Live, attenuated viral and bacterial vaccines are sensitive to environmental stress, especially elevated temperatures, and often require lyophilization to preserve potency during long-term storage and distribution (see Table 1 and Table 2). This is not surprising since intact viruses and bacteria replicate in vivo and must maintain their ability to infect cells. This requires intact native structures of proteins, DNA/RNA and lipid bilayers. Freeze-dried (lyophilized) viruses and bacterial vaccines usually contain a mixture of cell culture components after isolation of the attenuated virus/bacteria as well as excipients that permit successful freeze-drying, stabilization of microorganisms, and acceptable pharmaceutical properties such as intact physical appearance (no cake collapse) and rapid reconstitution.

The lyophilized vaccine product is subsequently stored for months to years, under appropriate conditions, and then reconstituted immediately before administration to the patient. Reconstitution refers to the process of mixing of the freeze-dried vaccine powder with the diluent (liquid). Diluents for lyophilized vaccines vary in their composition and volume and are specifically designed to meet the pH, volume and chemical requirements of each vaccine [41]. While some diluents consist of sterile water, others may contain ingredients that not only help dissolve the freeze-dried powder but also stabilize (e.g., saline) and/or maintain the sterility of the reconstituted vaccine (e.g., preservatives). The volume of the diluent provided with a vaccine product is dependent on whether a single or multi-dose vial of vaccine will be reconstituted. The stability of reconstituted lyophilized vaccines is limited, both in terms of potency and ensuring sterility, and usually is used within hours or must be discarded. Therefore, once reconstituted, these vaccines should be stored under appropriate conditions (lower temperatures, protect from light, etc.) to better maintain the minimum required potency and sterility.

Although lyophilization technology has resulted in the development of many successful live, attenuated viral and bacterial vaccines, most of these vaccines still require storage at 2–8 °C or below which necessitates a cold chain for acceptable long term stability. A better understanding of the molecular basis or the origin of the instabilities of viruses and bacteria should result in the more rational design of stable vaccine formulations. In addition, alternative methods of processing such as spray drying may also lead to improved vaccine stability [40].