Lysozyme Formulations Research Articles

Influence of water and trehalose on α- and β-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 Tgβ. Based on an overview of applied methods to study beta relaxations, Dynamic Mechanical analysis was used to measure Tgα and Tgβ 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 Tgβ also was observed. On the other hand, an increasing amount of trehalose lowered Tgα but increased Tgβ showing that Tgα and Tgβ 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.

Bioanalytical Workflow for Qualitative and Quantitative Assessment of Hot-Melt Extruded Lysozyme Formulations.

Structural and functional integrities of formulated proteins are key characteristics that provide a better understanding of influencing factors and their adjustment during formulation development. Here, the procedures commonly used for protein analysis were applied and optimized to obtain a higher degree of accuracy, reproducibility, and reliability for the analysis of lysozyme extracts from hot-melt extrudates (HME). The extrudates were prepared with polyethylene glycol 20 000. The test lysozyme HMEs were subjected to extraction procedures and analytical methods following the International Council of Harmonization guidelines for testing the active protein ingredient Q 1 A (R2) in its pure and formulated form. Therefore, reversed-phase high-pressure liquid chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, matrix-assisted laser desorption ionization mass spectrometry, and fluorescence-based activity measurements were applied to study lysozyme stability and function after formulation. Long-term accelerated stability studies were performed for the pure and formulated protein. Our findings revealed a high degree of stability for lysozyme toward different temperatures and storage times, confirming that HME is a suitable formulation alternative that preserves lysozyme's properties and stability. The presented methods and workflow are recommended to be exploited for further protein drugs to assess usability and compatibility concerning different pharmaceutical applications.

Influence of a Small Amount of Glycerol on the Trehalose Bioprotective Action Analyzed In Situ During Freeze-Drying of Lyzozyme Formulations by Micro-Raman Spectroscopy

Micro-Raman spectroscopy gives the original opportunity to monitor simultaneously the operating process and the protein structure from in situ investigations along the 3 stages of the freeze-drying (FD) process. This opportunity was used for determining how a small amount of glycerol enhances the bioprotective efficiency of trehalose during FD of lysozyme formulations. Three lysozyme formulations were analyzed: lysozyme dissolved in D2O (wt% 1:9), in trehalose-D2O mixture (wt% 1:1:8), and in the trehalose-glycerol-D2O mixture (wt% 1:1:0.17:7.93). Raman mapping performed during each stage of the FD process has provided important information about the preferential interaction between water, trehalose, and lysozyme in relation to the protein stability. It was found that the addition of a small amount of glycerol had a plasticizing effect on the glassy trehalose-water matrix during the primary drying stage and then reduced the bioprotective effect of trehalose. By contrast, during the secondary drying stage, glycerol significantly enhanced the stabilizing effect of trehalose in the same sample, by replacing water-trehalose H-bonds with stronger glycerol-trehalose H-bonds and stiffening the amorphous trehalose matrix. The action of glycerol is also related to its capability to prevent aggregation of trehalose, making the structure of the frozen product more homogeneous, by changing the hydrogen-bond networks in the liquid formulation before the freezing stage.

Influences of copolymers (Copovidone, Eudragit RL PO and Kollicoat MAE 30 DP) on stability and bioactivity of spray-dried and freeze-dried lysozyme

Protein stability is the most crucial factor in protein pharmaceutical preparations. Various techniques were applied for producing stable protein formulations such as spray-drying and freeze-drying. However, heating and freezing stresses are disadvantages for proteins using these methods, respectively. Accordingly, excipients have been used to preserve therapeutic effects of proteins during processing and for long period of time. Therefore, influences of Copovidone, Eudragit® RL-PO and Kollicoat® MAE-30 DP (as excipients) on stability and integrity of lysozyme (as a model protein) in spray-dried and freeze-dried forms were investigated. Protein formulations in both dried forms were prepared without and with the addition of mentioned excipients at different concentrations. Protein formulations were characterized for yield determination, morphology using scanning electron microscopic (SEM), thermal analysis by Differential Scanning Calorimetry (DSC), secondary structure stability using Fourier transform infrared (FT-IR) spectroscopy and biological activity. All protein formulations were subjected to a stability study as solid protein formulations for 3 weeks at 24 °C/76% relative humidity and aqueous protein samples were stored at 50 °C for 30 min in a water bath. Results showed that Copovidone successfully preserved integrity and biological activity of lysozyme before and after storage in both spray-dried and freeze-dried forms with more advantage for using higher concentration of the same excipient. Smooth spheres of spray-dried lysozyme formulations with Copovidone were smaller than spray-dried lysozyme without and with Kollicoat® MAE-30 DP, which affected %yield produced. Copovidone has demonstrated valuable protection ability for lysozyme.

Scalable organic solvent free supercritical fluid spray drying process for producing dry protein formulations

In this study, we evaluated the influence of supercritical carbon dioxide (scCO2) spray drying conditions, in the absence of organic solvent, on the ability to produce dry protein/trehalose formulations at 1:10 and 1:4 (w/w) ratios. When using a 4L drying vessel, we found that decreasing the solution flow rate and solution volume, or increasing the scCO2 flow rate resulted in a significant reduction in the residual water content in dried products (Karl Fischer titration). The best conditions were then used to evaluate the ability to scale the scCO2 spray drying process from 4L to 10L chamber. The ratio of scCO2 and solution flow rate was kept constant. The products on both scales exhibited similar residual moisture contents, particle morphologies (SEM), and glass transition temperatures (DSC). After reconstitution, the lysozyme activity (enzymatic assay) and structure (circular dichroism, HP-SEC) were fully preserved, but the sub-visible particle content was slightly increased (flow imaging microscopy, nanoparticle tracking analysis). Furthermore, the drying condition was applicable to other proteins resulting in products of similar quality as the lysozyme formulations. In conclusion, we established scCO2 spray drying processing conditions for protein formulations without an organic solvent that holds promise for the industrial production of dry protein formulations.

