Physical characterisation of formulations for the development of two stable freeze-dried proteins during both dried and liquid storage (original) (raw)
Related papers
Archives of biochemistry …, 1996
Together the results of these studies document that to obtain optimum stability of dried rhIL-1ra it was The effects of glass transition of, and protein confornecessary to inhibit conformational change during lymation in, the dried solid on the storage stability of ophilization and to store at temperatures below the Tg freeze-dried recombinant human interleukin-1 recepof the dried formulation. ᭧ 1996 Academic Press, Inc. tor antagonist (rhIL-1ra) were examined. Glass transition is a temperature-dependent phenomenon. Amorphous materials become hard and brittle at temperatures below their characteristic glass transition tempera-There are numerous unique, critical applications for tures (Tg) such that diffusion of molecules along the proteins in human health care. However, even the most matrix is not sufficient to cause large-scale structural promising protein therapeutic will not be useful, if its changes. To ascertain the importance of the glass transtability cannot be maintained during shipping and sition in protein storage stability, we compared 10 diflong-term storage (1, 2). The inherent instability of proferent lyophilized rhIL-1ra formulations, with Tgs teins often precludes preparation of formulations as ranging from 20 to 56ЊC, during several weeks of storaqueous solutions (2). However, if the water is removed age at temperatures above and below the samples' Tgs. by freeze-drying (lyophilization), the dehydrated pro-Protein degradation, both deamidation and aggregatein theoretically should be much more resistant to tion, was greatly accelerated at temperatures above Tg, but for some formulations also arose below Tg. damage, even at ambient temperatures (3, 4). We have Thus, storage of dried proteins below the Tg is neces-recently achieved such long-term stability (5) with sary but not sufficient to ensure long-term stability. freeze-dried recombinant human interleukin-1 recep-To examine the effects of protein structure in the dried tor antagonist (rhIL-1ra). 3 An optimum formulation, solid, we prepared formulations with various sucrose containing 100 mg/ml rhIL-1ra, 2% (wt/vol) glycine, 10 concentrations, all of which had a Tg Å 66 { 2.5ЊC. With mM sodium citrate (pH 6.5), and 10% (wt/vol) sucrose, infrared spectroscopy, we determined that the protein could be stored for 56 weeks at 30ЊC with no detectable lyophilized with £1% sucrose was unfolded in the inidamage to the protein and at 50ЊC with only a 4% loss tial dried solid. In contrast, in those formulations with of native protein due to deamidation. The purpose of §5% sucrose, conformational change was inhibited the current study was to investigate the physical bases during lyophilization. When stored at 50ЊC, degradafor this stability and, in so doing, test rigorously the tion of the freeze-dried protein varied inversely with proposed mechanisms for storage stability of freezesucrose concentration. These results indicate that dried proteins. structural changes arising during the lyophilization Two physical criteria have been proposed to be improcess led to damage during subsequent storage, portant for long-term stability of dried proteins. First, even if the storage temperature was less than the Tg.
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 Food Engineering, 2011
In this study, to preserve the integrity of plasma protein, protective agents, such as saccharides are added to produce a glassy (vitrified) state. Differential scanning calorimetry (DSC) was used to measure the glass transition (T g ), crystallization temperatures (T c ) of the solid freeze-dried bovine plasma protein and the glass transition temperature (T 0 g ) of the protein freeze solution, with the addition of inulin as protective agent, comparing the behavior with glucose and sucrose. The results indicated that transition temperatures increased with the molecular weight of the saccharide, conferring inulin a stabilizing effect at higher storage temperature. The T 0 g and the water plasticizing effect were estimated by means of two theoretical models: Miller/Fox and Gordon/Taylor extended for multi-component systems. The determination of the glass transition temperatures is useful in defining a freeze-drying cycle and storage stability of plasma protein concentrates.
The physical behaviour of food protein's supramolecular structures during freeze- drying
2011
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Journal of Pharmaceutical Sciences, 2002
This work investigates the use of spray freeze-drying (SFD) to produce protein loaded particles suitable for epidermal delivery. In the first part of the study, the effects of formulation and process conditions on particle properties are examined. Aqueous solutions of trehalose produce SFD particles in the size range 20-80 mm, with a smooth, textured surface, but having high internal porosity. The latter was visualized using SEM and a novel particle embedding and sectioning technique. Use of an annealing step during the freeze-drying cycle caused the particles to shrink, reducing hereby porosity and also the measured rate of moisture uptake into these amorphous particles. SFD pure mannitol was approximately 40% amorphous, but not hygroscopic. Incorporation of dextran 37,500 into a combined amorphous trehalose/mannitol formulation led to increased particle shrinkage and lower particle porosity on annealing. The model protein trypsinogen lost approximately 15% activity during SFD of solutions containing 50 mg/mL protein, but was only marginally aggregated (1.4%). It is suggested that trypsinogen forms an irreversible partially unfolded state or molten globule on SFD/rehydration. The pure protein was also partially inactivated without aggregation during atomization into air. Surprisingly, neither activity loss nor aggregation were detected on atomization of the protein solution into liquid nitrogen. Quench-freezing of small droplets may reverse the partial unfolding of trypsinogen occurring on atomization into air. The origin of the trypsinogen inactivation during SFD must therefore be the subsequent freeze-drying step of this multistep process. Isolated freeze drying of trypsinogen produces strong aggregation and equivalent inactivation. This result suggests that trypsinogen behaves differently during freeze drying from frozen droplets and from bulk solution in a vial. In the former case the protein forms an irreversible partially unfolded state, whereas in the latter case aggregates are formed. Trypsinogen inactivation during SFD could be completely prevented by the presence of trehalose in the formulation. Electron Spectroscopy for Chemical Analysis (ESCA) showed a high surface excess of the protein in the SFD particles, which was reduced on inclusion of Polysorbate 80, but not trehalose. Taken together, these results help to elucidate the complex destabilization behavior of trypsinogen during SFD.
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
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.
Archives of Biochemistry and Biophysics, 1998
Freeze-drying is often used to improve storage stability of therapeutic proteins. In order to obtain a product with optimal storage stability it is important to understand the mechanisms by which solutes protect the protein against freeze-drying-induced stresses and also against damage induced during subsequent storage. The objective of the current study was to examine the importance of various mechanisms proposed to account for acute and long-term storage stability using recombinant human Factor XIII (rFXIII) 4 as a model protein. Initially, for acute stability during freeze-drying, it was found that solutes which formed an amorphous phase stabilized rFXIII to a greater degree than solutes which crystallized during freezedrying. However, only amorphous solutes which were able to hydrogen bond to the protein, and thus preserve the native protein structure in the dried solid, provided optimal acute stability. Thus, in addition to forming an amorphous phase, it was also important to possess the ability to hydrogen bond to the protein. Long-term storage stability was found to be optimal in the presence of solutes which formed and maintained amorphous phases with T g values above the storage temperature and which also preserved the native protein structure during freeze-drying. Solute crystallization during storage compromised storage stability.