Freezing-induced protein aggregation - Role of pH shift and potential mitigation strategies.

The aggregation behavior of two model proteins- i) bovine serum albumin (BSA) and ii) ß-galactosidase (ß-gal), was investigated by micro-flow imaging (MFI) during freeze-thaw cycling in phosphate buffered solutions. The pH shift was measured upon cooling the solutions from 20 to -25 °C. When the buffer concentration was 100 mM, cooling caused a pH decrease of 3.1 and 2.7 units (for BSA and ß-gal, respectively) attributed to selective crystallization of disodium hydrogen phosphate as a dodecahydrate. The crystallizing solute phase was characterized by low temperature powder X-ray diffractometry. The pH shift resulted in protein aggregation, evident from the pronounced increase in particle count (by MFI). The addition of cellobiose attenuated the pH shift on cooling (pH decrease of ~1.0 unit), and no evidence of either buffer salt crystallization or protein aggregation was observed. Decreasing the buffer concentration to 10 mM, also prevented protein aggregation. The protein, by inhibiting buffer crystallization, prevented the pH shift and then the buffer, by maintaining the pH, enhanced protein stability.

Characterization of Phosphate Buffered Saline (PBS) in Frozen State and after Freeze-Drying.

PURPOSE: To study the effect of mannitol or trehalose on the crystallization behavior of solutes in phosphate buffered saline (PBS) when the solutions were frozen and freeze-dried. METHODS: PBS (pH 7.5 at RT) either alone, or with trehalose (5% w/v) or mannitol (1% w/v), were frozen and characterized using low temperature differential scanning calorimetry (DSC), X-ray diffractometry (XRD), and pH measurement. Freeze dried lyophiles were characterized by XRD. RESULTS: In the absence of cosolutes, upon freezing PBS, a pH shift of ~ 4 units was observed due to crystallization of Na2HPO4•12H2O. XRD indicated sequential crystallization of Na2HPO4•12H2O, NaCl•2H2O and KCl during cooling. When the frozen solutions were heated, two eutectics were observed - the first at ~ -24°C (ternary, NaCl•2H2O-KCl-ice) and the second at ~ -22°C (binary, NaCl•2H2O-ice). Trehalose completely inhibited buffer salt crystallization, whereas mannitol suppressed it partially thereby attenuating the magnitude of pH shift. The two eutectic meltings were also suppressed by the cosolutes. XRD of final lyophiles from PBS alone revealed peaks of anhydrous Na2HPO4, NaCl, and KCl. Trehalose rendered the lyophiles completely XRD amorphous, whereas in presence of mannitol, all the solutes except KH2PO4 crystallized. CONCLUSIONS: Freezing of PBS solution caused a pronounced pH shift due to selective crystallization of Na2HPO4•12H2O. The addition of trehalose or mannitol suppressed the buffer salt crystallization and attenuated the magnitude of pH shift. The potential instability of biologics due to pH shift in PBS, can be potentially mitigated with the cosolutes.

Effects of Mono-, Di-, and Tri-Saccharides on the Stability and Crystallization of Amorphous Sucrose.

Amorphous sucrose is a component of many food products but is prone to crystallize over time, thereby altering product quality and limiting shelf-life. A systematic investigation was conducted to determine the effects of two monosaccharides (glucose and fructose), five disaccharides (lactose, maltose, trehalose, isomaltulose, and cellobiose), and two trisaccharides (maltotriose and raffinose) on the stability of amorphous sucrose in lyophilized two-component sucrose-saccharide blends exposed to different relative humidity (RH) and temperature environmental conditions relevant for food product storage. Analyses included X-ray diffraction, differential scanning calorimetry, microscopy, and moisture content determination, as well as crystal structure overlays. All lyophiles were initially amorphous, but during storage the presence of an additional saccharide tended to delay sucrose crystallization. All samples remained amorphous when stored at 11% and 23% RH at 22 °C, but increasing the RH to 33% RH and/or increasing the temperature to 40 °C resulted in variations in crystallization onset times. Monosaccharide additives were less effective sucrose crystallization inhibitors relative to di- and tri-saccharides. Within the group of di- and tri-saccharides, effectiveness depended on the specific saccharide added, and no clear trends were observed with saccharide molecular weight and other commonly studied factors such as system glass transition temperature. Molecular level interactions, as evident in crystal structure overlays of the added saccharides and sucrose and morphological differences in crystals formed, appeared to contribute to the effectiveness of a di- or tri-saccharide in delaying sucrose crystallization. In conclusion, several di- and tri-saccharides show promise for use as additives to delay the crystallization kinetics of amorphous sucrose during storage at moderate temperatures and low RH conditions. PRACTICAL APPLICATION: Amorphous sucrose is desirable in a variety of food products, wherein crystallization can be problematic for texture and shelf-life. This study documents how different mono-, di-, and tri-saccharides influence the crystallization of sucrose. Monosaccharide additives were less effective sucrose crystallization inhibitors relative to di- and tri-saccharides. These findings increase the understanding of how different mono-, di-, and tri-saccharide structures and their solid-state properties influence the crystallization of amorphous sucrose and show that several di- and tri-saccharides have potential for use as sucrose crystallization inhibitors.

