Developing a safe lyophilised adjuvant vaccine

There are a variety of vaccine types, each varying in safety and efficacy (Table 1), and each possessing its own formulation challenges. To overcome potential instabilities when developing vaccines, one formulation strategy is to produce a dried product. Lyophilisation (freeze drying) removes the water content and maintains the vaccine in a dried state until it is reconstituted when required for vaccination.

Although live and inactivated vaccines can be freeze dried, each micro‑organism or virus varies markedly in terms of its sensitivity to the lyophilisation process. Gram-positive bacteria and spores exhibit a high survival rate after undergoing the process, whereas gram-negative bacteria exhibit poor levels of survival. The type and state of the bacteria can have a profound impact on vaccine stability.

Because of the possible side effects and associated toxicities, it can be difficult to obtain approval for live attenuated vaccines, or even for dead whole cell vaccines, because the overall safety of the vaccine is more important than efficacy — especially if the efficacy of safe sub‑unit vaccines can be enhanced through the application of an adjuvant system.

Freeze‑drying liposomal adjuvants
Liposomal adjuvants are spherical bilayer constructs manufactured from lipid molecules. As a pharmaceutical product, the liposomal formulation should be stable over a long period of time and possess a relatively long shelf‑life. However, many liposomal formulations exhibit inherent physical and chemical instabilities during storage; for example, aqueous formulations tend to be thermodynamically unstable because the presence of water surrounding the lipid preparations cause lipid oxidation and hydrolytic degradation.¹ These instabilities result in vesicle aggregation or fusion and potential loss of antigen, which leads to undesirable alterations of the final vaccine.An ideal approach to overcome these associated problems is to develop a sterile, freeze‑dried liposome product, whereby water is removed from the system to produce a dry lipid vaccine product that can be reconstituted when vaccination is required. The key attributes of a successful liposomal vaccine can be analysed and determined by:

• excipients (i.e., lipids and antigen)
• freeze‑dried product — water content and long‑term stability

• characteristics of the final reconstituted product used for vaccination, including size, surface charge, antigen content, antigen release kinetics and overall biological activity.

During freeze drying, with the removal of the surrounding aqueous solution, in the absence of a lyoprotectant and with a phase transition from the gel to liquid state, the lipid bilayer is disrupted and the vesicles fuse and aggregate. This creates larger liposomal structures after reconstitution compared with before (e.g., small unilamellar vesicles), as illustrated in Figure 1. This destabilising effect can be positively applied to effectively entrap antigens within liposomal vesicles — a technique known as the dehydration–rehydration procedure.²

For the development of a freeze‑dried adjuvant vaccine product, however, the liposomal preparation should maintain the original characteristics. Lyoprotectants, such as saccharides (e.g., sucrose or trehalose) or amino acids (e.g., lysine),¹ can be added to the formulation prior to the freeze‑drying process as they act as a protective barrier, immobilising the lipid membrane and reducing membrane–membrane fusion. Consequently, this stabilises and maintains the characteristics of the final product and, most importantly, preserves the vaccine’s immunogenicity.

Disaccharides are the preferred option compared with monosaccharides because they are non‑reducing sugars; thus avoiding damage to the dried product by Maillard reactions. Including disaccharides is said to maintain the lipid bilayer membranes in a liquid crystalline state upon rehydration, preventing phase transitions occurring from the gel to liquid phase. The disaccharides interact with the polar groups of the phospholipids by hydrogen bonds and stabilise the vesicles during freeze drying.³

Moisture must be maintained
It is mandatory to state the water content of freeze‑dried vaccines distributed for clinical use because high‑moisture content can lead to poor stability. However, as demonstrated by a case study examining the stability of a freeze‑dried influenza A virus vaccine, both over drying and under drying can compromise vaccine stability, and reduce the efficacy of the vaccine.⁴Moisture content can be analysed by various methods, including Karl Fischer titration, thermogravimetric and gas chromatography. To maintain the level of moisture stated on the vaccine product, the vials should be sealed using chemically inert gases, such as argon, and the sealing integrity should be confirmed on all batches of vaccines issued for clinical use.

When developing a new freeze‑dried adjuvant vaccine, each stage throughout the process needs to be optimised — from formulation through to freeze drying and reconstitution. Each system is different from the next and not all excipients are universal protectors.

References
1. A.R. Mohammed, A.G.A. Coombes and Y. Perrie, Eur. J. Pharm. Sci., 30(5), 406–413 (2007).
2. C. Kirby and G. Gregoriadis, Biotechnology, 2, 979–984 (1984).
3. J.H. Crowe et al., Biochimica et Biophysica Acta (BBA) — Reviews on Biomembranes, 947(2), 367–384 (1988).
4. P.R.W. Baker, Epidemiology and Infection, 53(04), 426–435 (1955).

Based on a contribution by Sarah E. McNeil, Senior Research Fellow, and Yvonne Perrie, Professor in Pharmaceutics and Drug Delivery, both from Aston Pharmacy School (UK).