Design and evaluation of a two‐stage, cyclic, recombinant fermentation process

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Abstract
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°C, but at 42°C the promoter is derepressed. The first or growth, stage of the process was maintained at 28°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°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.