This study examined the drying kinetics of catalase protein solutions using acoustic levitation. Catalase droplets containing trehalose dried more slowly and formed denser particles compared to catalase-only droplets. Trehalose increased the residual enzyme activity of catalase to 95% while droplets dried at higher humidity showed lower activity. Acoustic levitation is a useful technique for studying single droplet drying kinetics but may not replicate conditions of spray drying.

1. Determination of Sin g le Dro p let Dr y in g Kinetics of Protein Solutions via Acoustical Levitation Heiko A. Schiffter & Geoffrey Lee Department of Pharmaceutics - Institute for Pharmacy and Food Chemistry Friedrich–Alexander-University Erlangen-Nuremberg [E-mail correspondence of the presenting author: schiffter@pharmtech.uni-erlangen.de] Spray-drying is a well known method to produce protein and peptide loaded powders for pulmonary or parenteral application. Unfortunately, there are effects like shearing stress in the nozzle, thermal stress in the spray-drying tower, aggregation and unfolding of the protein at the air-liquid interface [1]. To investigate influences of the formulation excipients on the drying kinetics and the particle formation, single droplet drying methods have been brought forward in the last years [2]. Acoustic levitation is the containerless processing of small liquid or solid samples in the pressure nodes of a standing acoustic wave between an ultrasonic transducer and a reflector at a distance of a multiple half wavelength [3]. The characteristics of the acoustic levitator enable the determination of the drying kinetics of a single droplet within the constant rate and the falling rate of the drying process [4] (Fig.1). In the presented work the levitation system was used to examine the influence of trehalose on the drying kinetics of catalase solutions, the particle morphology and the residual activity of the enzyme. Materials: Catalase from bovine liver and trehalose-dihydrate (Sigma, Germany) were dissolved in different ratio in a 50mM tris buffer pH 8.0 with a total solid content of 100mg/ml. Levitation s y stem: A 58kHz levitator (tec5-AG, Germany) was used together with a controlled evaporation mixer (Bronkhorst) and a CCD-camera (Jai AG) connected to a PC and an imaging software (ImageProPlus, Media Cybernetics) (Fig.1). Experiments were performed at 60°C and different humidity of the drying air The evaporation rate in the first stage was determined using the change of size of droplet volume with time (Equation 1). In the second drying stage the evaporation rate was calculated using the rise of the particle in the stationary ultrasonic field (Equation 2) [4]. S p ra y -dryin g : Spray-drying was performed in a Buchi B-190 Mini Spray-Dryer (Buchi, Switzerland) at 100°C inlet and 60°C outlet temperature with 54m 3 /h drying air flow rate. Liquid feed was 3.0ml/min with an atomizing air flow rate of 700l/min. Enz y me activit y : The rate of disappearance of H 2 O 2 is followed by monitoring the decrease in absorbance at 240 nm in 50mM phosphate buffer at pH 7.0 using a PerkinElmer Lambda 25 UV/VIS spectrometer [5]. SEM p ictures: SEM pictures of the particles were taken using an Amray 1810T scanning electron microscope. The samples were spluttered with gold at 20mA/5kV for 1.5 minutes. Evaporation rate: The evaporation rate is plotted in mass per time and surface area with evaporation time divided by the squared initial droplet radius to compensate different initial droplet volumes in still air experiments (Fig.2). The influence of trehalose leads to an increase in the duration of the first drying stage and to larger values of the evaporation rate in the second. The critical point marking the transition between the two drying stages shifts to later points of time in the drying process (Fig.2). The evaporation rate decreases with increasing humidity of the drying air at 60°C for all formulations (Fig.3). Particle formation: Pure catalase solutions form hollow particles that rip open after the formation of a solid crust during the falling rate period (Fig.2; Pic.1/2). Influences of the axial levitation force of the standing acoustic wave causes impressions of the top of the particle and facilitate the damage of the new formed particle surface. Holes in the surface lead to an apparent increase of the evaporation rate in this phase due to the formation of a larger surface area. Increasing trehalose content forms massive particle structures and a decreasing evaporation rate after the critical point (Fig.2; Pic.3). Particle damage or surface rupture are not observed. The particle size decreases with increasing trehalose fraction of the mixture. Therefore, a decrease in density of the resulting particles can be observed. A longer evaporation time and slower particle formation at higher humidity results in denser particles than observed at 60°C and 5% humidity. Enzyme activity: The residual enzyme activity of catalase increases with addition of trehalose to the catalase solution. 20% trehalose of the solid content was sufficient to achieve a residual enzyme activity of 95%. Further increase in the trehalose content has no additional effect. An increasing evaporation time due to an increasing drying air humidity leads to lower values of the residual catalase activity (Fig.5). The surface temperature of the droplet increases from 24.9°C at 5% humidity to 43.2°C at 40% humidity. Different examinations on the determination of enzyme activity in levitated water droplets show, that a influence of the standing acoustic wave on the residual activity can be excluded [6]. In contrast, untreated dissolved catalase shows a 15% loss in activity in 3 hours at 25°C. Particles collected 5 minutes after the actual end of the drying process show the same residual enzyme activity. Values for catalase dried in the levitator are lower than for all spray-dried solutions (Fig.5). Acoustic levitation is a possibility to investigate the influences of formulations on the single droplet drying kinetics and the particle morphology. Due to the long evaporation time at 60°C and the large droplet size of the samples in the levitation system, a direct correlation of the residual enzyme activity to spray-drying experiments of the same catalase-trehalose formulations could not be shown. Furthermore, influence of the standing acoustic wave on the particle formation during the drying process can lead to different particle shape and morphology and in worst case to a change in surface area that influences the drying kinetics. In this study the morphological similarity to the spray-dried product seems very good. [1] Lee, G. (2002): Spray-drying of proteins. Pharm Biotechnol. 13, 135-158 [2] Walton, D.E.; Mumford, C.J. (1999): The morphology of spray-dried particles. Trans IChemE 77, 442-460 [3] Lierke, E.G. (2002): Deformation and displacement of liquid drops in an optimized acoustic standing wave levitator. Acta Acoustica united with Acoustica. 88, pp. 206-207 [4] Kastner, O.; Brenn, G.; Rensink; D., Tropea; C. (2001): The acoustic levitator – A novel device for determining the drying kinetics of single droplets. Chem. Eng. Technol. 24, pp.335-339 [5] Beers, R.F.J.; Sizer, I.W. (1952): A spectrometric method for measuring the breakdown of hydrogen peroxide by catalase. Journal of Biological Chemistry. 195, pp. 133-140 [6] Weis, D.D.; Nardozzi, J.D. (2005): Enzyme kinetics in acoustically levitated droplets of supercooled water: a new approach to Cryoenzymology. Anal. Chem. 77, 2558-2563 Introduction Materials and Methods Results and Discussion Conclusion References Fig.1: Set-up of the levitation system Equ.1/2: Calculation of the evaporation rate Fig.2: Evaporation rate at 60°C and 20% humidity Fig.6: Particle size and density at 60°C Fig. 3: Evaporation rate at different humidity Pic.1: Pure catalase Pic.2: Pure catalase Pic.3: Catalase-trehalose mixture (6:4) Pic.4: Spray-dried pure catalase Pic.5: Spray-dried catalase-trehalose (6:4) Fig.5: Residual catalase activity at 60°C. Fig. 4: Particle size and density.