Structural attributes of model protein formulations prepared by rapid freeze-drying cycles in a microscale heating stage

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.

Uncomplicated oocyte donation pregnancies are associated with a higher incidence of human leukocyte antigen alloantibodies

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.

Constant size, variable density aerosol particles by ultrasonic spray freeze drying

This work provides a new understanding of critical process parameters involved in the production of inhalation aerosol particles by ultrasonic spray freeze drying to enable precise control over particle size and aerodynamic properties. A series of highly porous mannitol, lysozyme, and bovine serum albumin (BSA) particles were produced, varying only the solute concentration in the liquid feed, cs, from 1 to 5wt%. The particle sizes of mannitol, BSA, and lysozyme powders were independent of solute concentration, and depend only on the drop size produced by atomization. Both mannitol and lysozyme formulations showed a linear relationship between the computed Fine Particle Fraction (FPF) and the square root of cs, which is proportional to the particle density, ρ, given a constant particle size dg. The FPF decreased with increasing cs from 57.0% to 16.6% for mannitol and 44.5% to 17.2% for lysozyme. Due to cohesion, the BSA powder FPF measured by cascade impaction was less than 10% and independent of cs. Ultrasonic spray freeze drying enables separate control over particle size, dg, and aerodynamic size, da which has allowed us to make the first experimental demonstration of the widely accepted rule da=dg(ρ/ρo)1/2 with particles of constant dg, but variable density, ρ (ρo is unit density).

Correlation of protein-protein interactions as assessed by affinity chromatography with colloidal protein stability: A case study with lysozyme

Lysozyme-lysozyme interactions were assessed in the native state at 25°C as well in the denatured state at 80°C by affinity chromatography in order to measure the osmotic second virial coefficient (B). This parameter allows us to better understand protein aggregation pathways and colloidal protein stability. Repulsive interactions (B > 0) were weakened for both protein states by increasing salt concentration and by increasing the pH value toward lysozyme pI. This decrease was more pronounced in the denatured state, most likely caused by changes in electrostatic interactions and the formation of hydrophobic clusters. The lysozyme formulations presenting the more repulsive conditions (B > 0), as derived from the osmotic second virial coefficient, showed better colloidal stability under mechanical and thermal stresses. As expected, B values are much more negative for the interactions in the denatured state compared to the data obtained for the native state, reflecting a strong tendency of denatured lysozyme to aggregate. Thus, measurement of protein interactions by affinity chromatography allows us to gain information on protein interactions in both native and denatured states as well as to predict solution conditions prone for improving protein colloidal stability.

Investigation of the physical properties of spray-dried stabilised lysozyme particles.

The aim of this study was to investigate the effect of the composition of formulations on the physical properties, including glass-transition temperatures (Tg) and aerodynamic-related characteristics, of spray-dried lysozyme particles. The Tg, as determined by differential scanning calorimetry, of spray-dried lysozyme formulations was found to be dependent upon the type and amount of excipient(s) included in the formulation. In addition, the Tg of sucrose-containing particles appeared to be raised markedly by the inclusion of trehalose, but not by dextran. The surfaces of all spray-dried particles were shown by scanning electron microscopy to be smooth with some containing characteristic dimples, typical of spray-dried material, and the morphology appeared to be independent of variation in excipient composition. However, the volume median diameters (VMD) of spray-dried powders, as determined by laser diffraction, were found to depend upon the amounts of excipients. The fine particle fraction of enzyme delivered to the lower stage of a twin-stage impinger from lysozyme-trehalose 1:1 powders appeared to be greater than that from lysozyme-sucrose 1:1 particles (22.5% vs 15.9%) when dispersed via a Rotahaler although a similar dispersibility of the two formulations (39.6% vs 36.7%) was found from a glass inhaler. In general, spray-drying was demonstrated to be feasible to produce respirable particles of the stabilised model protein, with Tg of the formulations being > 30 degrees C higher than room temperature.

A solid-state NMR study of molecular mobility and phase separation in co-spray-dried protein–sugar particles

Molecular mobility and physical form of co-spray-dried sugar–lysozyme formulations were evaluated. Co-spray-dried trehalose:lysozyme and sucrose:lysozyme formulations in 1:9, 1:1 and 9:1 ratios (w:w) were stored at 0% RH and 75% RH for 5–6 days. Molecular mobility and physical form of the co-spray-dried formulations after storage were determined by using 13C and 1H solid-state NMR as well as X-ray powder diffractometry. The results showed that increasing sugar content in co-spray-dried formulations stored at 0% RH decreased molecular mobility of the amorphous formulations indicating a close association of the protein and sugar. Exposure of sugar–lysozyme 1:1 and 9:1 formulations to 75% RH led to separation of sugar and protein phases, where the sugar phase was crystalline. The intimate sugar–lysozyme interaction of the formulations stored at 0% RH and the phase separation of the sugar-rich formulations stored at 75% RH were also confirmed by using 13C solid-state NMR spin-lattice relaxation time-filter (T 1-filter) measurements. The propensity of sucrose and trehalose to crystallise was similar; however, the results suggest that part of the sugar in the phase-separated formulations remained amorphous and in close association with lysozyme.