Effects of Chloride and Sulfate Salts on the Inhibition or Promotion of Sucrose Crystallization in Initially Amorphous Sucrose-Salt Blends.

The effects of salts on the stability of amorphous sucrose and its crystallization in different environments were investigated. Chloride (LiCl, NaCl, KCl, MgCl2, CaCl2, CuCl2, FeCl2, FeCl3, and AlCl3) and sulfate salts with the same cations (Na2SO4, K2SO4, MgSO4, CuSO4, Fe(II)SO4, and Fe(III)SO4) were studied. Samples (sucrose controls and sucrose:salt 1:0.1 molar ratios) were lyophilized, stored in controlled temperature and relative humidity (RH) conditions, and monitored for one month using X-ray diffraction. Samples were also analyzed by differential scanning calorimetry, microscopy, and moisture sorption techniques. All lyophiles were initially amorphous, but during storage the presence of a salt had a variable impact on sucrose crystallization. While all samples remained amorphous when stored at 11 and 23% RH at 25 °C, increasing the RH to 33 and 40% RH resulted in variations in crystallization onset times. The recrystallization time generally followed the order monovalent cations < sucrose < divalent cations < trivalent cations. The presence of a salt typically increased water sorption as compared to sucrose alone when stored at the same RH; however, anticrystallization effects were observed for sucrose combined with salts containing di- and trivalent cations in spite of the increased water content. The cation valency and hydration number played a major role in dictating the impact of the added salt on sucrose crystallization.

Ultrasound-assisted modulation of concomitant polymorphism of curcumin during liquid antisolvent precipitation.

Curcumin polymorphs were found to precipitate concomitantly during liquid antisolvent precipitation. While, commercially available curcumin exists in a monoclinic form, the curcumin particles when precipitated in presence of additives and ultrasound were either found to be the mixtures of orthorhombic (Form 3) and monoclinic form (Form 1) or were found to be in orthorhombic form (Form 3) or monoclinic form (Form 1). The experimentally observed particle morphologies did not match clearly with the predicted BFDH morphologies of curcumin and the experimentally observed morphologies were more elongated as compared to the predicted BFDH morphologies. At lower ultrasonic irradiation times, the monoclinic form (Form 1) was found to dominate the mixture of particles. However, an increase in ultrasonic irradiation time was found to increase the percentage of orthorhombic form (Form 3) in the particles indicating that the increase in ultrasonic energy facilitates formation of orthorhombic form over the monoclinic form, irrespective of the additive used. These results therefore suggest that the ultrasonic energy can be effectively used to manipulate the polymorphic outcome of the precipitation.

Simple criterion for stability of aqueous suspensions of ultrafine particles of a poorly water soluble drug.

In this work, a simple criterion is proposed for prediction of a long-term stability of aqueous suspensions of ultrafine particles of a poorly water soluble drug, curcumin. A new "stability parameter" (γ0ε/γε0) has been defined, which is a ratio of nondimensional mechanical (mainly ultrasonic) energy (ε/ε0) to nondimensional solid-liquid interfacial energy (γ/γ0). The stability of aqueous suspensions of curcumin particles over a period of 1 year and 9 months has been correlated with this parameter. In order to calculate this parameter, solid-liquid interfacial energies were first estimated, from nucleation rates, which in turn were calculated from size distributions of curcumin particles precipitated using water as antisolvent. The mechanical energy was then estimated from the intensity of ultrasound and mechanical agitation used during precipitation. It was found that precipitations carried out with higher values of γ0ε/γε0 (more than 100) result in aqueous suspensions with particle size less than 1 µm. It was further observed that these suspensions remain stable (i.e., no or negligible change in average particle size) for a period of 1 year and 9 months. On the other hand, the suspensions of particles precipitated at lower values of γ0ε/γε0 (less than 10) were found to be highly unstable (i.e., the average particle size changes drastically). These results suggest that γ0ε/γε0 can be used as a parameter to engineer stable aqueous suspensions of curcumin particles. Further, it was found that the use of the Mersmann equation to estimate solid-liquid interfacial surface tensions can help in making this criterion predictive.