mode of use. The relatively high MAb capacity of Prosep A 'high capacity' was further reflected in a superior dynamic isotherm. Frontal analysis, however, generally resulted in a greater SpA contamination of the purified MAbs. Under these conditions the lowest levels of SpA contamination were found for the Prosep A 'high capacity', and Repligen solid phases (12 ppm) on purifying 12.8 and 4.3 mg of MAb respectively. For the large scale downstream processing of a MAb for therapeutic applications, Prosep A 'high capacity', would appear to be the most appropriate of the solid phases tested.

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