Predicting the Stability of Lyophilized Human Serum Albumin Formulations Containing Sucrose and Trehalose Using Solid-State NMR Spectroscopy: Effect of Storage Temperature on 1H T1 Relaxation Times.

In a lyophilized protein/disaccharide system, the ability of the disaccharide to form a homogeneous mixture with the protein and to slow the protein mobility dictates the stabilization potential of the formulation. Human serum albumin was lyophilized with sucrose or trehalose in histidine, phosphate, or citrate buffer. 1H T1 relaxation times were measured by solid-state NMR spectroscopy and were used to assess the homogeneity and mobility of the samples after zero, six, and twelve months at different temperatures. The mobility of the samples decreased after 6 and 12 months storage at elevated temperatures, consistent with structural relaxation of the amorphous disaccharide matrix. Formulations with sucrose had lower mobility and greater stability than formulations with trehalose.

Targeting Mycobacterium tuberculosis Persistence through Inhibition of the Trehalose Catalytic Shift.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is the leading cause of death worldwide by infectious disease. Treatment of Mtb infection requires a six-month course of multiple antibiotics, an extremely challenging regimen necessitated by Mtb's ability to form drug-tolerant persister cells. Mtb persister formation is dependent on the trehalose catalytic shift, a stress-responsive metabolic remodeling mechanism in which the disaccharide trehalose is liberated from cell surface glycolipids and repurposed as an internal carbon source to meet energy and redox demands. Here, using a biofilm-persister model, metabolomics, and cryo-electron microscopy (EM), we found that azidodeoxy- and aminodeoxy-d-trehalose analogues block the Mtb trehalose catalytic shift through inhibition of trehalose synthase TreS (Rv0126), which catalyzes the isomerization of trehalose to maltose. Out of a focused eight-member compound panel constructed by chemoenzymatic synthesis, the natural product 2-trehalosamine exhibited the highest potency and significantly potentiated first- and second-line TB drugs in broth culture and macrophage infection assays. We also report the first structure of TreS bound to a substrate analogue inhibitor, obtained via cryo-EM, which revealed conformational changes likely essential for catalysis and inhibitor binding that can potentially be exploited for future therapeutic development. Our results demonstrate that inhibition of the trehalose catalytic shift is a viable strategy to target Mtb persisters and advance trehalose analogues as tools and potential adjunctive therapeutics for investigating and targeting mycobacterial persistence.

Trehalose-polyamine/DNA nanocomplexes: impact of vector architecture on cell and organ transfection selectivity.

A novel family of precision-engineered gene vectors with well-defined structures built on trehalose and trehalose-based macrocycles (cyclotrehalans) comprising linear or cyclic polyamine heads have been synthesized through procedures that exploit click chemistry reactions. The strategy was conceived to enable systematic structural variations and, at the same time, ensuring that enantiomerically pure vectors are obtained. Notably, changes in the molecular architecture translated into topological differences at the nanoscale upon co-assembly with plasmid DNA, especially regarding the presence of regions with short- or long-range internal order as observed by TEM. In vitro and in vivo experiments further evidenced a significant impact on cell and organ transfection selectivity. Altogether, the results highlight the potential of trehalose-polyamine/pDNA nanocomplex monoformulations to achieve targeting transfection without the need for any additional cell- or organ-sorting component.

Formulation factors affecting foam properties during vacuum foam-drying.

This paper explores how vacuum foam-drying of a protein is influenced by formulation parameters by investigating the foam structure, physical properties of the foam, and the stability of the protein. Recombinant human bile salt-stimulated lipase was used as a model of a protein drug. The stability of the lipase was evaluated through activity measurements. Two disaccharides (sucrose and trehalose), strongly tending to an amorphous form, were used as matrix formers, and the physical properties were assessed through residual water content, glass transition temperature, and crystalline state. Moreover, some formulations included surfactants with different sizes and structures of the head group. The alkyl chain length was kept constant to only investigate the impact of the surfactant head group, in the presence of the lipase, on the foamability and surface coverage of the lipase. The study demonstrated that the lipase allowed for a dry, solid foam with a foam overrun of up to 2600 %. The wall thickness of the dry, solid foam was estimated to be 20-50 µm. Clear differences between sucrose and trehalose as matrix former were identified. The lipase showed no tendency to lose activity because of the drying and rehydration, despite a proportion of the lipase covering the surfaces of the dry material.

