HUMAN IL-1 RECEPTOR ANTAGONIST FROM ESCHERICHIA-COLI - LARGE-SCALE MICROBIAL-GROWTH AND PROTEIN-PURIFICATION

Interleukin-l receptor antagonist (IL-1ra) is a recently discovered cy tokine which specifically inhibits IL-I pro-inflammatory activities in various experimental conditions. In this work, the growth conditions of a recombinant E. coli strain which in laboratory studies expressed human IL-1ra mostly in insoluble form, have been optimized at the leve l of 6-1 bioreactors and then scaled up to a 50-1 process. As a result , at high amount (0.43 g l(-1) of microbial culture) of soluble, activ e IL-1ra has been directly obtained in the large-scale cell lysate wit h no need for protein solubilization. Also, an efficient purification procedure has been developed for the soluble protein, based on cation exchange expanded bed adsorption directly followed by anion exchange c hromatography. This process, which does not include any intermediate d ialysis step or gradient elutions, can be easily scaled up to larger p roduction volumes and is therefore well-suited for manufacturing. As a result of the overall optimization study, more than 12 g of pure IL-1 ra have been obtained from a single 50-1 fermentation run, without any denaturation/renaturation process. The final product, whose identity and purity have been checked also by MALDI-TOF and ESI-MS, shows full biological activity both in cellular assays and in in vivo experiments with Cynomolgus monkeys. (C) 1998 Elsevier Science B.V. All rights re served.