Collapse Temperature of Freeze-Dried Lactobacillus bulgaricusSuspensions and Protective Media

Abstract

Optimization of the freeze-drying process needs to characterize the physical state of frozen and dried products. A protocol to measure the collapse temperature of complex biological media such as concentrated lactic acid bacteria using freeze-drying microscopy was first elaborated. Afterward, aqueous solutions of one or several components as well as concentrated lactic acid bacterial suspensions were analyzed in order to study how the structure of these materials is degraded during freeze-drying. A similar behavior toward collapse was observed for all aqueous solutions, which was characterized by two temperatures: the "microcollapse" temperature (T(microc), beginning of a local loss of structure) and the "collapse" temperature (T(c), beginning of an overall loss of structure). For aqueous solutions, these two temperatures were close, differing by less than 3 degrees C. Nevertheless, when lactic acid bacteria were added to aqueous solutions, the collapse temperatures increased. Moreover, the interval between microcollapse and collapse temperatures became larger. Lactic acid bacterial cells gave a kind of "robustness" to the freeze-dried product. Finally, comparing glass transition, measured by differential scanning calorimetry (DSC) and collapse temperature for aqueous solutions with noncrystallizable solutes, showed that these values belonged to the same temperature range (differing by less than 5 degrees C). As suggested in the literature, the glass transition temperature can thus be used as a first approximation of the collapse temperature of these media. However, for lactic acid bacterial suspensions, because the difference between collapse and glass transition temperatures was about 10 degrees C, this approximation was not justified. An elegant physical appearance of the dried cakes and an acceptable acidification activity recovery were obtained, when applying operating conditions during freeze-drying in vials that allowed the product temperature to be maintained during primary drying at a level lower than the collapse temperature of lactic acid bacterial suspensions. Consequently, the collapse temperature T(c) was proposed as the maximal product temperature preserving the structure from macroscopic collapse and an acceptable biological activity of cells.

Freeze-Dry Microscopy: Impact of Nucleation Temperature and Excipient Concentration on Collapse Temperature Data

“…Clearly, a great difference between T oc and T fc may be translated into a higher temperature tolerance of the product matrix. For example, Fonseca and coworkers reported in 2004 that a lactic acid bacterial suspensions showed a surprisingly strong bias between T g ′ and T c (up to 10°C) which implied a significant "robustness" of the formulation to temperature during the freeze-drying process (11). A statistical analysis for both PVP 40 kDa and HP-ß-CD showed that T oc and T fc are highly correlated for both datasets, with correlation coefficients of 0.91 for PVP and 0.98 for HP-ß-CD.…”

“…Note that the rational behind the use of a 10°C/min cooling rate in this study is to simply make the experimental time more appropriate for the amount of data required for statistical analysis. Further, a much slower cooling rate was not expected to show a significant difference in the results obtained, based on a recent report which showed for 5% (w/w) aqueous solutions of sucrose no difference in collapse temperature measured by FDM when using a cooling rate of 1°C/min and of 10°C/min (11). These results might be perfectly plausible if the variability of the nucleation temperature during an FDM experiment is taken into consideration, even when using the same sample and identical experimental conditions (i.e., T n data were found exactly in the same temperature range for a 1°C/min and 10°C/min cooling rate) (12).…”

Freeze-Dry Microscopy of Protein/Sugar Mixtures: Drying Behavior, Interpretation of Collapse Temperatures and a Comparison to Corresponding Glass Transition Data

“…Note that most authors refer to this temperature as the ''collapse temperature'' when T c values are published in the literature. 35 Next, a T c-50 value was newly introduced in this work. This parameter is used as a simple way to estimate a temperature where roughly 50% of structural loss in the product is present.…”

Physical Characterization of <i>meso</i>-Erythritol as a Crystalline Bulking Agent for Freeze-Dried Formulations

“…Starting the vacuum drying before sufficient crystal growth resulted in a partially collapsed solid due to a higher product temperature than T c of the freeze-concentrated amorphous erythritol region, as observed in the FDM analysis. [31][32][33] The results indicate that meso-erythritol crystallizes as a stable-form anhydride in the freeze-drying process. Absence of the metastable form and/or hydrate crystals is favorable to prevent physical changes of the excipient or chemical degradation of co-lyophilized APIs during storage.…”