Chung C. Hsu

Protein Inhalation Powders: Spray Drying vs Spray Freeze Drying

AbstractPurpose. To develop a new technique, spray freeze drying, for preparing protein aerosol powders. Also, to compare the spray freeze-dried powders with spray-dried powders in terms of physical properties and aerosol performance. Methods. Protein powders were characterized using particle size analysis, thermogravimetric analysis, scanning electron microscopy, X-ray powder diffractometry, and specific surface area measurement. Aerosol performance of the powders was evaluated after blending with lactose carriers using a multi-stage liquid impinger or an Anderson cascade impactor. Two recombinant therapeutic proteins currently used for treating respiratory tract-related diseases, deoxyribonuclase (rhDNase) and anti-IgE monoclonal antibody (anti-IgE MAb), were employed and formulated with different carbohydrate excipients. Results. Through the same atomization but the different drying process, spray drying (SD) produced small (∼3 μm), dense particles, but SFD resulted in large (∼8−10 μm), porous particles. The fine particle fraction (FPF) of the spray freeze-dried powder was significantly better than that of the spray-dried powder, attributed to better aerodynamic properties. Powders collected from different stages of the cascade impactor were characterized, which confirmed the concept of aerodynamic particle size. Protein formulation played a major role in affecting the powders aerosol performance, especially for the carbohydrate excipient of a high crystallization tendency. Conclusions. Spray freeze drying, as opposed to spray drying, produced protein particles with light and porous characteristics, which offered powders with superior aerosol performance due to favorable aerodynamic properties.

Protein denaturation by combined effect of shear and air‐liquid interface

The effect of shear alone on the aggregation of recombinant human growth hormone (rhGH) and recombinant human deoxyribonuclease (rhDNase) has been found to be insignificant. This study focused on the synergetic effect of shear and gas-liquid interface on these two model proteins. Two shearing systems, the concentric-cylinder shear device (CCSD) and the rotor/stator homogenizer, were used to generate high shear (> 10(6)) in aqueous solutions in the presence of air. High shear in the presence of an air-liquid interface had no major effect on rhDNase but caused rhGH to form noncovalent aggregates. rhGH aggregation was induced by the air-liquid interface and was found to increase with increasing protein concentration and the air-liquid interfacial area. The aggregation was irreversible and exhibited a first-order kinetics with respect to the protein concentration and air-liquid interfacial area. Shear and shear rate enhanced the interaction because of its continuous generation of new air-liquid interfaces. In the presence of a surfactant, aggregation could be delayed or prevented depending upon the type and the concentration of the surfactant. The effect of air-liquid interface on proteins at low shear was examined using a nitrogen bubbling method. We found that foaming is very detrimental to rhGH even though the shear involved is low. The use of anti-foaming materials could prevent rhGH aggregation during bubbling. The superior stability exhibited by rhDNase may be linked to the higher surface tension and lower foaming tendency of its aqueous solution. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 503-512, 1997.

The Effect of Formulation Excipients on Protein Stability and Aerosol Performance of Spray-Dried Powders of a Recombinant Humanized Anti-IgE Monoclonal Antibody1

AbstractPurpose. To study the effect of trehalose, lactose, and mannitol on the biochemical stability and aerosol performance of spray-dried powders of an anti-IgE humanized monoclonal antibody. Methods. Protein aggregation of spray-dried powders stored at various temperature and relative humidity conditions was assayed by size exclusion chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis. Protein glycation was determined by isoelectric focusing and affinity chromatography. Crystallization was examined by X-ray powder diffraction. Aerosol performance was assessed as the fine particle fraction (FPF) of the powders blended with coarse carrier lactose, and was determined using a multiple stage liquid impinger. Results. Soluble protein aggregation consisting of non-covalent and disulfide-linked covalent dimers and trimers occurred during storage. Aggregate was minimized by formulation with trehalose at or above a molar ratio in the range of 300:1 to 500:1 (excipient:protein). However, the powders were excessively cohesive and unsuitable for aerosol administration. Lactose had a similar stabilizing effect, and the powders exhibited acceptable aerosol performance, but protein glycation was observed during storage. The addition of mannitol also reduced aggregation, while maintaining the FPF, but only up to a molar ratio of 200:1. Further increased mannitol resulted in crystallization, which had a detrimental effect on protein stability and aerosol performance. Conclusions. Protein stability was improved by formulation with carbohydrate. However, a balance must be achieved between the addition of enough stabilizer to improve protein biochemical stability without compromising blended powder aerosol performance.

