(M1330-05-30) Investigating Partial Trehalose Crystallization During Lyophilization of Proteins

Purpose: Trehalose is one of the cryoprotectants used in pharmaceutical formulation to stabilize of proteins during lyophilization and storage. 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. 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 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: We studied samples containing trehalose (50-96%w/w) that were annealed for 0-72 hours. BSA was used as a model protein for this study. Formulation was frozen to -45°C and annealed at -15°C for different durations. Primary and secondary drying was carried out at -27°C (70mT) and 30°C (60mT), respectively. The monomer (%) was analyzed using Size exclusion chromatography (SEC) on an Agilent infinity 1200ii system with a Tosoh 2000 PWXL column. Solid-state Nuclear Magnetic Resonance (SSNMR) data were collected using a Bruker Avance 400 and a PhoenixNMR MAS HX probe. All spectra were acquired using cross-polarization, magic angle spinning at 4 kHz, and total sideband suppression (TOSS). Molecular mobility was measured using a ¹³C-detected saturation recovery pulse sequence with TOSS. 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 a sample containing 70% trehalose and 30% BSA was mostly amorphous, with the amount of trehalose crystallinity increasing as a function of trehalose concentration. After 24-hour annealing prior to primary drying, the samples containing 84% or higher trehalose concentration were crystalline at time zero. The ¹H T₁ relaxation times, indicative of molecular mobility, was lowest for 70% trehalose compared to the higher concentrations (84%, 88%). The ¹H T₁ relaxation times between the protein and trehalose were different, indicating that the samples were no longer homogeneous. The SEC data showed that the protein was stable in all formulations containing greater than 70% trehalose concentration for up to 12 months at 50°C. This showed that the presence of crystalline trehalose did not affect long-term protein stability. However, protein degradation was observed in 30% BSA 70% trehalose at 12 months’ time point. The loss in monomer increased as a function of annealing time prior to primary drying. The reason for the loss of monomer for these samples could be due to phase separation of trehalose that had hydrogen bonded with BSA, which leads us to believe there is a minimum protein to trehalose ratio needed to stabilize the protein, which may be exceeded during annealing. Surprisingly, a 72hr annealing prior to primary drying showed maximum stability at 3 and 4 months’ time-point for all investigated protein/trehalose ratios.The impact of protein to trehalose ratio on protein stability were more pronounced after a post-secondary drying annealing at 90°C. The samples with 30 and 35% BSA maintained over 95% monomer even after 1 week at 90°C and 6 weeks at 70°C while the samples with 40% and over protein concentration lost around 10% monomer during 1 week at 90°C. While 40 and 45%w/w samples did not continue to degrade for 6 weeks at 70°C, 50%w/w samples continued to lose monomer as a function of time.

Conclusion: The ¹H T₁ relaxation time, which correlates with molecular mobility, increased as a function of trehalose concentration, annealing duration and upon storage. Initial results from the SEC indicated that there was no further aggregation of BSA at storage except for 30% BSA 70% trehalose. Investigating this further showed there is an effective protein to trehalose ratio and effective annealing duration which stabilizes protein formulation during the long-term storage. Exploration of different protein to trehalose ratios and annealing durations has shown that samples with less than 30%w/w protein concentration and 72 hr annealing have better long-term stability.

References: 1. 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


Figure 1: BSA\Trehalose formulations at different concentrations lyophilized with different annealing duration post-freezing at-15°C and stored at 50 °C for 12 months A) PXRD patterns showing crystalline peaks of trehalose at high trehalose concentration at 0-time point B) 1HT1 of trehalose and BSA at 0hr and 24hr annealing durations at 0 and 6 months time point C) SEC data showing monomer concentration remaining the same till 12 months except for 30%BSA 70%Trehalose


Figure 2: Protein stability of samples annealed post-secondary drying for a week at 90°C stored and stored at 70°C measured by SEC over 6weeks


Figure 3: Protein stability of samples annealed prior to primary drying for different durations at -15°C stored at 70°C measured by SEC over 4 months time point A. 30%BSA 70%Tre B. 35%BSA 70%Tre C. 40%BSA 60%Tre D. 45%BSA 65%Tre E. 50%BSA 50%Tre