# Explorative Supercooling Technology for Prevention of Freeze Damages in Vaccines
## 3 Results and Discussion  
Supercooling operating parameters such as field strength, frequency, duty cycles, and sample volumes (10 mL as a default vial volume) were tested and optimized. It was previously found that the probability of supercooling over long periods was significantly increased with OMF intensities of 50 mT. Therefore, OMF strength was optimized at 50 mT, 60 V, and 1 Hz with a 1 s on/off cycle. It should be noted the Food and Drug Administration (FDA) in 1987 limited magnetic field magnitudes on biomaterials at a maximum of 2000 mT, while our projected OMF strength was exercised at 50 mT [23]. Figure 3 shows that the supercooled vaccine was successfully stored at \(-\)14 \({}^{\circ}\)C, and the negative control without OMF treatment experienced the phase transition at 2.5 h into the trial and froze. Temperature profiles for other control vaccine samples (freezer at \(-\)20 \({}^{\circ}\)C and refrigerator at 7 \({}^{\circ}\)C) showed unremarkably expected results, with samples freezing in the freezer and remaining unfrozen in the refrigerator. The shake test was designed based on the apparent differences in sedimentation rates of previously frozen and unfrozen vaccines to understand whether freeze-sensitive vaccines are damaged by freezing. Figure 4 shows the appearance of sample vials 3 min after they were vigorously shaken; these vials were previously stored at four different conditions for 24 h. Test vials (a) and (b) were stored at \(-\)20 \({}^{\circ}\)C and \(-\)14 \({}^{\circ}\)C, freezing in their respective storage units and then thawed before commencing the shake tests. Vials (c) and (d) were left unfrozen in their respective conditions, with vial (d) being subject to supercooling treatment during the 24 h. Sedimentation in frozen-then-thawed vaccines was quick; a definite, clear supernatant formed gradually above a white disk of coagulates at the bottom. However, much similar to the refrigerated test vaccine, the supercooled vials showed no sedimentation and remained a cloudy, homogeneous mixture after the shaking. It is known that ice crystals formed during freezing force aluminum particles  
Figure 3: Temperature profiles of supercooled and control vaccines. The control vaccine sample (without OMF treatment) was frozen after 2.5 h of storage as indicated by the temperature overshoot. In general, as the temperature of the sample is lowered below its freezing point, ice nucleation occurs. This phase change causes the release of latent heat with the freezing point being revealed. Once all latent heat has been released, the temperature of the sample continues to decline until it matches the temperature of its ambient surroundings. to overcome repulsion, thereby producing strong interparticle attraction. This ultimately results in aluminum particle coagulation and agglomeration [10]. Consequently, these particles become heavier and sediment faster in their respective suspensions. Figure 5 shows microscopic snapshots of supercooled vaccines vs. other controls under light microscopy. Freeze-damaged vaccine samples (i.e., frozen (\(-20\)\({}^{\circ}\)C) and frozen (\(-14\)\({}^{\circ}\)C)) appeared to consist of large conglomerates of massed precipitates with amorphous, crystalline, solid, and needlelike structures; however, the supercooled vaccine showed fine grain structures (in both \(10\times\) and \(40\times\) magnifications), which is identical to vaccine stored under the refrigeration (\(7\)\({}^{\circ}\)C). Aggregates in frozen vaccines measured up to \(500\)\(\upmu\)m and \(350\)\(\upmu\)m on average. Particles in undamaged TT vaccine samples were measured at 2 and \(20\)\(\upmu\)m. The concordance in establishing the status of a TT vaccine as frozen or non-frozen was \(100\%\) between the light microscopy and the shake test method. Therefore, the vaccines kept supercooled at \(-14\)\({}^{\circ}\)C were not freeze-damaged and most likely retained their full potency and immunogenicity during storage.