Mechanisms of aggregate formation and carbohydrate excipient stabilization of lyophilized humanized monoclonal antibody formulations.

The purpose of this study was to evaluate the mechanisms of aggregate formation and excipient stabilization in freeze-dried formulations of a recombinant humanized monoclonal antibody. Protein degradation was measured using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) and native size exclusion chromatography, and protein structure was studied using Fourier transform-infrared spectrometry and circular dichroism. The results showed that protein aggregates present following reconstitution were composed of native antibody structure and a reduced amount of free thiol when compared to protein monomer, which implied that intermolecular disulfides were involved in the aggregation mechanism. An excipient-free formulation resulted in reversible solid-state protein structural alteration and increased aggregation during storage. This correlated with dehydration to an extent that the amount of water was less than the estimated number of surface-accessible hydrogen-bonding sites on the protein. Improved native-like solid-state protein structure and reduced aggregation were obtained by formulation with enough carbohydrate to fulfill the hydrogen-bonding sites on the surface of the protein. Carbohydrate in excess of this concentration has less of an influence on protein aggregation. Reduced aggregation during storage was obtained by the addition of sufficient excipient to both stabilized solid-state protein structure and provide an environment that consisted of an amorphous glassy state matrix.

Effect of Spray Drying and Subsequent Processing Conditions on Residual Moisture Content and Physical/Biochemical Stability of Protein Inhalation Powders

AbstractPurpose. To understand the effect of spray drying and powder processing environments on the residual moisture content and aerosol performance of inhalation protein powders. Also, the long-term effect of storage conditions on the powders physical and biochemical stability was presented. Methods. Excipient-free as well as mannitol-formulated powders of a humanized monoclonal antibody (anti-IgE) and recombinant human deoxyribonuclease (rhDNase) were prepared using a Buchi 190 model spray dryer. Residual moisture content and moisture uptake behavior of the powder were measured using thermal gravimetric analysis and gravimetric moisture sorption isotherm, respectively. Protein aggregation, the primary degradation product observed upon storage, was determined by size-exclusion HPLC. Aerosol performance of the dry powders was evaluated after blending with lactose carriers using a multi-stage liquid impinger (MSLI). Results. Spray-dried powders with a moisture level (~ 3%) equivalent to the freeze-dried materials could only be achieved using high-temperature spray-drying conditions, which were not favorable to large-male manufacturing, or subsequent vacuum drying. These dry powders would equilibrate with the subsequent processing and storage environments regardless of the manufacturing condition. As long as the relative humidity of air during processing and storage was lower than 50%, powders maintained their aerosol performance (fine particle fraction). However, powders stored under drier conditions exhibited better long-term protein biochemical stability. Conclusions. Manufacturing, powder processing, and storage environments affected powders residual moisture level in a reversible fashion. Therefore, the storage condition determined powders overall stability, but residual moisture had a greater impact on protein chemical stability than on powder physical stability.

Effect of high shear on proteins

Shear is present in almost all bioprocesses and high shear is associated with processes involving agitation and emulsification. The purpose of this study is to investigate the effect of high shear and high shear rate on proteins. Two concentric cylinder‐based shear systems were used. One was a closed concentric‐cylinder shear device (CCSD) and the other was a homogenizer with a rotor/stator assembly. Mathematical modeling of these systems allowed calculation of the shear rate and shear. The CCSD generated low shear rates (a few hundred s−1), whereas the homogenizer could generate very high shear rates (> 105 s−1). High shear could be achieved in both systems by increasing the processing time. Recombinant human growth hormone (rhGH) and recombinant human deoxyribonuclease (rhDNase) were used as the model proteins in this study. It was found that neither high shear nor high shear rate had a significant effect on protein aggregation. However, a lower melting temperature and enthalpy were detected for highly sheared rhGH by using scanning microcalorimetry, presumably due to some changes in proteins conformation. Also, SDS‐PAGE indicated the presence of low molecular‐weight fragments, suggesting that peptide bond breakage occurred due to high shear. rhDNase was relatively more stable than rhGH under high shear. No conformational changes and protein fragments were observed.

Performance of sonication and microfluidization for liquid-liquid emulsification.

