(T1130-03-16) Deaeration Enhances the Cryoprotective Effect of Tween 80 on Denaturation of Bovine IgG during Thin-Film Freezing

Purpose: The objective of this study is to enhance the stability of therapeutic proteins in aqueous preparations, freeze-drying has been employed as a manufacturing method¹. However, this process can induce protein denaturation^2-6. Dao et al. showed that faster cooling rates increased formation of air microbubbles compared to slower cooling rates, and removing these air microbubbles can substantially reduce protein denaturation⁶. Tween 80, a nonionic surfactant, can compete with protein for binding to the ice-water interface^3,5,7 and potentially the air-water interface of the microbubbles during freezing, thereby improving protein stability. However, high content of Tween 80, a viscous liquid at 25°C⁸, alters the physical properties of the powders such as: decreasing aerodynamic performance, flowability and brittleness. This study aimed to investigate the effect of Tween 80 and deaeration to provide sufficient protection for the IgG model protein, while maintaining high aerosol performance of the resulting powder.

Methods: Sample preparation: Bovine IgG (MP Biomedicals, New Zealand) solutions 1 mg/mL with 0.02-0.24%w/v of Tween 80 (Fisher Scientific, USA) in phosphate buffer (Signa-Aldrich®, Germany) pH 7.4 were prepared and divided into two groups. One group was subjected to freeze-drying with deaeration and the other without deaeration. Deaeration process: The sample solutions were placed in a vacuum chamber followed by stepwise reduction of air pressure to 4 KPa to prevent formation of air-bubbles and boiling of water. Samples were kept at 4 KPa for 2 hours and only taken out of vacuum chamber immediately before processing by thin-film freezing. Thin-film freeze-drying: The sample solutions were dropped onto the freezing rotating drum (150 RPM) at -100± 10°C. The frozen samples were collected in liquid nitrogen and lyophilized as follows: (1) the shelf temperature was held at -40°C for 1200 minutes at 100 mTorr, (2) ramped up to 25°C at 100 mTorr, (3) and held at 25°C, 100 mTorr for 1200 minutes. Analytical testing: The small and large soluble aggregate formation was determined by Dynamic light scattering (DLS) Zetasizer Nano-ZS (Malvern analytical, UK) at 173° and 13° scattering angles, respectively. The number of subvisible aggregates was analyzed by micro-flow imaging (MFI) MFI^TM 5100 (ProteinSimple, USA). The specific surface area of dried powders was measured with single-point Braummer-Emmett-Teller (BET) method by Monosorb^TM rapid surface area analyzer (Quantachrome, USA).

Results: Micro-flow imaging (MFI): The MFI results (Fig.1) revealed the expected positive impact of Tween 80 on the total aggregate count of IgG. Increasing the concentration of Tween 80 from 0.02% to 0.04% resulted in a substantial reduction of 94% and 96% in total aggregate count for the non-deaerated and deaerated samples, respectively. However, Tween 80 concentration beyond 0.04% did not provide a significant effect on the total aggregate count for either sample groups. Regarding the effect of deaeration, at 0.02% of Tween 80, deaeration caused a 13% reduction in total aggregate count compared to samples without deaeration. Dynamic light scattering (DLS): The derived count rate (DCR) results (Fig.2) from DLS analysis agreed with the MFI findings. In the non-deaerated samples, there was a significant reduction in DCR as the concentration of Tween 80 was increased. Specifically, increasing Tween 80 concentration from 0.02% to 0.04% resulted in a 70% decrease in DCR at 173^o and a substantial 97% decrease at 13^o. Similarly, in the deaerated group, a comparable reduction of 62% in DCR at 173^o and 86% in at 13^o was observed. However, beyond 0.04% of Tween 80, no further reduction in the DCR was observed. With respect to the impact of deaeration, at a concentration of 0.02% Tween 80, deaeration led to a 43% reduction in DCR at 173^o and a 78% reduction at 13^o compared to samples without deaeration. Specific surface area (SSA): Increasing concentration of Tween 80 led to a significant decrease in the SSA of the non-deaerated group (Fig.3). Furthermore, Fig.3 shows a significant reduction of SSA of the deaerated group at 0.02 and 0.04% Tween 80 compared to non-deaerated group by 30% and 22%, respectively. The decrease in SSA of the deaerated group at 0.02% and 0.04% Tween 80 agreed well with the results from MFI and DLS.

