DESIGN AND EVALUATION OF A 2-STAGE, CYCLIC, RECOMBINANT FERMENTATION PROCESS

A two-stage, cyclic fed-batch fermentation process to produce recombinant human lymphokine was designed. The organism used in the study was Escherichia coli K-12 containing a temperature-sensitive walkaway plasmid bearing an insert which codes for a human lymphokine. Transcription of the recombinant gene is controlled by a lambda repressor/pL promoter system. The lambda promoter is regulated by the temperature-sensitive product of the cl857 gene at 30-degrees-C, but at 42-degrees-C the promoter is derepressed. The first, or growth, stage of the process was maintained at 28-degrees-C and operated in the fed-batch mode. The vessel was fed at a rate which gives a constant specific growth rate using a media designed to maintain a constant optical density OD600 of 50. After the volume in the first stage reached the maximum working volume of the vessel (12 L), a portion of the vessel contents was transferred to the second stage. The second, or induction/product formation, stage also operated in the fed-batch mode, was kept at 42-degrees-C, and was fed with a media that is conducive to recombinant human lymphokine synthesis. An optical density of more than 100 was consistently achieved in the second stage. Thirty cycles were completed with a consistent yield of human lymphokine and cell density in each cycle. The process was used to produce 200 L of OD600 50 material from the first stage in 10 days. The volumetric productivity (g lymphokine/L . day) of the two-stage, cyclic fed-batch process is twice that of a single-stage, fed-batch fermentation process.