Role of Poloxamer 188 in Preventing Ice-Surface-Induced Protein Destabilization during Freeze–Thawing

Abstract

The phase behavior of poloxamer 188 (P188) in aqueous solutions, characterized by differential scanning calorimetry (DSC) and synchrotron X-ray diffractometry, revealed solute crystallization during both freezing and thawing. Sucrose and trehalose inhibited P188 crystallization during freeze−thawing (FT). While trehalose inhibited P188 crystallization only during cooling, sucrose completely suppressed P188 crystallization during both cooling and heating. Lactate dehydrogenase (LDH) served as a model protein to evaluate the stabilizing effect of P188. The ability of P188, over a concentration range of 0.003−0.800% w/v, to prevent LDH (10 μg/mL) destabilization was evaluated. After five FT cycles, the aggregation behavior (by dynamic light scattering) and activity recovery were evaluated. While LDH alone was sensitive to interfacial stress, P188 at concentrations of ≥0.100% w/v stabilized the protein. However, as the surfactant concentration decreased, protein aggregation after FT increased. The addition of sugar (1.0% w/v; sucrose or trehalose) improved the stabilizing function of P188 at lower concentrations (≤0.010% w/v), possibly due to the inhibition of surfactant crystallization. Based on a comparison with the stabilization effect of polysorbate (both 20 and 80), it was evident that P188 could be a promising alternative surfactant in frozen protein formulations. However, when the surfactant concentration is low, the potential for P188 crystallization and the consequent compromise in its functionality warrant careful consideration.

Solid-State NMR Spectroscopy to Probe State and Phase Transitions in Frozen Solutions

“…For example, hexagonal ice is observed in Figure C, while the ice peaks in the other patterns could not be reliably attributed to a specific physical form of ice. The same problem was reported recently . Despite the lack of reproducibility in ice phase formation, once formed, the ice phase did not exhibit any further transformation upon cooling.…”

“…The same problem was reported recently. 29 Despite the lack of reproducibility in ice phase formation, once formed, the ice phase did not exhibit any further transformation upon cooling. This enabled us to characterize the phase behavior of the solutes in frozen solutions.…”

Dual Functionality of Poloxamer 188 in Freeze-Dried Protein Formulations: A Stabilizer in Frozen Solutions and a Bulking Agent in Lyophiles

“…We had previously observed that upon cooling P188 aqueous solution (4.0% w/v), in addition to ice, the solute had also crystallized. 13 During heating, several thermal events were observed, including (i) glass transition of P188 freeze concentrate (∼−70 °C), (ii) crystallization of P188 and the accompanying unfrozen water (∼−55 °C), and (iii) P188−ice eutectic melting (∼−18 °C).…”

“…11, 12 We had previously documented the ability of P188 to prevent ice- surface-induced protein destabilization. 13 However, P188 crystallization in frozen solutions undermined its stabilization effect. On the other hand, the high crystallization propensity of P188 may bring about an additional functionality as a bulking agent in freeze-dried formulations.…”

Toward Clinical Applications: Transforming Nonantibiotic Antibacterials into Effective Next-Generation Supramolecular Therapeutics

“…33 Therefore, there is a need to evaluate their potential in future studies as alternative excipients in frozen NAA agent formulations and stabilization. Most recently, using lactate dehydrogenase as a model protein, Li et al 40 demonstrated that P188 could be full of promise for frozen NAA agent formulation. In summary, the current evaluations show a promising future for developing powder-based formulations for NAA agents to treat bacterial infections.…”