(T1230-10-55) The Influence of Combined Deaeration and Polysorbate 80 on IgG Denaturation during Thin-Film Freeze-Drying

Purpose: Freeze-drying processes are widely employed in protein therapeutics production. However, protein denaturation during the freeze-drying process remains a significant concern. The stress induced by interfacial surfaces (i.e., ice/freeze concentrate and air/freeze concentrate) plays a crucial role in protein denaturation during freezing, leading to protein aggregation, potential immunogenicity, and compromised therapeutic effects^1–3. To mitigate protein denaturation, polysorbate 80 (PS80) is commonly used as a cryoprotectant. However, excessive PS80 can cause dry powder stickiness, reducing production yield and altering powder properties^4,5. Additionally, PS80’s oxidation potential poses a risk of destabilizing proteins in formulations^6,7. Therefore, this study aimed to investigate the synergistic cryoprotective effect of deaeration and PS80 in IgG dry powders using the thin-film freeze-drying (TFFD) technique. Furthermore, the impact of PS80 on the aerodynamic performance and surface area of IgG dry powder for inhalation (DPI) produced by TFFD was investigated.

Methods: Sample preparation: Bovine IgG (MP Biomedicals, New Zealand) and PS80 (Fisher Scientific, USA) were dissolved in phosphate-buffered saline at pH 7.1-7.5 (Sigma-Aldrich^®, USA) to obtain the solutions of 1 mg/mL IgG and PS80 concentrations ranging from 0.0025% to 0.0400% w/v. Samples were deaerated by stirring at 100 rpm for 2 hours at 25 °C in a vacuum chamber at 4 kPa (0.04 atm). The dissolved oxygen content was quantified with Milwaukee^® MW600 PRO Dissolved Oxygen Meter (Rocky Mount, USA). The deaerated solutions exhibited approximately 35% dissolved oxygen level. IgG dry powders were prepared using TFFD. IgG solutions were dispensed dropwise onto a cryogenic roller at -100 ± 5 °C, followed by a collection of frozen sample pellets in liquid nitrogen. Subsequently, a lyophilizer was used for drying, with three stages: 1) constant shelf temperature at -40°C for 1200 mins, 2) ramping from -40°C to 25°C over 1200 mins, and 3) maintaining a shelf temperature of 25°C for an additional 1200 mins. IgG monomers and aggregates assessment: The IgG monomer content was quantified using an HPLC system equipped with a TSKgel G3000SWXL column, mobile phase consisted of a sodium phosphate buffer (150 mM, pH 7) with 15 mM NaCl, flowing at 0.7 mL/min. Detection occurred at 220 nm UV wavelength. The soluble aggregates of IgG were assessed using dynamic light scattering (Malvern Analytical, UK) at 173° and presented as the derived count rate (DCR). Surface area analysis: The specific surface area (SSA) of IgG TFFD powders was determined with Monosorb^TM rapid surface area analyzer MS-21 (Quantachrome, USA). After outgassing with helium for 24 hours, the SSA was analyzed using a mixture of nitrogen and helium (30:70 v/v) as an adsorbate. Evaluation of aerodynamic performance: The aerodynamic performance (i.e., fine particle fraction (FPF) and mass median aerodynamic diameter (MMAD)) of IgG TFFD powders was assessed with a next-generation pharmaceutical impactor (MSP Corporation, USA) with a high resistance RS00 inhaler device (Plastiape^®). The Micro BCA^TM Protein Assay Kit (Thermo Scientific, USA) was utilized to quantify IgG deposition at each NGI stage, and FPF and MMAD were calculated using the Copley Inhaler Testing Data Analysis Software (CITDAS).

Results: Synergistic cryoprotective effect: Deaeration reduced the minimum PS80 concentration required to achieve maximum IgG stability (~90% recovered IgG monomer) in TFFD by 0.0025%, from 0.0050% to 0.0025% as observed in SEC (Figure 1), and from 0.0075% to 0.0050% as observed in DLS 173° (Figure 2). This highlights the synergistic impact of deaeration and PS80. PS80 completely prevents IgG aggregation during TFF: The SEC results (Figure 1) and DLS 173° (Figure 2) for both non-deaerated and deaerated samples illustrate that PS80 effectively prevents IgG denaturation during freezing, even at concentrations as low as 0.0025%. However, while the presence of PS80 can decrease denaturation during the drying step, complete prevention of denaturation is not attainable even at 0.0040% concentration. PS80 does not affect the SSA and aerodynamic performance of DPI produced by TFFD: The BET analysis reveals that the SSA (Table 1) of IgG dry powders produced by TFFD remains unchanged (~20 m²/g) across PS80 concentrations ranging from 0% to 0.0400% w/v. Consequently, the aerodynamic performance of TFFD powders containing PS80 (0%-0.0400%) remains consistent with FPF of approximately greater than 70% and MMAD of 2.2-2.5 μm (Table 1).

