Aprendizajes del docente.

Rituximab particles were prepared by incubating 1.5 mL of the 1 mg/mL rituximab solution for 30 minutes at 71 °C in a thermomixer (Eppendorf, Hamburg, Germany). HSA particles were generated by heating 50 mL of the 1 mg/mL formulation in a 50 mL tube (Greiner bio-one, Frickenhausen, Germany) for 30 minutes at 70 °C in a water bath (HSA (heating)) or by spray- drying 50 mL of the 5% formulation using a Büchi Mini Spray-Dryer B-290 (HSA spray-dried (Mini)) or Büchi Nano Spray-Dryer B-90 (HSA spray-dried (Nano)) (Büchi Labortechnik, Flawil, Switzerland) with subsequent cross-linking. For the Mini Spray-Dryer, process parameters were chosen based on previous studies by Schüle7 _{and Fuhrherr}8_{: inlet temperature (T}

in) 130 °C, outlet temperature (Tout) 60-70 °C, liquid feed flow rate 3 mL/min (9%), atomizing air volumetric flow rate 670 L/min (40 mmHg), aspirator flow rate 35 m3_{/h (100%), cooled two-fluid} nozzle (0.7 mm). For the Nano Spray-Dryer, the following process parameters were applied: Tin 55 °C, Tout 25 °C, gas flow 115 L/min, pressure 37 hPa, pump mode 1, spray cap diameter 5.5 µm, spray head temperature 33 °C, spray rate 100%, spray angle 45°. After the spray-drying process, 150 mg of the particles generated by the two different approaches were suspended in 30 mL acetone and cross-linked by the addition of 500 µL glutaraldehyde (8%) under stirring at 400 rpm (Heidolph MR 3001K, Schwabach, Germany). After stirring for additional 30 minutes at 400 rpm, the suspensions were centrifuged for 10 minutes at 7,000 g (centrifuge 5810 R, Eppendorf AG, Hamburg, Germany) and the supernatants were discarded. Subsequently, the sediments were resuspended in 4 mL water and filtered through a coarse tea filter to remove large agglomerates.

Gelatin microparticles were prepared by a two-step desolvation method, which was originally developed for preparing nanoparticles.9 _{The first precipitation was} triggered by the addition of 25 mL acetone at 500-600 rpm to 25 mL of a 5%

146

gelatin solution at room temperature with a precipitation time of 2 minutes. The supernatant was discarded and the deposit was redissolved in 25 mL water at 50 °C. The pH was adjusted to 3.9 with 1 M HCl. The second precipitation was initiated by adding 50 mL acetone at about 9-10 mL/min using a burette. After 10 minutes of stirring at 500-600 rpm, 500 µL glutaraldehyde (8%) were added as a cross-linker and the suspension was stirred for another 30 minutes. The particles were harvested by centrifugation at 10,000 g for 10 minutes at room temperature. The supernatant was discarded and the pellet was resuspended in 5 mL water and filtered through a coarse tea filter to remove large agglomerates.

For the evaluation of particle density, the preparation process was optimized to maximize the number of particles in the target size range of 2-8 µm. The cross- linking time with glutaraldehyde was extended to 40 minutes and the centrifugation speed for particle harvesting was decreased to 5,000 g. After filtration through a coarse tea filter to remove large agglomerates an additional purification step was introduced to minimize the number of particles below 2 µm: The pH of the filtrate was adjusted to pH 3 to provoke electrostatic repulsion between the particles prior to an additional centrifugation step (200 g, 20 minutes). The pellet was discarded and the supernatant was used.

HSA starch particles were produced by an emulsion-based process according to previous studies by Heritage et al.5 _{1 g starch was dissolved in 2 mL DMSO under} stirring at 85 °C, cooled down to room temperature and subsequently 1 mL 10% (w/v) aqueous HSA solution was added drop-wise. This solution was emulsified drop-wise in 20 mL rape oil under stirring at 1250 rpm (Heidolph MR 3001K) and sonication. Afterwards this emulsion was added drop-wise to 400 mL acetone containing 0.5 mL polysorbate 80, again under stirring at 1250 rpm. The generated microparticles were then collected by filtration (0.22 µm Durapore® (PVDF) membrane filter, Millipore) under vacuum, washed with 1 L acetone, and dried on the filter by vacuum. The powder was sieved through a 100 µm mesh to exclude large agglomerates and the particles were stored under desiccation at 5±3 °C. 30 mg of the particles were suspended in 4 mL water for analysis.

147

2.3

Light obscuration (LO)

Subvisible particles in a size range between 1 and 200 µm were analyzed by LO using a PAMAS SVSS-C (Partikelmess- und Analysesysteme GmbH, Rutesheim, Germany) equipped with an HCB-LD-25/25 sensor. Particle suspensions were diluted with the according buffer (filtered by a 0.22 µm cellulose acetate/nitrate membrane filter, MF-Millipore®_{) or water in order to adhere to the concentration} limit of the system of 120,000 particles/mL > 1 µm. Three measurements of a volume of 0.3 mL of each sample were performed with a pre-run volume of 0.5 mL at a fixed fill rate, emptying rate and rinse rate of 10 mL/min and the mean particle concentration per mL was reported by the system. Samples were measured in triplicates and mean and standard deviation were calculated.

2.4

Micro-Flow Imaging (MFI)

Subvisible particles in a size range between 1 and 70 µm were analyzed by MFI using an MFI4100 (ProteinSimple, Santa Clara, CA) equipped with a high- resolution 100 µm flow cell. Particle suspensions were diluted with the according buffer (filtered by a 0.22 µm cellulose acetate/nitrate membrane filter) or water in order to adhere to the concentration limit of the system of 1,200,000 particles/mL > 0.75 µm. Samples were analyzed with a sample volume of 0.65 mL and a pre-run volume of 0.3 mL at a flow rate of 0.1 mL/min. Prior to each sample run the appropriate diluting buffer was flushed through the system to provide a clean flow cell and to perform “optimize illumination”. Particles stuck to the flow cell wall were only counted once and edge particles were ignored for analysis. Samples were measured in triplicates and mean and standard deviation were calculated. Results were analyzed using the MFI view application software (version 1.2, ProteinSimple).