GB1108956A - Process for the manufacture of bcg vaccines - Google Patents

Abstract

Storage stable cultures of Bacterium Calmette Guerin (BCG), for use as a vaccine are selected from normal cultures by subjecting a lyophilized normal culture to storage under defined conditions, determining the survival rate of the bacteria from the lyophilisate, and selecting the bacteria having a survival rate of 80-100%. A normal BCG culture, defined as a culture which has not bee subjected to the inventive process, is centrifuged and the sediment washed with albumin solution. The cells are suspended in a drying medium containing dextran, sodium glutamate, and one or more of sucrose, lactose, glucose and/or fructose, and the suspension deep frozen at -50 DEG C. and then lyophilized at -15 DEG to -20 DEG C. and further dried in a low vacuum dessiccator gassed with dry nitrogen. The lyophilisate is stored for 1 week to 6 months at 20 DEG -50 DEG C. (i.e. 1-6 months at 30 DEG -40 DEG C. or 8 days at 50 DEG C.). The lyophilisate is then suspended in water or albumin solution and plated out on solid medium to give colonies of single cell origin. 9-20 of such colonies are individually inoculated into shaken liquid cultures containing specified nutrients, glycerin as an energy and carbon source and a wetting agent, and grown up. The survival rate of the cells from each colony is determined by lyophilizing the centrifuged sediment from a first part of these liquid cultures, under the same conditions as before, plating out the lyophilisate and comparing the number of colonies on the plate with that provided on a similar plate by an equivalent quantity of a second part of the liquid culture which has not been lyophilized. The cultures having a survival rate of 80-100% are selected for further cultivation in the aforsaid glycerine medium, on a larger scale in industrial fermenters.ALSO:BCG vaccine is prepared from a BCG live culture which has been selected for stability under storage by (1) lyophilising a normal aqueous BCG culture in a sugar-containing drying medium; (2) storing the lyophilisate under the conditions to be used for storing the final vaccine; (3) culturing the stored lyophilisate to give colonies originating from single cells; (4) selecting organisms from the colonies showing the highest survival rate and re-cultivating on a larger scale for use in the vaccine. The initial culture of BCG is an aqueous BCG suspension which has not been previously subjected to the inventive process. The sugar-containing drying medium is suitably an aqueous solution of dextran, sodium glutamate and a sugar, e.g. glucose, fructose, lactose and/or sucrose, lyophilisation being performed at a temperature not above -15 DEG C. The storage conditions are defined as being one week to six months at 20 DEG - 50 DEG C., (e.g. 1-6 months at 30 DEG - 40 DEG C. or 8 days at 50 DEG C). The stored lyophilisate is suspended in water or e.g. albumin solution, and the suspension plated out on solid medium to give colonies of monocellular origin. 9-20 colonies are individually transferred to shaken liquid cultures, incubated, re-inoculated into further liquid cultures and grown up. The cells are centrifuged down and re-suspended in the sugar-containing drying medium. The survival rates of the cultures are determined by lyophilising a first part of the drying medium suspension, re-suspending and plating out to give single colonies, the number of which is compared with the number given on a similar plate by a corresponding volume of a second part of the suspension which has not been lyophilised. Only those cultures having a survival rate of 80-100% are selected for preparing the vaccine. The vaccine is prepared by cultivating the selected BCG organisms on a larger scale and lyophilising the organisms under the same conditions as before. Suitable vaccines contain at least 4 x 107 live germs/mg. dry weight, and may be contained in sealed unit dose ampoules, for reconstitution with saline solution.