Conclusion: Deaeration reduced formation of air microbubbles during the freezing process, which led to a decrease in the air-water interfacial surface area, resulting in lower SSA of the dried powders. With a reduced air/water interface for proteins to adsorb, total particle count and DCR were decreased. However, above 0.04%w/v, there is a high probability of micelle formation that could trap air microbubbles leading to the observed lower SSA and correspondingly low aggregation as observed with MFI and DLS of both non-deaerated and deaerated groups. Our data suggests that the accumulation of Tween 80 at the air/water interface can improve the stability of protein. Therefore, reducing air/water interface of air microbubbles by deaeration can improve the efficiency of Tween 80 to protect IgG from denaturation, allowing it to be used at lower levels.

References: [1] Schiffter, H. A., 2011, Pharmaceutical proteins – structure, stability, and formulation: Comprehensive Biotechnology, p. 521–541, doi:10.1016/b978-0-08-088504-9.00468-2.
[2] Chirmule, N., V. Jawa, and B. Meibohm, 2012, Immunogenicity to therapeutic proteins: Impact on PK/PD and efficacy: The AAPS Journal, v. 14, no. 2, p. 296–302, doi:10.1208/s12248-012-9340-y.
[3] Sane, R., and M. Sinz, 2017, Introduction of drug metabolism and overview of disease effect on Drug Metabolism: Drug Metabolism in Diseases, p. 1–19, doi:10.1016/b978-0-12-802949-7.00001-8.
[4] Bhatnagar, B. S., R. H. Bogner, and M. J. Pikal, 2007, Protein stability during freezing: Separation of stresses and mechanisms of protein stabilization: Pharmaceutical Development and Technology, v. 12, no. 5, p. 505–523, doi:10.1080/10837450701481157.
[5] Arsiccio, A., J. McCarty, R. Pisano, and J.-E. Shea, 2020, Heightened cold-denaturation of proteins at the ice–water interface: Journal of the American Chemical Society, v. 142, no. 12, p. 5722–5730, doi:10.1021/jacs.9b13454.
[6] Dao, H. M., S. Sahakijpijarn, R. Chrostowski, H.-H. Peng, C. Moon, H. Xu, F. Mangolini, H. H. Do, Z. Cui, and R. O. Williams, 2022, Entrapment of air microbubbles by ice crystals during freezing exacerbates freeze-induced denaturation of proteins: International Journal of Pharmaceutics, v. 628, p. 122306, doi:10.1016/j.ijpharm.2022.122306.
[7] Wang, Z., L. Li, G. Ren, X. Duan, J. Guo, W. Liu, Y. Ang, L. Zhu, and X. Ren, 2022, A comprehensive review on stability of therapeutic proteins treated by freeze-drying: Induced stresses and stabilization mechanisms involved in processing: Drying Technology, v. 40, no. 16, p. 3373–3388, doi:10.1080/07373937.2022.2048847.
[8] National Center for Biotechnology Information, 2023, PubChem Compound Summary for, Polysorbate 80. Retrieved July 16, 2023 from https://pubchem.ncbi.nlm.nih.gov/compound/Polysorbate-80.

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).

Fig 1. Total particle count per mL after TFF of non-deaerated and deaerated solutions containing 1 mg/mL bovine IgG solution.

Fig 2. Derived count rate of non-deaerated and deaerated 1 mg/mL bovine IgG solution after TFF, including A) measured at 173° scattering angle and B) measured at 13° scattering angle.

Fig 3. Specific surface area of TFF processed powders of bovine IgG.