The physical behaviour of food protein's supramolecular structures during freeze- drying (original) (raw)
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European Journal of Pharmaceutics and Biopharmaceutics, 2005
The development of stable freeze-dried proteins requires maintaining the physical and biological integrity of the protein as well as increasing the efficiency of the manufacturing process. Our objective was to study the effects of various excipients on both the physical characterisation and the dried and liquid stability of two proteins. Thermo-physical properties of 13 formulations were determined using both differential scanning calorimetry and freeze-drying microscopy. The antigenic activity was evaluated immediately after freeze-drying and after subsequent storage in both dried and liquid state. From the comparison between glass transition (T 0 g) and collapse (T coll) temperatures, we concluded that the collapse temperature was a more relevant parameter than T 0 g for freeze-drying cycle development and optimisation. One crystalline formulation composed of 4% mannitol and 1% of sucrose protected efficiently both proteins during subsequent storage in dried state (6 months at 25 8C) and in liquid state (3 months at 4 8C after rehydration). However, the freeze-drying behaviour of this crystalline formulation remained difficult to predict and control. On the other hand, two amorphous formulations composed of 4% of maltodextrin and 0.02% of Tween 80, or 5% of BSA preserved antigenic activity during storage in dried state. The glassy character of these formulations as well as their high collapse temperature values (K9 and K12 8C, respectively) should allow simplification and shortening of freeze-drying process.
International Journal of Pharmaceutics, 2015
In-line near infrared spectroscopy during freeze-drying as a tool to measure efficiency of hydrogen bond formulation between protein and sugar, predictive of protein storage stability. International Journal of Pharmaceutics 496 792-800 Abstract 18 Sugars are often used as stabilizers of protein formulations during freeze-drying. However, 19 not all sugars are equally suitable for this purpose. Using in-line near-infrared spectroscopy 20 during freeze-drying, it is here shown here that hydrogen bond formation during freeze-21 drying, under secondary drying conditions in particular, can be related to the preservation of 22 the functionality and structure of proteins during storage. The disaccharide trehalose was best 23 capable of forming hydrogen bonds with the model protein, lactate dehydrogenase, thereby 24 stabilizing it, followed by the molecularly flexible oligosaccharide inulin 4 kDa. The 25 molecularly rigid oligo-and polysaccharides dextran 5 kDa and 70 kDa, respectively, formed 26 the least amount of hydrogen bonds and provided least stabilization of the protein. It is 27 concluded that smaller and molecularly more flexible sugars are less affected by steric 28 hindrance, allowing them to form more hydrogen bonds with the protein, thereby stabilizing it 29 better. 30 Keywords 31 Near-infrared (NIR) spectroscopy, water-replacement, vitrification, molecular flexibility, 32 solid-state stability, Fourier transform infrared (FTIR) spectroscopy 33 34
Journal of Pharmaceutical Sciences, 2009
The purpose of this study is to investigate the impact of sucrose level on storage stability of dried proteins and thus better understand the mechanism of protein stabilization by disaccharides in lyophilized protein products. Five proteins were freeze dried with different amounts of sucrose, and protein aggregation was quantified using Size Exclusion Chromatography. Protein secondary structure was monitored by FTIR. The global mobility was studied using Thermal Activity Monitor (TAM), and fast local dynamics with a timescale of nanoseconds was characterized by neutron backscattering. The density of the protein formulations was measured with a gas pycnometer. The physical stability of the proteins increased monotonically with an increasing content of sucrose over the entire range of compositions studied. Both FTIR structure and structural relaxation time from TAM achieved maxima at about 1:1 mass ratio for most proteins studied. Therefore, protein stabilization by sugar cannot be completely explained by global dynamics and FTIR structure throughout the whole range of compositions. On the other hand, both the fast local mobility and free volume obtained from density decreased monotonically with an increased level of sucrose in the formulations, and thus the local dynamics and free volume correlate well with protein storage stability.
Accelerated Formulation Studies for Frozen Storage of Proteins
Freezing of protein solutions is required for many applications such as storage, transport, or lyophilization; however, freezing has inherent risks for protein integrity. It is difficult to study protein stability below the freezing temperature because phase separation constrains solute concentration in solution. In this work, we developed an isochoric method to study protein aggregation in solutions at −5, −10, −15, and −20°C. Lowering the temperature below the freezing point in a fixed volume prevents the aqueous solution from freezing, as pressure rises until equilibrium (P,T) is reached. Aggregation rates of bovine hemoglobin (BHb) increased at lower temperature (−20°C) and higher BHb concentration. However, the addition of sucrose substantially decreased the aggregation rate and prevented aggregation when the concentration reached 300 g/L. The unfolding thermodynamics of BHb was studied using fluorescence, and the fraction of unfolded protein as a function of temperature was determined. A mathematical model was applied to describe BHb aggregation below the freezing temperature. This model was able to predict the aggregation curves for various storage temperatures and initial concentrations of BHb. The aggregation mechanism was revealed to be mediated by an unfolded state, followed by a fast growth of aggregates that readily precipitate. The aggregation kinetics increased for lower temperature because of the higher fraction of unfolded BHb closer to the cold denaturation temperature. Overall, the results obtained herein suggest that the isochoric method could provide a relatively simple approach to obtain fundamental thermodynamic information about the protein and the aggregation mechanism, thus providing a new approach to developing accelerated formulation studies below the freezing temperature.
