Production of human interleukin-8 expressed in Escherichia coli: from a laboratory scale for in vitro tests via a technical scale for animal studies to a process scale for a GMP-compatible production.
An Escherichia coli K 12 strain has been constructed for efficient expression of recombinant biologically active human IL-8 (Interleukin-8). The development of a fermentation and purification process from the laboratory scale (cells from 15 l fermentation broth) to a production scale (cells from 200 l fermentation broth) is described. Material obtained from the laboratory scale was used for initial in vitro studies and for the development of a biological assay. An upscale purification process starting from 80 l fermentation broth resulted in larger amounts of IL-8 needed for preclinical studies. This process includes a fully automated control of the initial affinity chromatography step. Finally, a production process which differed markedly from the small-scale processes was tailor-made for GMP conformity and economic considerations. It consists of a cell disruption step followed by two crossflow diafiltrations with different molecular weight cut offs and filtration rates, one cation exchange chromatography and a final dialysis step. In order to enhance the overall yield of biologically active IL-8, conditions for a resolubilisation of insoluble IL-8 present in the remaining pellet after cell disruption were worked out.
Koltermann
A
A
Max-Planck-Institute of Biochemical Chemistry, Department of Biochemical Kinetics, Göttingen, Germany.
Boidol
W
W
Daum
J
J
Scholz
P
P
Donner
P
P
D019149:Bioreactors; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D016209:Interleukin-8 / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D009504:Neutrophils / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D012995:Solubility; D014508:Urea / Q000494:pharmacology
D016209:Interleukin-8; D011994:Recombinant Proteins; D014508:Urea