AG001

For the normal shelf-ramped freezing at -1°C/min, microscopy revealed a sponge-like matrix with spherulitic pores at the top of the cake and the pore size between 70 and 170 µm depending on the substance. Valine and cromolyn sodium exhibited the smallest and phenylalanine the biggest pore diameters. The valine lyophilisate demonstrated a whitish crystalline skin on the top where crystals were partly arranged in rosettes. In comparison to the top, pores at the bottom of the cake were smaller with pore diameters of 30 to 40 µm except for the phenylalanine lyophilisate, which demonstrated both small pores and areas with pores of approximately 90 µm. Freezing with annealing caused a complete loss of the ordered spherulitic pore structure for lactose (Figure 1a) and trehalose lyophilisates with a translucent

Table 1: Tabular overview of the freezing process variation results -1°C/min -1°C/min

with hold annealing precooled shelf liquid nitrogen vacuum induced

Valine

Physical state crystalline crystalline crystalline crystalline crystalline crystalline Lyophilisate

morphology spherulitic spherulitic - spherulitic lamellar spherulitic Pore size

[µm] top: 65 bottom: 40 - - top: 70 bottom: 55 - bottom: 60 Particle

morphology porous agglomerates - porous agglomerates - porous agglomerates - Particle size 61 µm - 42 µm - 80 µm - Mechanical

testing plateau at 0.004 N plateau at 0.003 N - plateau at 0.003 N slope of 0.001 N/mm plateau at 0.006 N Disintegration

characteristics fast and complete fast and complete - fast and complete fast and complete fast and complete ED 73% 77% 50% 71% 85% 67% FPF (ED) 8% 9% 9% 9% 19% 10% FPF (MD) 6% 7% 5% 6% 16% 7% Lactose

Physical state amorphous amorphous amorphous amorphous amorphous amorphous Lyophilisate

morphology spherulitic spherulitic consolidated spherulitic lamellar spherulitic Pore size

[µm] top: 80 bottom: 40 - top: 100 top: 104 bottom: 35 - - Particle

morphology porous agglomerates - Different shaped fragments

- porous

agglomerates - Particle size 35 µm - 8 µm - 82 µm - Mechanical

testing peak of max. 0.089 N peak of max. 0.088 N plateau at 0.006 N peak of max. 0.105 N slope of 0.035 N/mm plateau at 0.025 N Disintegration

characteristics separation into subunits and scaling off1 separation into subunits and scaling off1 fast and complete (finished after 100 ms) separation into subunits and scaling off fast and

complete fast and complete ED 46% 46% 18% 40% 78% 46% FPF (ED) 32% 34% 14% 31% 34% 26% FPF (MD) 15% 16% 2% 12% 27% 12% Trehalose

Physical state amorphous amorphous amorphous amorphous amorphous amorphous Lyophilisate

morphology spherulitic spherulitic consolidated spherulitic lamellar spherulitic Pore size

[µm] top: 110 bottom: 30 - top: 140 top: 90 bottom: 25 - - Particle

morphology - - - - Particle size - - - - Mechanical

testing peak of max. 0.113 N peak of max. 0.088 N plateau at 0.006 N peak of max. 0.118 N slope of 0.041 N/mm plateau at 0.025 N Disintegration

characteristics separation into subunits and scaling off1 separation into subunits and scaling off1 fast and complete (finished after 100 ms) separation into subunits and scaling off fast and

complete fast and complete ED 41% 44% 13% 26% 56% 44% FPF (ED) 38% 33% 19% 44% 41% 34% FPF (MD) 16% 14% 2% 10% 23% 15%

-1°C/min -1°C/min

with hold annealing precooled shelf liquid nitrogen vacuum induced

Phenylalanine

Physical state crystalline crystalline crystalline crystalline crystalline crystalline Lyophilisate

morphology spherulitic spherulitic spherulitic spherulitic lamellar spherulitic Pore size