Journal of Pharmaceutical Sciences, 2009
The purpose of this study is to investigate protein-sugar interactions in dried protein solids as a function of sucrose level using water sorption isotherm data and secondary structure information from Fourier transform infrared (FTIR) spectroscopy. Three IgG1 fusion proteins and two cytokines were freeze-dried with sucrose at different sucrose/protein mass ratios. The water monolayer of the colyophilized sucrose/protein samples, as determined by BET analysis of water sorption data, was found to be lower than that expected based on additive contributions of pure protein and pure sucrose. This negative deviation suggests the presence of a solid-state interaction between protein and sucrose that reduces the availability of total water-binding sites. The difference in water monolayer between colyophilized and a physical mixture of protein and sucrose reached a maximum value at sucrose/protein mass ratio of 1/1 for these proteins, suggesting saturation of the protein-sugar interaction at this ratio. In addition, for four proteins studied, the normalized peak height of the major band in the FTIR spectra reached a plateau at about a 1/1 mass ratio. Therefore, it appears that there is a coupling between the preservation of protein secondary structure and the protein-sugar interaction as measured by water sorption isotherms. ß
Downscaled freeze-drying was demonstrated to be a valuable alternative for formulation development and optimization. Although the pore structure is known to exert a major influence on the freeze-drying cycle, little is known about the ones of microscale preparations. This study describes morphology evaluation methods for lysozyme formulations prepared in one microscale processing option and the assessment of fundamental product quality criteria. Scanning electron microscopy (SEM) revealed cooling rate dependent pore size variations at the nucleation site which diminished as the rate increased. Micro-X-ray computed tomography (μ-CT) showed that porosity generally increased in the sample from bottom to top, the pore size fractions shifted toward larger pores in elevated sample levels, and horizontal homogeneity was found throughout each sample with minor deviations in the bottom region. Furthermore, the event of microcollapse could be identified and quantified. Low residual moisture was achieved repeatedly and the procedure did not influence the post freeze-drying bioactivity. This microscale heating stage is a valuable option to reduce overall cycle times and cost, and to prepare freeze-drying formulations with high reproducibility. The mapping tools permit a quick but detailed insight into the structural features resulting from the process environment and processing conditions.
Journal of Pharmaceutical Sciences
Freezing is widely used during the manufacturing process of protein-based therapeutics, but it may result in undesired loss of biological activity. Many variables come into play during freezing that could adversely affect protein stability, creating a complex landscape of interrelated effects. The current approach to the selection of freezing conditions is however nonsystematic, resulting in poor process control. Here we show how mathematical models, and a design space approach, can guide the selection of the optimal freezing protocol, focusing on protein stability. Two opposite scenarios are identified, suggesting that the ice-water interface is the dominant cause of denaturation for proteins with high bulk stability, while the duration of the freezing process itself is the key parameter to be controlled for proteins that are susceptible to cold denaturation. Experimental data for lactate dehydrogenase and myoglobin as model proteins support the model results, with a slow freezing rate being optimal for lactate dehydrogenase and the opposite being true for myoglobin. A possible application of the calculated design space to the freezing and freeze-drying of biopharmaceuticals is finally described, and some considerations on process efficiency are discussed as well.
Heliyon
Freezing is widely used in food preservation, but if not carried out properly, ice crystals can multiply (nucleation) or grow (recrystallization) rapidly. This also affects thawing, causing structural damage and affecting overall quality. The objective of this review is to comprehensively study the cryoprotective effect of antifreeze proteins (AFPs), highlighting their role in the freeze-thaw process of food. The properties of AFPs are based on their thermal hysteresis capacity (THC), on the modification of crystal morphology and on the inhibition of ice recrystallization. The mechanism of action of AFPs is based on the adsorption-inhibition theory, but the specific role of hydrogen and hydrophobic bonds/residues and structural characteristics is also detailed. Because of the properties of AFPs, they have been successfully used to preserve the quality of a wide variety of refrigerated and frozen foods. Among the limitations of the use of AFPs, the high cost of production stands out, but currently there are solutions such as the use the production of recombinant proteins, cloning and chemical synthesis. Although in vitro, in vivo and human studies have shown that AFPs are non-toxic, their safety remains a matter of debate. Further studies are recommended to expand knowledge about AFPs, to reduce costs in their large-scale production, to understand their interaction with other food compounds and their possible effects on the consumer. ☆ This article is a part of the "Structure and function of food proteins and peptides" Special issue.
Journal of Pharmaceutical Sciences
The bioprotective properties of 2 disaccharides (sucrose and trehalose) were analyzed during the freezedrying (FD) process and at the end of the process, to better understand the stabilization mechanisms of proteins in the solid state. In situ Raman investigations, performed during the FD process, have revealed that sucrose was more efficient than trehalose for preserving the secondary structure of lysozyme during FD, especially during the primary drying stage. The lower bioprotective effect of trehalose was interpreted as a consequence of a stronger affinity of this disaccharide to water, responsible for a severe phase separation phenomenon during the freezing stage. Dielectric spectroscopy investigations on the freezedried state of protein formulations have shown the capabilities of trehalose assisted by residual water to reduce the molecular mobility of the vitreous matrix, suggesting that trehalose is more efficient to preserve the protein structure during long-term storage.