(M1330-12-77) Dual Functionality of Poloxamer 188 in Freeze Dried Protein Formulations: A Stabilizer in Frozen Solution and a Bulking Agent

Purpose: Poloxamer 188 (P188) is an alternative to polysorbates in preventing ice surface-induced protein denaturation. It has a high crystallization propensity in frozen state, rendering its role as a potential bulking agent in freeze-dried formulations. Multifunctional excipients can potentially reduce the complexity during formulation design and prevent undesirable drug-excipient and excipient-excipient interactions. We hypothesize that P188 can be a dual functional excipient – a cryoprotectant in frozen systems and a bulking agent in freeze-dried formulations. As the crystallization of P188 can potentially undermine its stabilization effect, sugars (sucrose and trehalose) can be added to inhibit its crystallization. However, as the crystallization of P188 can undermine its stabilization effect, the impact of its crystallization on protein stability, both during processing and storage, warrants careful investigation.

Methods: The phase behavior of P188-sugar in frozen aqueous solutions was characterized using differential scanning calorimetry. Lactate dehydrogenase (LDH) was used as the model protein (200 μg/mL). The total solute concentration in the prelyophilzation solution was 3% (w/v). The freeze-drying parameters were: (i) Freezing: -45 °C, 3 h; (ii) Primary drying: -25 °C, 200 mTorr for 12 h; (iii) Secondary drying: 20 °C, 200 mTorr, 6 h; (iv) Annealing (optional): -30 °C, 6 h. P188 crystallinity in the final lyophiles was determined by X-ray diffractometry. Besides, the freeze-dried products were also evaluated based on their appearance, reconstitution time and water content. The LDH stability in the final lyophiles as well as the reconstituted solution was determined.

Results: Sucrose, in a concentration-dependent manner, inhibited P188 crystallization in freeze-dried formulations. However, trehalose did not have such an effect. Annealing did not improve P188 crystallinity in the final lyophiles. Cake collapse was observed in the 1:2 (w/w) P188-sucrose lyophile. All the lyophiles exhibited a rapid reconstitution time of ~ 30 s. The water content of pure P188 cake was < 1% (w/w). The addition of sugar increased the water content to 2% ~ 4% (w/w). Surfactants played a substantial role in preventing ice surface induced LDH destabilization during freeze-drying. The low LDH recovery in pure P188 cake indicated its poor stabilization effect during drying. P188-sugar systems effectively stabilized LDH in its native state, revealing the dual functionality of P188.

Conclusion: P188 could serve as a cryoprotectant as well as a bulking agent in freeze-dried formulations.

Acknowledgements: We acknowledge funding from the Kildsig Center for Pharmaceutical Processing Research and William and Mildred Peters Endowment Fund. The X-ray diffractometry study was carried out in the Characterization Facility, University of Minnesota, which receives partial support from the NSF through the MRSEC (Award Number DMR-2011401) and the NNCI (Award Number ECCS-2025124) programs.

Dual Functionality of Poloxamer 188