The purpose of this research was to evaluate and compare liquid-liquid emulsions (water-in-oil and oil-in-water) prepared using sonication and microfluidization. Liquid-liquid emulsions were characterized on the basis of emulsion droplet size determined using a laser-based particle size analyzer. An ultrasonic-driven benchtop sonicator and an air-driven microfluidizer were used for emulsification. Sonication generated emulsions through ultrasound-driven mechanical vibrations, which caused cavitation. The force associated with implosion of vapor bubbles caused emulsion size reduction and the flow of the bubbles resulted in mixing. An increase in viscosity of the dispersion phase improved the sonicators emulsification capability, but an increase in the viscosity of the dispersed phase decreased the sonicators emulsification capability. Although sonication might be comparable to homogenization in terms of emulsification efficiency, homogenization was relatively more effective in emulsifying more viscous solutions. Microfluidization, which used a high pressure to force the fluid into microchannels of a special configuration and initiated emulsification via a combined mechanism of cavitation, shear, and impact, exhibited excellent emulsification efficiency. Of the three methodologies, sonication generated more heat and might be less suitable for emulsion systems involving heat-sensitive materials. Homogenization is in general a more effective liquid-liquid emulsification method. The results derived from this study can serve as a basis for the evaluation of large-scale liquid-liquid emulsification in the microencapsulation process.

Water sorption behavior of lyophilized protein–sugar systems and implications for solid-state interactions

Abstract This study examines the water sorption behavior of proteins co-lyophilized with sugar/polyol excipients. Gravimetric sorption analysis (GSA) was used to measure water sorption of the lyophilized mixtures and these data allowed for calculation of the water monolayer ( M 0 ). Lyophilized protein–mannitol mixtures behaved as predicted from the data for the pure components. Mannitol was shown to crystallize upon lyophilization. For protein co-lyophilized with sucrose or trehalose, which remain amorphous upon lyophilization, M 0 tended to be lower than that expected based on contributions of the pure protein and sugar. This negative deviation supports the view that amorphous sugars and pharmaceutical proteins interact in the solid state in such a way as to reduce the availability of water-binding sites. At high relative humidities (rh), sucrose and trehalose were susceptible to moisture-induced crystallization. When co-lyophilized protein was present, the GSA data revealed that this crystallization required a higher rh, or did not occur. For the temperature-induced (non-isothermal) sucrose crystallization, which was studied by differential scanning calorimetry, it was found that the temperature of crystallization tended to increase with an increasing amount of protein. The tendency to crystallize rose in the presence of elevated moisture, whether or not protein was present, likely due to the ability of water to plasticize the solid phase.

Spray-drying performance of a bench-top spray dryer for protein aerosol powder preparation

The objective of this work was to improve a bench-top spray dryers efficiency in both production recovery and throughput for preparing protein aerosol powders. A Büchi mini-spray dryer was used to prepare the powders of recombinant humanized anti-IgE antibody. The resulting powders physical properties such as particle size, residual moisture, and morphology, along with its recovery and production rate was the basis of this development work. Mass balance suggests that approximately 10-20% of powder was lost in the exhaust air, consisting primarily of particles less than 2 micrometer. Also, significant loss (20-30%) occurred in the cyclone. Attempts were made to improve product recovery in the receiving vessel using dual-cyclone configurations, different cyclone designs, cyclones with anti-static treatment, and different receiver designs. System modifications such as replacing the original bag-filter unit with a vacuum system effectively reduced drying air flow resistance, allowing the protein to be dried at a lower inlet air temperature and the production scale to be increased. We concluded that the modified spray-drying system is advantageous over the original bench-top spray dryer. This improvement will be beneficial to early-stage research and development involving high-valued protein powders.

Aggregation of recombinant human growth hormone induced by phenolic compounds

Phenolic compounds can be used as antimicrobial preservative agents in pharmaceutical formulations. Unfortunately, these compounds often adversely affect proteins, triggering aggregation in particular. In this study, a variety of phenolic compounds and structurally similar non-aromatic alcohols were investigated for their role in causing the aggregation of a model therapeutic protein, recombinant human growth hormone (rhGH). As determined by various methodologies, most of the phenolic compounds caused rhGH aggregation, especially at high concentrations. Stress studies under freezing, high-temperature incubation, and agitation suggest that the destabilizing influence of the compounds tested increases in the order of benzyl alcohol < phenol × resorcinol < catechol < meta-cresol < 2-chlorophenol. Non-aromatic alcohols, except 2,6-dimethylcyclohexanol, have a much less adverse effect. Determination of the thermal transition temperature by microcalorimetry studies also reflected this trend. From our study, we conclude: (1) the phenolic additive-induced rhGH aggregates were held by non-covalent forces; (2) no significant physical binding occurred between the protein and these compounds; (3) the aggregation tendency of the phenolic compounds failed to correlate with their hydrogen bonding strength; (4) the presence of a phenolic additive caused conformational changes in rhGHs structure; (5) the effect of these phenolic compounds on rhGH aggregation decreased at high and low pHs.