PROCESS-DEVELOPMENT FOR THE RECOVERY AND PURIFICATION OF RECOMBINANT PROTEIN-G

The domains of protein G from streptococcus which bind immunoglobulin G have been cloned and expressed in Escherichia coli (Fahnestock et al., 1986). Because protein G binds to several animal immunoglobulin G's, it has many immunochemical applications. This report describes process development for large-scale production of this recombinant protein G (also known as GammaBind(R) G). In 200 1 cultures of E. coli, this protein G variant was released from the cell into the culture medium by heating at 80-degrees-C for 10 min. The concentration was monitored by either a competitive enzyme-linked immunoassay or a liquid chromatographic assay. Cross-flow microfiltration with 0.22 mum membrane was used to remove the cells. The protein G-rich permeate from the crossflow microfilter was purified by affinity chromatography using a 5 l column of IgG-Sepharose 6 Fast Flow, which yielded 16-18 g of protein G per column cycle. The pools of purified protein G were concentrated and desalted using ultrafiltration. The salt-free protein G was then lyophilized as bulk product. The overall recovery through the entire process was 50-64%. The analysis of the final product included sodium dodecyl sulfate polyacrylamide gel electrophoresis, UV-visible spectrum, high performance gel filtration, endotoxin level and binding efficiency to human IgG Sepharose.