(M0930-06-40) Investigating the Outcomes of Trehalose Crystallization in Lyophilized Protein Systems

Purpose: Trehalose is a key cryoprotectant used in pharmaceutical formulations to stabilize proteins during lyophilization and storage. The physical state of trehalose, both during and after lyophilization, plays a critical role in preserving the stability of lyophilized proteins. Literature has shown that trehalose crystallizes during annealing but typically loses its crystalline form during secondary drying due to dehydration, resulting in an amorphous product. (1) However, trehalose crystallization can cause phase separation, where regions of crystalline trehalose fail to stabilize the protein effectively. This phase separation can reduce the overall effectiveness of the cryoprotectant, compromising protein stability. The purpose of this study is to investigate the impact of trehalose phase separation on the stability of BSA (Bovine Serum Albumin) and mAb (monoclonal Antibodies) using SSNMR (solid-state Nuclear Magnetic Resonance).

Methods: Three protein–trehalose systems were studied: BSA formulations with 60% or 95% monomer, and mAb formulations with ~98% monomer at initial time point. The highest purity of BSA that could be obtained during COVID was 60%monomer BSA. That is why it was used to prepare the first set of samples. All formulations contained 70–96% (w/w) trehalose and were annealed at –15 °C for 0–72 hours following freezing at –45 °C. Primary and secondary drying were conducted at –27 °C (70 mT) and 30 °C (60 mT), respectively. Post-lyophilization, samples were stored at 50 °C. Monomer content was analyzed by SEC using an Agilent 1200 II system. A Tosoh G2000 PWXL column was used for BSA, and a Sephax column for mAb. Solid-state NMR data were collected on a Bruker Avance 400 with a PhoenixNMR MAS HX probe. PXRD was performed on lyophilized samples using continuous scanning mode at 1°/min.

Results: BSA–trehalose formulations with 60% monomer BSA: In BSA–trehalose formulations with 60% monomer, trehalose crystallized after 24 hours of annealing. PXRD showed that a 70% trehalose/30% BSA formulation remained mostly amorphous, with crystallinity increasing at higher trehalose concentrations. After 24 hours annealing, samples with ≥84% trehalose were crystalline at time zero (Figure 1A). ¹H T₁ relaxation times were lowest for 70% trehalose and higher for 84% and 88%, indicating differences in molecular mobility (Figure 2A). The observed differences in ¹H T₁ times between protein and trehalose suggested loss of homogeneity. Despite crystallinity, SEC analysis showed protein stability was maintained in formulations with >70% trehalose for up to 12 months at 50 °C (Figure 3A). BSA–trehalose formulations with 95% monomer BSA: In a second BSA–trehalose set, trehalose remained amorphous after 24 hours of annealing. However, PXRD revealed crystallization after 72 hours of annealing (Figure 1B). Formulations annealed for 24 hours remained stable at 50 °C, while those annealed for 72 hours showed monomer loss, contradicting earlier results. mAb–trehalose formulations with ~98% monomer: In mAb–trehalose formulations, crystallization occurred only after 72 hours of annealing (Figure 1C); 24-hour samples remained amorphous. ¹H T₁ relaxation times were highest in 96% trehalose and decreased with lower trehalose concentrations. After 72-hour annealing, differences in ¹H T₁ values between protein, buffer, and trehalose indicated phase separation, which became more pronounced at the 6-month time point (Figure 2B). SEC data confirmed monomer loss in these samples. In contrast, formulations annealed for 24 hours remained homogeneous and stable at 50 °C for up to 6 months (Figure 3B, 3C).

Conclusion: Formulations with higher trehalose content or extended annealing (72 hours) exhibited trehalose crystallization and phase separation, as indicated by differences in ¹H T₁ relaxation times. This inhomogeneity was associated with reduced protein stability, observed in both BSA and mAb formulations. Notably, trehalose crystallized after 24 hours of annealing in BSA formulations with 60% monomer, likely because the BSA was not interacting sufficiently with the trehalose. In contrast, formulations with 95% monomer BSA remained mostly amorphous, even after 24 hours of annealing. This showed that protein aggregates at initial time point influences the crystallization kinetics. Formulations with lower trehalose content or shorter annealing durations remained amorphous and preserved protein stability. These results suggest that the physical state of trehalose plays a critical role in maintaining protein integrity in lyophilized formulations. This study shows that trehalose crystallization is difficult to induce; it can occur only under extreme conditions, and a higher fraction of trehalose is required for crystallization. Furthermore, molecular mobility, measured through ¹H T₁ relaxation times, was a good predictor of protein stability and provided mechanistic insight into how trehalose crystallization compromises protein integrity in lyophilized systems.

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

Acknowledgements: The mAb was supplied by Merck & Co., Inc., Rahway, NJ, USA, and is gratefully acknowledged


Figure 1: (A) PXRD pattern for BSA-Trehalose formulations with 60% monomer after 24 hours of annealing (B) PXRD pattern for BSA-Trehalose formulations with 95% monomer after 72 hours of annealing (C) PXRD pattern for mAb-Trehalose formulations after 72 hours of annealing


Figure 2: (A) ¹H T₁ relaxation times of BSA- trehalose with 60%monomer after no annealing and 24 hours of annealing at initial time point and 6-month time point (B) ¹H T₁ relaxation times of mAb- trehalose with after 24 and 72hours of annealing at initial time point and 6-month time point


Figure 3: (A) Monomer (%) for BSA-trehalose with 60% monomer annealed for upto 24 hours at initial, 6 and 12 months at 50C (B) Monomer (%) for mAb-trehalose annealed for 24 hours upto 6 months at 50C (C) Monomer (%) for mAb-trehalose annealed for 72 hours upto 6 months at 50C