The effects of dextran in residual impurity on trehalose crystallization and formula in food preservation.

The residual dextran impurities in the upstream process significantly impact the crystallization of starch-based functional sugar and the related food properties. This study intends to reveal the mechanism of dextran's influence on trehalose crystallization, and build a relationship among the dextran in syrup and the physicochemical and functional properties of trehalose. Instead of incorporating into the crystal lattice, dextran changes the assembly rate of trehalose molecules on crystal surface. The different sensitivity and adsorption capacity of the crystal surface to the chain length of dextran determines the growth rate of crystal surfaces, resulting in different crystal morphology. The bulk trehalose crystals, which were obtained from syrups with short chain dextran, have excellent powder properties, including best flowability (35◦), highest crystal strength (2.7 N), lowest caking rate (62.22 %), and the most uniform mixing with other sweeteners (sucrose/xylitol) in food formulations, achieving more stable starch preservation.

Effects of freezing and drying programs on IgY aggregation and activity during microwave freeze-drying: Protective effects and interactions of trehalose and mannitol.

The acquisition of high quality lyophilized IgY products, characterized by an aesthetically pleasing visage, heightened stability, and a marked preservation of activity, constitutes an indispensable pursuit in augmenting the safety and pragmatic utility of IgY. Within this context, an exploration was undertaken to investigate an innovative modality encompassing microwave freeze-drying (MFD) as a preparatory methodology of IgY. Morphological assessments revealed that both cryogenic freezing and subsequent MFD procedures resulted in aggregation of IgY, with the deleterious influence posed by the MFD phase transcending that of the freezing phase. The composite protective agent comprised of trehalose and mannitol engendered a safeguarding effect on the structural integrity of IgY, thereby attenuating reducing aggregation between IgY during the freeze-drying process. Enzyme-linked immunosorbent assay (ELISA) outcomes demonstrated a discernible correlation between IgY aggregation and a notable reduction in its binding affinity towards the pertinent antigen. Comparative analysis vis-à-vis the control sample delineated that when the trehalose-to-mannitol ratio was upheld at 1:3, a two-fold outcome was achieved: a mitigation of the collapse susceptibility within the final product as well as a deterrence of IgY agglomeration, concomitant with an elevated preservation rate of active antibodies (78.57 %).

Influence of water and trehalose on α- and ß-relaxation of freeze-dried lysozyme formulations.

Molecular mobility in form of alpha and beta relaxations is considered crucial for characterization of amorphous lyophilizates and reflected in the transition temperatures Tgα and Tgß. Based on an overview of applied methods to study beta relaxations, Dynamic Mechanical analysis was used to measure Tgα and Tgß in amorphous freeze-dried samples. Lysozyme and trehalose as well as their mixtures in varying ratios were investigated. Three different residual moisture levels, ranging from roughly 0.5-7 % (w/w), were prepared via equilibration of the freeze-dried samples. Known plasticising effects of water on Tgα were confirmed, also via differential scanning calorimetry. In addition and contrary to expectations, an influence of water on the Tgß also was observed. On the other hand, an increasing amount of trehalose lowered Tgα but increased Tgß showing that Tgα and Tgß are not paired. The findings were interpreted with regard to their underlying molecular mechanisms and a correlation with the known influences of water and trehalose on stability. The results provide encouraging hints for future stability studies of freeze-dried protein formulations, which are urgently needed, not least for reasons of sustainability.

Stabilization effects of saccharides in protein formulations: A review of sucrose, trehalose, cyclodextrins and dextrans.

