Several processing methods were evaluated to stabilize Ty21a, including freeze drying and spray drying, prior to developing the foam drying process. A commercial scale freeze drying process for Ty21a was developed by Clarke et al. in which a shelf-life of 18–24 months at 2–8°C was reported [32
]. However, at elevated temperatures, the vaccine potency of such freeze dried product decreased dramatically; e.g. the shelf-life decreased to several months or to several days, for storage at room temperature or 37°C respectively [15
]. Initial efforts to develop a stable lyophilized preparation of Ty21a from a variety of formulations resulted in similar storage stability; 0.8log10
process-associated loss and titer loss of 0.5log10
following 1 week of storage at 25°C (). Although the exact mechanism of Ty21a inactivation is unknown, the various processing methods were expected to result in differing process recovery, as the stresses involved in desiccation vary widely. For freeze drying, low temperature stress, ice crystal formation, and phase separation have been suggested to be significant contributors to inactivation of a wide range of biomolecules [44
], while for spray drying, high temperature stress and shear stress (i.e., atomization of liquid feed into droplets) are two of the most significant contributing factors leading to inactivation [47
]. Furthermore, the time scale involved for dehydration is significantly different for these processes; freeze drying requires several days whereas spray drying is completed on a millisecond time scale [50
]. Thus, both the rate of water removal and the thermal history of the biomolecule during processing are quite different and are expected to lead to differing results.
Research has led to a spray drying method that effectively stabilizes Mycobacterium smegmatis
]. Specifically, salts and other traditional cryoprotectants, such as glycerol, were removed from the formulation which was then air dried. Contrary to expectations, the activity of the recovered bacteria was improved by up to 2log10
. The same strategy was utilized with Ty21a, however, neither the process loss nor the storage stability was as high as those reported by Wong et al. [34
]; the process loss for Ty21a formulated in un-buffered solution containing 7% (w/v) sucrose was 0.7log10
and demonstrated loss in titer of 1.7log10
CFU following 1 week of storage at 25°C ().
Compared to the two processes described above, foam drying is conducted at a moderate rate of water removal (process complete in hours to days). Furthermore, the entire process is conducted at a mild temperature, generally in the range of 15–25°C. The foaming process reported here is modestly different from that originally developed in the 1960s by Annear for use in the preservation of vaccines and bacteria [54
]. The overall method involves boiling, or foaming, of the solution under lowered vapor pressure followed by rapid evaporation, leaving a structure resembling solidified foam (). The temperature is carefully controlled so as to avoid freezing due to evaporative cooling. More recently, the foam drying method has been adapted to be conducted under both non-freezing [21
] and freezing [24
] conditions followed by rapid evaporation and/or sublimation of water. Foam drying is conducted using a conventional freeze dryer and the process can be thought of as a freeze drying method conducted under a cake collapse condition. It should be noted, however, that it is difficult to directly compare various processes because different formulation components may be required for the processes being compared and optimization can be conducted to varying degrees. For the purpose of this study, a rigorous process comparison was not conducted as the focus has been on foam drying process and formulation development, based on publications which reported superiority of this dehydration technique over freeze drying and spray drying [35
The foam drying cycle was first optimized to minimize process loss. For processing methods in which the system pressure is decreased too quickly, the solution has a greater tendency to freeze and will not foam effectively. Conversely, foaming will be inhibited by decreasing the pressure too slowly, with the sample solution resembling a slurry. Examination of activity loss during processing and the water content of the samples suggest an inverse relationship between the residual water content and the remaining activity of Ty21a (). It is evident that the majority of activity loss as well as water removal was completed within the first 100min of processing. A similar study using other process cycles suggested that the rate of systemic pressure decrease, particularly during the early stages of foam drying, has a significant effect on the viability of Ty21a. By decreasing the systemic pressure in a stepwise manner, the process-associated loss was decreased from 0.8 to 0.3log10
. This suggests that the rate of water removal during the initial stages of the foam drying cycle (i.e., the rate at which the physical structure of the sample is set prior to desiccation), is crucial in determining the activity Ty21a. This may be related to the increase in solute concentration encountered during dehydration and the associated changes in osmotic stress across the bacterial membrane. The rate of water desorption is expected to be slower for foam dried material in relation to a similar formulation processed by freeze drying, due to the lower specific surface area of the former [35
]. Thus, longer secondary drying process may be required to reduce the residual water content to similar levels as achieved by freeze drying.
