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Affinity cross‐flow filtration: Purification of IgG with a novel protein a affinity matrix prepared from two‐dimensional protein crystals
Author(s) -
Weiner Christian,
SáRa Margit,
Dasgupta Gautam,
Sleytr Uwe B.
Publication year - 1994
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.260440109
Subject(s) - carbodiimide , cross flow filtration , chemistry , glutaraldehyde , chromatography , peptidoglycan , affinity chromatography , adsorption , size exclusion chromatography , protein a , s layer , biochemistry , cell wall , membrane , organic chemistry , antibody , biology , immunology , enzyme , gene
In this article, we describe the use of 1‐ to 2‐μm 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. © 1994 John Wiley & Sons, Inc.