Saccharides are a popular group of stabilizers in liquid, frozen and freeze dried protein formulations. The current work reviewed the stabilization mechanisms of three groups of saccharides: (i) Disaccharides, specifically sucrose and trehalose; (ii) cyclodextrins (CDs), a class of cyclic oligosaccharides; and (iii) dextrans, a class of polysaccharides. Compared to sucrose, trehalose exhibits a more pronounced preferential exclusion effect in liquid protein formulations, due to its stronger interaction with water molecules. However, trehalose obtains higher phase separation and crystallization propensity in frozen solutions, resulting in the loss of its stabilization function. In lyophilized formulations, sucrose has a higher crystallization propensity. Besides, its glass matrix is less homogeneous than that of trehalose, thus undermining its lyoprotectant function. Nevertheless, the hygroscopic nature of trehalose may result in high water absorption upon storage. Among all the CDs, the ß form is believed to have stronger interactions with proteins than the α- and γ-CDs. However, the stabilization effect, brought about by CD-protein interactions, is case-by-case - in some examples, such interactions can promote protein destabilization. The stabilization effect of hydroxypropyl-ß-cyclodextrin (HPßCD) has been extensively studied. Due to its amphiphilic nature, it can act as a surface-active agent in preventing interfacial stresses. Besides, it is a dual functional excipient in freeze dried formulations, acting as an amorphous bulking agent and lyoprotectant. Finally, dextrans, when combined with sucrose or trehalose, can be used to produce stable freeze dried protein formulations. A strong stabilization effect can be realized by low molecular weight dextrans. However, the terminal glucose in dextrans yields protein glycation, which warrants extra caution during formulation development.

Insight into the effect of osmosis agents on macro- and micro- texture, water distribution, and thermal stability of instant controlled pressure drop drying peach chips.

To investigate the influence of sugar structure on the quality of peach chips produced using osmotic dehydration (OD) in combination with instant controlled pressure drop (DIC) drying, erythritol, glucose, maltose, and trehalose were selected as osmotic agents. The properties of the osmotic solutions, as well as the macro- and micro-texture, water distribution, and thermal stability of peach chips were investigated. Results showed that OD pretreatments inhibited the formation of large cavity structures. The highest hardness (101.34 N) and the lowest hydrophobicity (0°) were obtained in erythritol-OD samples. Trehalose-OD samples with the most homogeneous pore structure exhibited the highest crispness (1.05 mm) and the highest glass transition temperature (52.06 °C). Various absorption peaks of peach chips pretreated with different OD methods, characterized by Raman spectroscopy, suggested changes in composition and functional groups due to the diffusion of sugars into the cells of peach tissues, which also contributed to the higher Tg.

Enhancement of cryopreservation with intracellularly permeable zwitterionic polymers.

A novel copolymer containing zwitterionic and methylsulfinyl structures was developed, which enhanced cryoprotective efficacy by enabling intracellular cytoplasmic permeation without relying on mediated endocytosis and diffused out of the cells within approximately 30 min, making it more advantageous than polymeric nanoparticles for the transport of membrane-impermeable cryoprotectants such as trehalose.

Tableting properties of freeze-dried trehalose: Physico-chemical and mechanical investigation.

Freeze-drying of biopharmaceutical products is the method of choice in order to improve their stability and storage conditions. Such freeze-dried products are usually intended for parenteral route administration. However, many biopharmaceutical materials administered by parenteral route are used to treat local diseases particularly in the gastro-intestinal tract. Therefore, many studies concentrate nowadays their effort on developing alternative dosage forms to deliver biopharmaceutical molecules by the oral route. Tablets are the most popular solid pharmaceutical dosage form used for oral administration since they present many advantages, but poor informations are available on the possibility of tableting freeze-dried powders. In this study, we evaluate the compaction behavior of freeze-dried trehalose powder since trehalose is one of the most used cryo and lyoprotectant for the lyophilisation of biopharmaceutical entities. Results show that freeze-dried trehalose powder can be tableted while remaining amorphous and the obtained compacts present very specific properties in terms of compressibility, tabletability, brittleness and viscoelasticity compared to the crystalline trehalose and compared to classical pharmaceutical excipients.

