Studies on the Stability of Lyophilized BCG Vaccine *†

The application of lyophilization to the preservation of BCG, in an effort to combat the rapid loss in allergenic potency of the liquid vaccine, has presented important technical difficulties. Although the effectiveness of lyophilized BCG in producing tuberculin conversion appears to be as great as that of the fresh vaccine,"118,2 clinical experience has shown that its use engenders quantitatively lessened tuberculin reactions"'5 that frequently are temporally delayed."7'18'2' More important, however, is the fact that the protective value of lyophilized vaccines against challenge infection with virulent tubercle bacilli is less than that produced by fresh, liquid vaccines."' Inasmuch as the allergenic potency of the vaccine has been shown to be a function of the viability of the suspension5'9 the failure of dried vaccines has been attributed to the large decrement in viable cells that occurs during the period of lyophilization itself,"5 and the smaller decrease that occurs in the storage period which follows. Initial attempts at prolonging the effective life of BCG vaccine were made in Russia in 1937.1' From the start, workers have experienced difficulty, and the tendency has been to use accessory substances along with the vaccine suspension to enhance its stability. Excipients such as glucose,""' starch," lactose,' " 16 gelatin,' denatured gelatin,'5 serum,6'2 sucrose, gum arabic,"1 peptone solution,9 sodium ammonium pectate, and glutamic, malic, and succinic acids' have been tried. It has been suggested recently that Tweenalbumin medium, as used for diffuse, submerged growth of Mycobacterium tuberculosis, be used as the vehicle for lyophilization.'5 Rosenthal'8 has reported excellent results using a mixture of lactose, asparagin, buffered salts, and glycerine as a suspending menstruum. Reported s-urvival rates vary from 100 per cent2'" to about 14 per cent,'0 immediately following lyophilization. Birkhaug' reports a survival rate of 2.83 to 3.26 per cent three to twelve months after freeze-drying. Preliminary experiments carried out in this laboratory having supported the reports of low survival rates following lyophilization and, further, having indicated that BCG could not be successfully stabilized in the absence of drying excipients a survey has been made of sixty-two adjuvants, embracing