(T1430-11-73) Investigating Phase Separation of Trehalose during Lyophilization of Proteins Using Solid State NMR

Purpose: Proteins and macromolecules are often formulated as lyophilized products. Cryoprotectants are used to increase the stability of macromolecules during lyophilization and storage. Trehalose is one of the cryoprotectants used in pharmaceutical formulation. The physical state of trehalose during and after lyophilization is crucial in maintaining the physical and chemical stability of proteins. Literature shows presence of crystalline trehalose during annealing, and disappearance of the crystalline form during primary drying due to dehydration, resulting in an amorphous final product. (1) Trehalose crystallization may result in phase separation, where there will be domains of trehalose that are not providing stability to the protein. This has the potential to lead to increased mobility of proteins and ultimately, lower effectiveness of the cryoprotectant. However, the amount of trehalose that crystallizes during annealing may not reduce its effectiveness as cryoprotectant if only a fraction of trehalose crystallizes. We have previously studied samples of HSA (Human Serum Albumin) and trehalose at different ratios of protein to disaccharide. At trehalose levels >80%, there was a difference in the NMR relaxation times of the trehalose and protein, indicating that phase separation had occurred in these samples. The purpose of this study is to investigate the impact of trehalose phase separation on the stability of BSA (Bovine Serum Albumin) using SSNMR (solid-state Nuclear Magnetic Resonance).

Methods: Lyophilization: We studied samples containing five concentrations of trehalose (70, 82, 84, 92, 96 %w/w) that were annealed for 0-24 hours. BSA was used as a model protein for this study. BSA, 2mM sodium phosphate buffer at pH 7 along with trehalose was frozen to -45°C and annealed at -15°C. Primary and secondary drying was carried out for 62.5 hr at -27°C (60mT) and 4hr at 30°C (200mT), respectively. Size exclusion chromatography (SEC): SEC was used to analyze the monomer (%). Agilent infinity 1200ii system with Tosoh 2000 PWXL column was used. 100 mM sodium phosphate buffer was used as Mobile phase with flow rate maintained at 0.5 ml/min. The retention time of the BSA monomer peak was 17 min. SSNMR: All SSNMR data were collected in Bruker Avance 400 using PhoenixNMR MAS HX probe. Samples were packed in 6mm zirconia rotors in a dry box with < 1% RH. All spectra were acquired using cross-polarization, magic angle spinning at 4 kHz, and total sideband suppression (TOSS). Proton spin–lattice relaxation time in the laboratory frame (¹H T₁) which is indicative of molecular mobility was measured using a 13C-detected saturation recovery pulse sequence with TOSS. Powder X-Ray Diffraction: Powder X-Ray Diffraction (PXRD) was done on samples after lyophilization using continuous scanning mode with 1deg/min speed.

Results: The PXRD pattern showed that sample with 70% trehalose was mostly amorphous and crystallinity increased as a function of trehalose concentration. At 24-hour annealing, most of the samples were crystalline at time zero. The ¹H T₁ relaxation time, indicative of molecular mobility, was lowest for 70% trehalose compared to the higher concentrations (84% ,88%) which correlates with the PXRD data; as amorphous substance have low ¹H T₁ relaxation time/high mobility compared to the crystalline lattice structure. ¹H T₁ relaxation time increased as a function of trehalose concentration indicating generation more crystalline domains at higher concentrations. The difference in ¹H T₁ relaxation time between the protein and trehalose increased with increase in annealing time for most of the samples. The ¹H T₁ relaxation time increased after the samples were stored at 50°C for six months indicating reduced molecular mobility. The difference in ¹H T₁ relaxation time between the protein and trehalose increased after storage at 50°C for six months indicating phase separation. SEC data showed that the monomer percentage of BSA was 62% after lyophilization and after storage. The monomer concentration of BSA when received was approximately 62% as well.

Conclusion: The ¹H T₁ relaxation time, which correlates with molecular mobility, increased as a function of trehalose concentration, annealing duration and upon storage. After six months of storage at 50°C, there was evident phase separation in samples compared to samples at time zero. Results from the size exclusion chromatography indicated that there was no further aggregation of BSA at storage. This shows that protein was stable at storage for six months at 50°C. While they had evident phase separation, the monomer percentage remained the same. The results show that the phase separation of trehalose in trehalose/BSA model system did not compromise the stability of BSA.

References: Sundaramurthi P, Suryanarayanan R. Trehalose Crystallization During Freeze-Drying: Implications On Lyoprotection. J Phys Chem Lett [Internet]. 2010 [cited 2023 Jan 19];1:510–4. Available from: https://pubs.acs.org/sharingguidelines

Acknowledgements: Munson Lab, Department of Industrial and Physical Pharmacy, College of Pharmacy, Purdue University

Fig:1 Powder X-ray Diffraction pattern of trehalose/BSA samples with different concentrations (70, 88, 84, 92, 96) of Trehalose showing crystalline peaks for most of the samples after 24 hours of annealing

Fig 2: 1H T1 relaxation times of samples at time point 0 and 6 months at zero hour and 24 hour annealing. 1H T1 relaxation times shows phase separation of trehalose at 6 months time point.

Fig3: Monomer (%) concentration of BSA before and after the storage at 50C for six months