Hierarchy of the Components in Spray-Dried, Protein-Excipient Particles Using DNP-Enhanced NMR Spectroscopy.

Protein-based drugs are becoming increasingly important, but there are challenges associated with their formulation (for example, formulating stable inhalable aerosols while maintaining the proper long-term stability of the protein). Determining the morphology of multicomponent, protein-based drug formulations is particularly challenging. Here, we use dynamic nuclear polarization (DNP) solid-state NMR spectroscopy to determine the hierarchy of components within spray-dried particles containing protein, trehalose, leucine, and trileucine. DNP NMR was applied to these formulations to assess the localization of the components within the particles. We found a consistent scheme, where trehalose and the protein are co-located within the same phase in the core of the particles and leucine and trileucine are distributed in separate phases at the surface of the particles. The description of the hierarchy of the organic components determined by DNP NMR enables the rationalization of the performance of the formulation.

New insights into the protein stabilizing effects of trehalose by comparing with sucrose.

Disaccharides are well known to be efficient stabilizers of proteins, for example in the case of lyophilization or cryopreservation. However, although all disaccharides seem to exhibit bioprotective and stabilizing properties, it is clear that trehalose is generally superior compared to other disaccharides. The aim of this study was to understand this by comparing how the structural and dynamical properties of aqueous trehalose and sucrose solutions influence the protein myoglobin (Mb). The structural studies were based on neutron and X-ray diffraction in combination with empirical potential structure refinement (EPSR) modeling, whereas the dynamical studies were based on quasielastic neutron scattering (QENS) and molecular dynamics (MD) simulations. The results show that the overall differences in the structure and dynamics of the two systems are small, but nevertheless there are some important differences which may explain the superior stabilizing effects of trehalose. It was found that in both systems the protein is preferentially hydrated by water, but that this effect is more pronounced for trehalose, i.e. trehalose forms less hydrogen bonds to the protein surface than sucrose. Furthermore, the rotational motion around dihedrals between the two glucose rings of trehalose is slower than in the case of the dihedrals between the glucose and fructose rings of sucrose. This leads to a less perturbed protein structure in the case of trehalose. The observations indicate that an aqueous environment closest to the protein molecules is beneficial for an efficient bioprotective solution.

Role of Poloxamer 188 in Preventing Ice-Surface-Induced Protein Destabilization during Freeze-Thawing.

The phase behavior of poloxamer 188 (P188) in aqueous solutions, characterized by differential scanning calorimetry (DSC) and synchrotron X-ray diffractometry, revealed solute crystallization during both freezing and thawing. Sucrose and trehalose inhibited P188 crystallization during freeze-thawing (FT). While trehalose inhibited P188 crystallization only during cooling, sucrose completely suppressed P188 crystallization during both cooling and heating. Lactate dehydrogenase (LDH) served as a model protein to evaluate the stabilizing effect of P188. The ability of P188, over a concentration range of 0.003-0.800% w/v, to prevent LDH (10 µg/mL) destabilization was evaluated. After five FT cycles, the aggregation behavior (by dynamic light scattering) and activity recovery were evaluated. While LDH alone was sensitive to interfacial stress, P188 at concentrations of ≥0.100% w/v stabilized the protein. However, as the surfactant concentration decreased, protein aggregation after FT increased. The addition of sugar (1.0% w/v; sucrose or trehalose) improved the stabilizing function of P188 at lower concentrations (≤0.010% w/v), possibly due to the inhibition of surfactant crystallization. Based on a comparison with the stabilization effect of polysorbate (both 20 and 80), it was evident that P188 could be a promising alternative surfactant in frozen protein formulations. However, when the surfactant concentration is low, the potential for P188 crystallization and the consequent compromise in its functionality warrant careful consideration.