Conclusion: The combination of deaeration and PS80 demonstrates a synergistic cryoprotective effect, allowing for a decrease in the minimum PS80 concentration required for optimal IgG protection during TFFD. This reduction minimizes the risk of protein instability due to PS80 degradation and mitigates foam formation and stickiness caused by high PS80 concentrations during manufacturing. Although deaeration significantly reduces IgG denaturation in TFFD processes at low PS80 concentrations, its impact diminishes as PS80 concentration increases. Furthermore, PS80 at 0.0025% fully prevents IgG denaturation during the freezing step of TFFD but only partially mitigates aggregation during the drying step. Additionally, PS80 (0.0025%-0.0400%) has no impact on the SSA and aerodynamic performance of IgG DPI produced by TFFD.

References: 1. Schwegman, J. J., Carpenter, J. F. & Nail, S. L. Evidence of partial unfolding of proteins at the ice/freeze-concentrate interface by infrared microscopy. J Pharm Sci 98, 3239–3246 (2009).
2. Strambini, G. B. & Gabellieri, E. Proteins in frozen solutions: evidence of ice-induced partial unfolding. Biophys J 70, 971–976 (1996).
3. Dao, H. M. et al. Entrapment of air microbubbles by ice crystals during freezing exacerbates freeze-induced denaturation of proteins. Int J Pharm 628, 122306 (2022).
4. De Pauw, E. et al. Drying behaviour and visualization of surfactants after co-spray drying of surfactant-stabilized aqueous suspensions. International Journal of Pharmaceutics 643, 123231 (2023).
5. Abdul‐Fattah, A. M., Lechuga‐Ballesteros, D., Kalonia, D. S. & Pikal, M. J. The Impact of Drying Method and Formulation on the Physical Properties and Stability of Methionyl Human Growth Hormone in the Amorphous Solid State. Journal of Pharmaceutical Sciences 97, 163–184 (2008).
6. Weber, J., Buske, J., Mäder, K., Garidel, P. & Diederichs, T. Oxidation of polysorbates – An underestimated degradation pathway? International Journal of Pharmaceutics: X 6, 100202 (2023).
7. Kerwin, B. A. Polysorbates 20 and 80 used in the formulation of protein biotherapeutics: structure and degradation pathways. J Pharm Sci 97, 2924–2935 (2008).

Acknowledgements: Conflicts of Interest: ROW reports financial support by TFF Pharmaceuticals, Inc. The terms have been reviewed and approved by UT Austin in accordance with its institutional policy on objectivity in research. ROW reports a relationship with TFF Pharmaceuticals, Inc. that includes consulting or advising, equity or stocks, and funding grants. ROW has patent(s) and/or patent applications related to thin-film freezing. This work was in part supported by a Sponsored Research Agreement and Technology Validation Agreements from TFF Pharmaceuticals Inc. (to ROW). CM has a consulting or advisory relationship with TFF Pharmaceuticals, Inc. CM has patent(s) and/or patent applications related to thin-film freezing technology, which is licensed to TFF Pharmaceuticals, Inc. from the University of Texas at Austin. SY and HMD are supported by a sponsored research agreement from TFF Pharmaceuticals Inc.

Figure 1. The percentage of IgG monomer recovery after thin-film freeze-drying (TFFD) and thin-film freeze-thawing (TFFT) from size exclusion chromatography (SEC) analysis (n=3). D indicates the deaeration step and ND indicates no deaeration step used to process samples.

Figure 2. The derived count rate (DCR) from dynamic light scattering (DLS) analysis at 173° of 1 mg/mL IgG samples (n=3). Thin-film freeze-drying (TFFD) and thin-film freeze-thawing (TFFT) are plotted with D (indicating deaeration) or ND (indicating no deaeration).

Table 1. The specific surface area (SSA) and aerodynamic performance of IgG dry powders produced by thin-film freeze-drying (TFFD) and spray drying (SD) (n=3).