[µm] top: 170 bottom: 30- 40 / 90

- top: 170

bottom: 110 top: 130 bottom: 35 - top: 4 bottom: 100 Particle

morphology porous agglomerates - porous agglomerates porous agglomerates porous agglomerates porous agglomerates Particle size 70 µm - 74 µm 68 µm 109 µm 91 µm Mechanical

testing plateau at 0.067 N plateau at 0.060 N plateau at 0.079 N plateau at 0.062 N slope of 0.005 N/mm peak of max. 0.108 N Disintegration

characteristics separation into subunits and scaling off1 separation into subunits and scaling off1 separation into subunits and scaling off1 separation into subunits and scaling off fast and

complete separation into subunits and scaling off1 ED 49% 43% 38% 63% 72% 68% FPF (ED) 57% 59% 61% 45% 42% 55% FPF (MD) 27% 25% 23% 29% 30% 36% Cromolyn sodium

Physical state amorphous amorphous amorphous amorphous amorphous amorphous Lyophilisate

morphology spherulitic spherulitic spherulitic lamellar (bottom to top) lamellar (outside to middle) spherulitic Pore size

[µm] top: 80 bottom: 30 - top: 50 bottom: 30 top: lamellar bottom: 14 - top: 15 bottom: 90 Particle

morphology porous agglomerates - porous agglomerates porous agglomerates porous agglomerates porous agglomerates Particle size 44 µm - 40 µm 75 µm 123 µm 58 µm Mechanical

testing plateau at 0.135 N plateau at 0.153 N plateau at 0.121 N slope of 0.062 N and 0.017 N

slope of

0.070 N peak of max. 0.124 N Disintegration

characteristics separation into subunits and scaling off1 separation into subunits and scaling off1 separation into subunits and scaling off1 fast and

complete fast and complete separation into subunits and scaling off1 ED 67% 48% 37% 64% 68% 50% FPF (ED) 28% 44% 45% 38% 18% 42% FPF (MD) 19% 21% 16% 24% 12% 21% 1_{incomplete disintegration}

skin on top of the cake. The structure of the valine lyophilisates could not be evaluated due to destruction during the ventilation process in the freeze-drier. For phenylalanine lyophilisates, the annealing process had no effect on the morphology except that the upper surface skin appeared thicker. Cromolyn sodium lyophilisates revealed a more homogenous pore size distribution after the annealing step (Figure 1b), with a mean pore diameter of 30 µm. After freezing on a -70°C precooled shelf most lyophilisates showed small spherulitic pores of 25- 35 µm at the bottom and bigger pores of 73-100 µm near the top of the cake. The top layer of valine lyophilisates was again composed of crystal rosettes, whereas phenylalanine lyophilisates showed needle shaped furrows and the sugars lactose and trehalose showed

a b

c d

e f

Figure 1: Microscopic appearance of lyophilisates: bottom of 12 mg/ml lactose frozen with annealing (a), bottom of 12 mg/ml cromolyn sodium frozen with annealing (b), cross section of 12 mg/ml cromolyn sodium frozen on a precooled shelf (c), cross section of 8 mg/ml phenylalanine frozen in liquid nitrogen (d), cromolyn sodium 12 mg/ml frozen vacuum induced (e) and lactose 12 mg/ml frozen vacuum induced (f).

rectangular pores on top of the cake. A cross sectional cut of cromolyn sodium lyophilisates demonstrated lamellar pores in the direction from bottom to top (Figure 1c) and also the upper surface showed a lamellar pore morphology, whereas the bottom of the cakes exhibited very small pores of around 14 µm. For all samples freezing by immersion into liquid nitrogen

resulted in a fine lamellar morphology in the direction from outside to inside. The cross sectional cut (exemplarily shown for phenylalanine in Figure 1d) showed directed narrow pores reaching from the walls of the vials towards the middle and finally upwards. Besides fine disruptions in lactose and trehalose lyophilisates, liquid nitrogen frozen cromolyn sodium cakes exhibited distinctive cracks directing from outside to the middle. Vacuum-induced freezing at -3°C resulted in a dense upper layer with small pores for cromolyn sodium (15 µm), phenylalanine (4 µm), and valine lyophilisates (not distinguishable). Lactose and trehalose lyophilisates demonstrated upward pointing villi on top of the cake and irregularly shaped oblong pores at the bottom. The inner pore structure of all lyophilisates is composed of spherulitic pores.