Physicochemical Mechanisms of Protection Offered by Agarose Encapsulation during Cryopreservation of Mammalian Cells in the Absence of Membrane-Penetrating Cryoprotectants.

During freeze/thaw, cells are exposed to mechanical, thermal, chemical, and osmotic stresses, which cause loss of viability and function. Cryopreservation agents such as dimethyl sulfoxide (DMSO) are deployed to minimize freeze/thaw damage. However, there is a pressing need to eliminate DMSO from cryopreservation solutions due to its adverse effects. This is of the highest priority especially for cryopreservation of infusible/transplantable cell therapy products. In order to address this issue, we introduce reversible encapsulation in agarose hydrogels in the presence of the membrane-impermeable cryoprotectant, trehalose, as a viable, safe, and effective cryopreservation method. Our findings, which are supported by IR spectroscopy and differential scanning calorimetry analyses, demonstrate that encapsulation in 0.75% agarose hydrogels containing 10-20% trehalose inhibits mechanical damage induced by eutectic phase change, devitrification, and recrystallization, resulting in post-thaw viability comparable to the gold standard 10% DMSO.

Impact of post-freeze annealing on shrinkage of sucrose and trehalose lyophilisates.

Freeze-drying of pharmaceuticals produces lyophilisates with properties that depend on both the formulation and the process. Characterisation of the lyophilisate in terms of appearance is necessary not only to produce a visually appealing product, but also to gain insight into the freeze-drying process. The present study investigates the impact of post-freeze annealing on the volume of lyophilisates. For this purpose, sucrose and trehalose solutions were freeze-dried with different annealing conditions and the resulting lyophilisates were analysed with a 3D structured light scanner. The external structure of the lyophilisates was found to be dependent on the bulk materials as well as the choice of vials, while the volume was influenced by the annealing time and temperature. Additionally, differential scanning calorimetry was used to determine glass transition temperatures of frozen samples. As a novelty, the volumes of the lyophilisates and their corresponding glass transition temperatures were compared. This resulted in a correlation supporting the theory that the shrinkage of lyophilisates depends on the amount of residual water in the freeze-concentrated amorphous phase before drying. Understanding the volume change of lyophilisates, in combination with material properties such as glass transition temperature, forms the basis for relating physicochemical properties to process parameters in lyophilisation.

Unraveling the Molecular Mechanisms of Trehalose-Mediated Protection and Stabilization of Escherichia coli Lipid Membrane during Desiccation.

Trehalose, a disaccharide renowned for its ability to stabilize biomolecular architectures under strenuous conditions, finds extensive use in the cryopreservation of probiotics. A profound comprehension of its molecular-level interactions is of great significance. It is notable that current research in the realm of lipid-sugar interactions primarily employs single-component lipid bilayers, which are far from being representative of real cell membranes. Our investigation, however, utilizes molecular dynamics simulations to delve into the specifics of a realistic Escherichia coli membrane that encompasses a diverse array of lipid types, comprising fourteen distinct species, subject to varying hydration levels. The results of our study showcase that the reduction of hydration levels induces lipid ordering and the formation of gel phases, yet trehalose, by forming hydrogen bonds with lipid headgroups, serves to uphold fluidity and supplant the role of water. Moreover, our findings evince that augmented trehalose concentrations lead to a slowdown in lipid motion and contribute to the maintenance of fluidity by way of endowing a viscous matrix. It is noteworthy that our conclusions lend support to the notion that water replacement and vitrification, despite their seemingly disparate nature, need not be considered mutually exclusive in a real bacterial membrane.

Thermophobic Trehalose Glycopolymers as Smart C-Type Lectin Receptor Vaccine Adjuvants.

