Thermal Stability of Vaccines

Publisher Summary This chapter describes the preservation of bacteria by lyophilization Lyophilization, or freeze-drying, is a term applied to the procedure of freezing and subliming water from frozen preparations “Lyophil method” is specifically applied to the condensation of water, sublimed from frozen preparations, on a cold surface as distinct from the use of desiccants As most biological materials dried from the frozen state are lyophilic, regardless of how water is removed, the term “lyophilization” is adopted by a number of workers to describe any process involving the sublimation of water from frozen preparations Plug Freezing, Shell Freezing, Evaporative Freezing, and Pelletizing are the general procedures of bacterial preservation by lyophilization In many instances lyophilization may be the preferred method but other methods, such as preservation at liquid nitrogen temperatures, may prove to be superior to lyophilization for the preservation of stock cultures However, as the reliability of preserving viability in lyophilized cultures is increased there will probably be an increased use of lyophilized organisms to be used directly as a reagent, as in microbiological assays

Preservation of bacteria by lyophilization.

The protective effects of glycerol, adonitol, and four other related polyhydric alcohols on lactic acid bacteria subjected to freeze-drying were examined. The presence of adonitol in the suspending medium markedly protected the viabilities of the 12 stains tested. Dulcitol, mannitol, m-inositol, and sorbitol were found to provide little or no protection.

Protective Effect of Adonitol on Lactic Acid Bacteria Subjected to Freeze-Drying

https://aem.asm.org/content/aem/9/3/256.2.full.pdf

Preservation of Microorganisms by Freeze-drying: II. The Destructive Action of Oxygen. Additional Stabilizers for Serratia marcescens. Experiments with Other Microorganisms.

Inasmuch as all living things usually require a high content of water either internally or externally, or both, it is remarkable that microorganisms, especially spore forms, can sometimes withstand an extreme desiccating environment. Any success achieved by chemical or physical means in modifying viability, that is in hastening or preventing death of the desiccated cell, has implications in many different fields. The various techniques exploited during the last 45 years in the preservation of bacteria by drying have been reviewed in an interesting and very useful manner by Fry (1954). Although he commends Naylor and Smith (1946) on the logical development of their stabilizing medium for the freezedrying of Serratia marcescens, he points out that its general applicability would have to be tested with more sensitive organisms. The present study of preservation by drying is also limited to S. marcescens but a considerable variety of new substances has been tried besides the Naylor-Smith components. Some of our results should find application in the preservation of other organisms freeze-dried under similar conditions. Experiments with sensitive strains of yeast and bacteria will be reported in a later paper.

Preservation of microorganisms by freeze-drying. I. Cell supernatant, Naylor-Smith solution, and salts of various acids as stabilizers for Serratia marcescens.

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

Kenneth Goodner

Papers

Studies on the stability of lyophilized B C G vaccine.