Patent GB-1138452-A (GB)
Title: Improvements in or relating to continuous phased culturing of cells
IPC: C12M1/04
Filed: 1966-03-04
Abstract: 1,138,452. Antibiotic-producing micro-organisms. CANADIAN PATENTS & DEVELOPMENT Ltd. 4 March, 1966 [8 March, 1965], No. 9635/66. Heading C2A. [Also in Division C6] The phasing of a cell culture is improved by growing the cell culture in a nutrient medium at a predetermined rate of growth, the quantity of medium being only sufficient for the cells to complete their cycle at that growth rate, and at the doubling time of the culture the cell culture is divided into two equal halves while further nutrient medium is added such that each of the halves has sufficient nutrient medium for the cells to complete their cycle. In the phased condition usually at least 70-80% of the cells are at an identical stage of growth over their cell cycle. The further nutrient medium may be added immediately prior to the doubling time in such an amount as to double the volume of the cell culture, and at the doubling time the cell culture is divided into two equal halves whereby the cells in the halves may complete a similar cell cycle. The procedure may be continually repeated with at least one half of the cell culture to provide for a continuing production of the phased cell culture, while the other half is employed for the harvesting of a desired cell metabolite (even one of transient production such as ribonucleic acid) at the time when it is produced in an optimum amount, as determined by analysis of the metabolite production on previous cell cycles of the phased cell culture; the metabolite may be harvested either during a repeating phased cycle or after the particular cell culture has proceeded beyond this to a post cycle for further growth under modified conditions. In the production of penicillin with batch culture, the penicillin is a secondary metabolite produced in the stationary stage which follows the primary growth. In the phased culture process, however, this metabolite may be harvested in the post cycle at the optimum time determined experimentally and due to the phasing of the cell culture the yield is stated to be greatly increased over that of the randomized batch process. The phased culture process may be applied to micro-organisms (bacteria yeasts and moulds) and to plant- and animal-tissue cells. The following species of micro-organisms are particularly mentioned: Candida utilis, Saccharomyces cerewisiae, Saccharomyces rouxii, Saccharomyces magnoliae, Streptomyces bovis, Streptomyces venezuleae, Aspergillus aerogenes, Aspergillus suboxydans and Pseudomonad sp. The process is described with reference to the cultivation of Candida utilis on nitrogen-limiting media based on glucose or glycerol. Detailed analyses are presented of the varying amount of metabolites produced during phases of differing durations. Referring to Fig. 1, a mixture 12 of cell culture and nutrient medium is maintained at a constant temperature for the cultivation by recirculation by a pump 13 from the cyclone column 10 to a water jacketed limb 11. It is arranged that at, or immediately prior to, the doubling time a dosing vessel 21 will have been filled with nutrient medium, delivered at a constant rate from reservoir 33, to such an extent that a constant volume of nutrient medium is syphoned off into the cyclone column 10 and that one-half of the resulting mixture either is syphoned off into a collector 39 or is withdrawn by manual operation of a clamp 41 into a second culturing system 10<SP>1</SP> from which product is collected in a harvesting vessel 46.