Kasper C, Reif O W, Freitag R
Institut für Technische Chemie, Hannover, Germany.
Bioseparation. 1996;6(6):373-82.
Affinity filters were investigated for their potential in the recovery of proteins from complex samples. The experiments covered membranes carrying high and low molecular weight affinity ligands as well as group and substance specific ones. For the ready-to-use affinity filters the specific protein binding capacity was determined and compared to that of the respective Sepharose affinity gels (Pharmacia). In the case of the pre-activated membranes the influence of the coupling chemistry on the affinity mediator concentration and the protein binding capacity were considered in the study. In the case of low molecular weight ligands (e.g. Cibacron Blue, Heparin) either type of membrane yielded stationary phases of a ligand concentration, binding capacity, resolution, and long term stability similar to that of the corresponding Pharmacia material. However, the membranes could be used at a higher flow rate than the columns, since they are less mass transfer limited and cause significantly less back pressure. The immobilization of high molecular weight ligands such as antibodies (immuno filtration) on the other hand, resulted in low ligand concentrations and worse antigen binding capacities whenever conventional immobilization procedures, e.g. epoxy group-based reactions, were used. In contradistinction, good results were obtained with tosyl- and tresyl activated membranes. Such membranes were successfully employed for the immobilization of monoclonal antibodies (mAb) and Concanavalin A. Concanavalin A and an anti gp 220/350 mAb were subsequently used to produce affinity filters for the isolation of a recombinant gp 220/350 Epstein-Barr virus surface antigen from culture supernatants of a Chinese hamster ovary cell line grown in protein-free medium.
研究了亲和过滤器从复杂样品中回收蛋白质的潜力。实验涵盖了携带高分子量和低分子量亲和配体以及基团和物质特异性配体的膜。对于即用型亲和过滤器，测定了其特异性蛋白质结合能力，并与相应的琼脂糖亲和凝胶（Pharmacia）进行了比较。对于预活化膜，研究中考虑了偶联化学对亲和介质浓度和蛋白质结合能力的影响。对于低分子量配体（如汽巴克隆蓝、肝素），两种类型的膜产生的固定相在配体浓度、结合能力、分辨率和长期稳定性方面与相应的Pharmacia材料相似。然而，这些膜可以在比柱更高的流速下使用，因为它们的传质限制较小，背压也明显较小。另一方面，当使用常规固定程序（如基于环氧基团的反应）时，将高分子量配体（如抗体，免疫过滤）固定会导致配体浓度较低和抗原结合能力较差。相比之下，使用甲苯磺酰基和三氟甲磺酰基活化的膜获得了良好的结果。这种膜已成功用于固定单克隆抗体（mAb）和伴刀豆球蛋白A。随后，伴刀豆球蛋白A和抗gp 220/350 mAb被用于制备亲和过滤器，以从在无蛋白培养基中生长的中国仓鼠卵巢细胞系的培养上清液中分离重组gp 220/350爱泼斯坦-巴尔病毒表面抗原。