Herein, this work reports the first synthetic vaccine adjuvants that attenuate potency in response to small, 1-2 °C changes in temperature about their lower critical solution temperature (LCST). Adjuvant additives significantly increase vaccine efficacy. However, adjuvants also cause inflammatory side effects, such as pyrexia, which currently limits their use. To address this, a thermophobic vaccine adjuvant engineered to attenuate potency at temperatures correlating to pyrexia is created. Thermophobic adjuvants are synthesized by combining a rationally designed trehalose glycolipid vaccine adjuvant with thermoresponsive poly-N-isoporpylacrylamide (NIPAM) via reversible addition fragmentation chain transfer (RAFT) polymerization. The resulting thermophobic adjuvants exhibit LCSTs near 37 °C, and self-assembled into nanoparticles with temperature-dependent sizes (90-270 nm). Thermophobic adjuvants activate HEK-mMINCLE and other innate immune cell lines as well as primary mouse bone marrow derived dendritic cells (BMDCs) and bone marrow derived macrophages (BMDMs). Inflammatory cytokine production is attenuated under conditions mimicking pyrexia (above the LCST) relative to homeostasis (37 °C) or below the LCST. This thermophobic behavior correlated with decreased adjuvant Rg is observed by DLS, as well as glycolipid-NIPAM shielding interactions are observed by NOESY-NMR. In vivo, thermophobic adjuvants enhance efficacy of a whole inactivated influenza A/California/04/2009 virus vaccine, by increasing neutralizing antibody titers and CD4+ /44+ /62L+ lung and lymph node central memory T cells, as well as providing better protection from morbidity after viral challenge relative to unadjuvanted control vaccine. Together, these results demonstrate the first adjuvants with potency regulated by temperature. This work envisions that with further investigation, this approach can enhance vaccine efficacy while maintaining safety.

Impact of Different Saccharides on the In-Process Stability of a Protein Drug During Evaporative Drying: From Sessile Droplet Drying to Lab-Scale Spray Drying.

OBJECTIVES: Solid biopharmaceutical products can circumvent lower temperature storage and transport and increase remote access with lower carbon emissions and energy consumption. Saccharides are known stabilizers in a solid protein produced via lyophilization and spray drying (SD). Thus, it is essential to understand the interactions between saccharides and proteins and the stabilization mechanism. METHODS: A miniaturized single droplet drying (MD) method was developed to understand how different saccharides stabilize proteins during drying. We applied our MD to different aqueous saccharide-protein systems and transferred our findings to SD. RESULTS: The poly- and oligosaccharides tend to destabilize the protein during drying. The oligosaccharide, Hydroxypropyl ß-cyclodextrin (HPßCD) shows high aggregation at a high saccharide-to-protein molar ratio (S/P ratio) during MD, and the finding is supported by nanoDSF results. The polysaccharide, Dextran (DEX) leads to larger particles, whereas HPBCD leads to smaller particles. Furthermore, DEX is not able to stabilize the protein at higher S/P ratios either. In contrast, the disaccharide Trehalose Dihydrate (TD) does not increase or induce protein aggregation during the drying of the formulation. It can preserve the protein's secondary structure during drying, already at low concentrations. CONCLUSION: During the drying of S/P formulations containing the saccharides TD and DEX, the MD approach could anticipate the in-process (in) stability of protein X at laboratory-scale SD. In contrast, for the systems with HPßCD, the results obtained by SD were contradictory to MD. This underlines that depending on the drying operation, careful consideration needs to be applied to the selection of saccharides and their ratios.

Glycosylated quantum dots as fluorometric nanoprobes for trehalase.

Trehalase is an important enzyme in the metabolic cascades of many organisms, catalysing the hydrolysis of the disaccharide trehalose. Herein we describe the first examples of fluorometric nanoprobes for detection of trehalase, based on trehalose-functionalised quantum dots (QDs). QDs cross-linked with trehalose form aggregates, which are released upon enzymatic cleavage of the trehalose glycosidic bond proportionally to the enzyme concentration, offering a unique and efficient approach for specific sensing of this biologically important enzyme.