Evaluation of affinity filters for protein isolation.
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.
Kasper
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Institut für Technische Chemie, Hannover, Germany.
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Freitag
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D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification; D000956:Antigens, Viral / Q000302:isolation & purification; D001665:Binding Sites; D016466:CHO Cells; D002478:Cells, Cultured; D003208:Concanavalin A / Q000737:chemistry; D006224:Cricetinae; D004852:Epoxy Compounds / Q000737:chemistry; D005374:Filtration / Q000379:methods*; D004854:Herpesvirus 4, Human / Q000276:immunology; D008024:Ligands; D008567:Membranes, Artificial; D011485:Protein Binding; D011506:Proteins / Q000302:isolation & purification*
D000911:Antibodies, Monoclonal; D000956:Antigens, Viral; D004852:Epoxy Compounds; D008024:Ligands; D008567:Membranes, Artificial; D011506:Proteins; D003208:Concanavalin A