Several groups have suggested that growth and harvesting conditions also have a significant effect on the physical stability of bacteria [55
]. Sodium chloride at various concentrations was incorporated in BHI broth to determine its effect on both the growth kinetics of Ty21a as well as its development of resistance to osmotic stress. In order to reach the same optical density (OD600nm
~ 1.6, corresponding to the stationary phase), Ty21a required incubation for 8 hours at 37°C in the presence of 400mM NaCl, compared to 4 hours in its absence. Thus, there appears to be no benefit of including osmotic stress-inducing agents in the growth media, at least from the perspective of growth kinetics. However, an improvement in desiccation tolerance was observed for Ty21a grown in the presence of salts (). Ty21a grown in the presence of 300mM NaCl demonstrated much improved process recovery compared to those grown in the absence of salt, <0.1log10
loss in titer, respectively. However, this effect was observed only for bacteria harvested in the stationary phase. For Ty21a harvested in the log phase, there was little-to-no effect of salt addition on the process recovery of bacteria following foam drying. Similar improvement in process recovery of Ty21a from the change in harvesting time to stationary phase was also noted in the absence of salt (). Thus, both the presence of osmolytes and the growth stage of bacteria appeared to play a role in determining the desiccation tolerance of bacteria. The increased osmolarity of the growth media may allow Ty21a to develop tolerance to osmotic stress, which would be encountered during desiccation. The higher titer of bacteria at stationary phase may benefit from a similar self-protective mechanism to desiccation encountered by proteins at high concentrations [59
Zeng et al. [40
] reported that Ty21a stability was improved at pH6 – 7 compared to that prepared at pH8. This enhanced stability was attributed to the difference in membrane fluidity. In the current study, both the process-associated loss and storage stability was poorer for Ty21a samples foam dried at pH6 compared to that prepared at pH8 (). Zeng presented stability conditions for Ty21a in the liquid phase, whereas the data presented here relates to stability in the dried state. The maintenance of membrane fluidity and distribution has been attributed to have an effect on the stability of a model membrane system, i.e., liposomes (i.e., leakage of encapsulated compounds) [60
]. Similarly, the avoidance of phase transition and phase separation of various membrane components may have an effect on Ty21a stability during desiccation. The inclusion of trehalose, as presented in this work, would be expected to lower the phase transition temperature of the bacterial membrane, as was reported for a variety of phospholipidic components [63
], and allow the membrane to remain in the fluid phase throughout the foam drying process as well as during reconstitution (i.e., thus avoiding the occurrence of phase transition). The formation of an amorphous glass during desiccation has also been demonstrated to resist the phase separation of membrane components, which would have occurred in the absence of sugars, perhaps contributing to the enhanced stability of Ty21a [66
]. Furthermore, the pH of the external media may affect the stability of Ty21a through the alteration of charge distribution on the bacterial membrane, as has been reported for liposomes [60
]. In addition to stabilizing the lipid components of the bacterial membrane, the membrane proteins also require stabilization, as has been demonstrated by the addition of trehalose to E. coli
and B. thuringiensis
Several amino acids, such as arginine, phenylalanine, and glycine, have been successfully used to stabilize a wide variety of complex biomolecules [69
]. In this study, methionine was demonstrated to be an effective stabilizer. The amino acid was incorporated into a trehalose-potassium phosphate formulation at pH8 in a concentration range of 0.5–2% (w/v). Optimum stability was observed with 0.5% methionine, and decreased at higher concentrations (). Typically, methionine has been employed as an antioxidant, as is the case for NovoSeven® RT, recombinant human coagulation factor VIIa, that demonstrates room temperature stability for up to 2 years [74
]. Similarly, in the present case, methionine may be acting as an antioxidant, but, there may be other contributing factors.
