Structural attributes of model protein formulations prepared by rapid freeze-drying cycles in a microscale heating stage (original) (raw)
Related papers
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.
Integrity of crystalline lysozyme exceeds that of a spray-dried form
International journal of pharmaceutics, 2002
The development of proteins as therapeutic agents is challenging partly due to their inherent instabilities. Consequently, crystallisation and spray drying techniques were assessed to determine their effects on protein integrity using lysozyme as a model protein. Unprocessed, crystallised and spray-dried lysozyme were characterised by: thermal analysis using hot stage microscopy (HSM), differential scanning calorimetry (DSC), high sensitivity differential scanning calorimetry (HSDSC) and thermogravimetry (TGA); and spectroscopic analysis employing Fourier transform Raman (FT-Raman). Moisture contents were determined by TGA and Karl Fisher titration (KFT). Enzymatic assay measured biological activity. HSM showed no changes in crystals until complete melting. TGA and KFT indicated that spray-dried lysozyme contained a lower moisture content than crystals, hence the higher apparent thermal stability was shown by DSC. HSDSC revealed that crystallisation and spray drying did not affect t...
Stability of crystallised and spray-dried lysozyme
International journal of pharmaceutics, 2004
Moisture and temperature promote protein degradation. The stabilities of commercial, crystallised and spray-dried lysozyme, a model protein, were assessed under these stresses to explore whether a crystalline protein had better storage stability than a conventionally produced one. Samples were maintained at different relative humidities (RH) and temperatures for 20 weeks and stabilities estimated in solid and aqueous states. Differential scanning calorimetry (DSC) and thermogravimetry (TGA) characterised solid samples. Fourier transform Raman (FT-Raman) spectroscopy analysed solid material and aqueous solutions. High sensitivity differential scanning calorimetry (HSDSC) and enzymatic assays were used to monitor solutions. DSC and HSDSC data revealed that crystals maintained thermal stability at high RH; spray drying appreciably changed melting characteristics. These results correlated with enzymatic assays that demonstrated good activity retention for crystals but less so for spray-...
Future Journal of Pharmaceutical Sciences
Background Process intensification is a major hurdle in pharmaceutical process scale-up. Solvent removal strategies have limited the effectiveness of the overall stability of pharmaceutical formulations. The main aim of present review article is to focus on the use of the freeze-drying process in pharmaceuticals, biopharmaceuticals and nanoderived therapeutics and their translation into commercial viable products. Unwavering efforts of scientists in the process intensification of lyophilization promote unique features of products for commercialization. Regulatory agencies are promoting the utilization of a quality-by-design approach to improve product characteristics. Among 300 FDA-approved pharmaceutical industries, 50% of products are freeze-dried. The freeze-drying process is costlier and requires more time than other drying methodologies. Unstable pharmaceutical dispersions and solutions can be preferably stabilized by using the freeze-drying method. Main text This review highli...
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.
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, 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.
The physical behaviour of food protein's supramolecular structures during freeze- drying
2011
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A new scaleable freeze–thaw technology for bulk protein solutions
Biotechnology and Applied Biochemistry, 2007
A new method of freeze-thaw is described using experimental data obtained from freezing of purified rhGH (recombinant human growth hormone). The method is based on freezing protein solutions in rectangular rather than cylindrical containers. It is hypothesized that the change in container geometry allows for linear scale-up of the freeze-thaw operation based on equivalency of temperature-time profile. The hypothesis is tested using freeze-thaw data from a miniature (30 ml) and a 2.4 litre container. Computational fluid dynamics techniques are used to simulate the freeze process and the simulations are compared with experimental results. Protein quality is assessed as a function of freeze conditions using dynamic light scattering, circular CD, size-exclusion and reverse-phase HPLC measurements. The results demonstrate the applicability of the new approach. Freezing of rhGH solution at concentrations of approx. 30 mg/ml is shown to be possible with no damage to the molecule for up to five cycles of freeze-thaw. A nitrogen blast chest-freezer is designed and evaluated as part of the process. The refrigeration system and the freezethaw method can be used to freeze-thaw bulk protein solutions for development work and has the potential for transfer to manufacturing.
Effect of Spray-Drying and Electrospraying as Drying Techniques on Lysozyme Characterisation
Electrospinning and Electrospraying - Techniques and Applications, 2019
The production of biopharmaceutical formulation incorporates several difficulties embracing their physical and chemical instabilities. In this study, two drying techniques, namely, spray-drying and electrospraying, were used to assess their application on lysozyme (as a model protein) without and with the use of betacyclodextrin. Samples were prepared in the ratio of 1:1 w/w (protein/ betacyclodextrin), and several characterisation methods were applied to study the percentage (%) yield, morphology of the produced partials, thermal stability and biological activity of the protein. The results show the two drying methods led to different particle morphology as spherical-like shape was produced by spray-drying, while rodlike shape was generated by electrospraying with larger particle size. Lysozyme formulations produced by electrospraying were stable just directly after preparation, but after few weeks, those formulations showed visible aggregates. The biological activity of lysozyme was preserved by both drying techniques. In conclusion, both drying methods have different effects on the protein integrity and biological activity in which spray-drying shows more promising results.