Godfrey M A, Kwasowski P, Clift R, Marks V
School of Biological Sciences, University of Surrey, Guildford, UK.
J Immunol Methods. 1993 Mar 15;160(1):97-105. doi: 10.1016/0022-1759(93)90013-w.
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.
为评估8种市售葡萄球菌蛋白A（SpA）亲和层析固定相用于大规模纯化鼠单克隆抗体（MAb，mIgG1）的潜力，开展了相关研究。该抗体是在内部的中空纤维生物反应器中无血清生产的。测试了固定相对盐浓度、pH值以及MAb的存在对配体泄漏和流速的影响。使用搅拌罐（滚动混合）和流通（填充床）操作模式下的固定相来比较这些影响。当固定相用于流通模式时，在无MAb情况下配体泄漏通常处于最低水平。在此操作模式下，将洗涤/吸附缓冲液的无机盐浓度和pH值从pH 8.6时的150 mM增加到pH 8.9时的3 M，通常会使固定相的MAb容量增加10%（Sepharose CL - 4B为20%）。然而，由于基质中背景配体泄漏水平升高，纯化抗体的污染也大幅增加。在亲和纯化的MAb中，SpA的残留污染水平在低盐（NaCl，150 mM）甘氨酸（1 M）吸附/洗涤缓冲液中最低。与脉冲使用模式相反，在前沿分析（突破曲线）中所有基质都达到了最大抗体容量。Prosep A“高容量”的容量最大（12 - 15 mg/ml柱体积），无论其使用模式如何，其容量都接近实验确定的理论容量（C/Co = 0.5）。Prosep A“高容量”相对较高的MAb容量在优越的动态等温线上也有进一步体现。然而，前沿分析通常会导致纯化的MAb中SpA污染更严重。在这些条件下，在分别纯化12.8 mg和4.3 mg MAb时，Prosep A“高容量”和Repligen固定相的SpA污染水平最低（12 ppm）。对于用于治疗应用的MAb的大规模下游加工，Prosep A“高容量”似乎是所测试固定相中最合适的。