Plasticizers, such as glycerol and DMSO, have been demonstrated to improve the storage stability of various proteins and enzymes even further than that possible by sugars alone [42
]. Typically, the use of plasticizers has been avoided due to its effect on lowering the glass transition temperature (Tg
) of the dried composition. On a global scale (i.e., translational motion), this may appear to be the overriding factor determining the stability of the biomolecule. However, locally (i.e., vibrational and rotational motion), plasticizers have been demonstrated to reduce molecular mobility by dampening the high frequency motion [43
]. It is this effect on the local motion, to which the authors attribute the enhanced storage stability. Both DMSO and glycerol were incorporated into the foam dried Ty21a formulation, but their effects on storage stability were quite different. While increasing glycerol composition resulted in decreased storage stability, DMSO demonstrated an optimal concentration at 1wt% (). Furthermore, the storage stability of foam dried Ty21a was better in formulations containing DMSO in comparison to glycerol at equal composition. For both plasticizers, the process loss associated with foam drying increased with higher plasticizer concentrations. Thus, the amount of plasticizer needs to be optimized to balance the titer loss associated with processing and subsequent storage.
The solids content of the formulation is an important parameter that requires optimization for efficient foaming. One key component is the viscosity of the solution, which can be enhanced through the incorporation of proteins or polymers. Furthermore, these large molecules can enhance the stability of labile biomolecules through their ability to interact, reduce aggregation, and to elevate the Tg
of the dried composition [75
]. Addition of 5% gelatin to methionine-trehalose formulation resulted in improved storage stability of foam dried Ty21a, reducing the rate of titer decrease from −1.06 to −0.27log10
(). Similarly, the storage stability was improved for plasticizer-containing formulations. Stabilization conferred to foam dried Ty21a by the individual components (i.e., methionine, glycerol, and DMSO) appears to be enhanced upon the addition of gelatin, perhaps through a complementary stabilization mechanism. The formulation that stabilized Ty21a the most in the foam dried format contained gelatin, methionine, and trehalose, demonstrating ~12 weeks of storage stability at 37°C. At lower storage temperatures, the foam dried sample demonstrated no loss in titer following 4 months of storage at 25 and 4°C. Other formulations of foam-dried Ty21a (not reported here) have been studied over 12–18 months real time for stability and have been found to have a projected shelf-life of >5 years at 4°C, stability at 25°C for 12 months and allowable temperature excursions to 37°C for 8 weeks. Another aspect of stability relates to the maintenance of vaccine titer during processing of the foam dried material, e.g. by jet milling, into powders. In fact, very insignificant loss in titer (< 0.2log10
) has been observed (data not shown).
Though not reported here, several other compounds were screened for their ability to stabilize Ty21a during foam drying and subsequent storage as well as to enhance the foam drying process. One class of compounds that was examined in detail was surfactants. Surfactants are very effective in enhancing the foaming process as well as in minimizing inactivation at the air-liquid interface. However, care must be taken to optimize their amount since surfactants can solubilize lipidic components, thus breaching the integrity of bacterial membrane, leading to decreased titer. Pluronic F68, a relatively mild non-ionic surfactant, was incorporated at concentrations ranging from 0.02–0.2wt% to the methionine-gelatin-trehalose formulation. For all cases, the storage stability was compromised, but their inclusion did facilitate the foaming process (i.e., foaming occurred at higher systemic pressure).
Finally, the ability of the foam dried Ty21a to elicit an immunogenic response was examined in mice. As illustrated in , mice vaccinated with the foam dried Ty21a (by both intranasal and intraperitoneal administration) elicited an enhanced level of anti-LPS IgG titers when compared to those for the unformulated vaccine as well as for Vivotif™, the commercial vaccine product. This demonstrates that not only does foam drying preserve the physical stability of Ty21a, but may enhance its immunogenic activity (i.e., potency).