Affinity cross-flow filtration: purification of IgG with a novel protein a affinity matrix prepared from two-dimensional protein crystals.
In this article, we describe the use of 1- to 2-mum sized affinity microparticles for the isolation and purification of IgG from artificial IgG-human serum albumin mixtures and clarified hybridoma cell culture supernatants by affinity cross-flow filtration. Affinity microparticles were prepared from cell wall fragments of Clostridium thermohydrosulfuricum L111-69, in which the peptidoglycan-containing layer was completely covered with a hexagonally ordered S-layer lattice. After crosslinking the S-layer protein with glutaraldehyde, carboxyl groups from acidic amino acids were activated with carbodiimide and used for immobilization of Protein. A. Quantitative determination confirmed that Protein A molecules formed a monomolecular layer on the outermost surface of the S-layer lattice. Affinity microparticles were found to withstand high centrifugal and shear forces and revealed no Protein A leakage or S-layer protein release under cross-flow conditions between pH 2 to 12. The IgG-binding capacity of affinity microparticles was investigated under crossflow conditions and compared with that obtained in batch adsorption processes.
Weiner
C
C
Zentrum für Ultrastruckturforschung and Ludwig Boltzmann-Institut für Molekulare Nanotechnologie, Gregor Mendel Strasse 33 Universität für Bodenkultur, A-1180 Vienna, Austria.
Sára
M
M
Dasgupta
G
G
Sleytr
U B
UB