A detailed and comprehensive map of the MAb solution frozen in the cryowedge was performed by taking cores at various positions
from the top and bottom halves as shown in Figure 3 and 4. Small portions of the frozen MAb solution block had to be thawed
from the sides (shown as white regions in the figures) to be able to remove the block from the wedge as discussed earlier
in the methods section. A total of 107 cores were taken from the top half and 95 from the bottom half. These samples were
analyzed for protein concentration and osmolality. Density also was measured of material collected from the region around
sampling ports 0–8.
Figure 3a shows a map of protein concentration distribution obtained for the top half of the frozen MAb solution block. The
measured concentration ranged from 3.8 to 21.7 mg/mL. The highest protein concentration was observed for core sample 5 from
position 5, followed by core sample 8 from position 3, with a concentration of 21 mg/mL. The lowest protein concentrations
were observed for the edge cores that were closest to the active heat transfer surfaces.
The greatest degree of solute exclusion occurred at the initial phase of ice formation because there is a larger liquid volume
for the solutes to diffuse into. At the same time, the linear velocity of the ice front is slower because ice is being formed
over a broader front. This reduces the amount of entrapped solute near the heat transfer surfaces. Progressive ice formation
eventually concentrates the solutes into the region near the last point to freeze, around position 3. The osmolality contour
map is shown in Figure 3b. The map for protein concentration and osmolality are broadly similar (Figures 3a and b). The highest
osmolality value was at core sample 8 for position 3 (339 mOsm/kg) followed by core 5 at position 5 (329 mOsm/kg) as compared
to the initial value of 270 mOsm/kg.
Protein concentration mapping for the bottom half of the frozen block is shown in Figure 4a. The relative spatial concentration
distribution in the bottom half is similar to the top, but a significant difference in the absolute protein concentrations
is seen between the two halves (Figure 3a compared to Figure 4a). The concentration at the edges was approximately 10 to 30
mg/mL, whereas on the top half, the edge concentration ranged from 2 to 12.5 mg/mL. The highest protein concentration measured
was 65.1 mg/mL (core 2, position 3), which was three times higher than the starting concentration as well as the highest concentration
observed for the top half. Osmolality values also demonstrated a similar pattern to protein concentration (Figure 4b). The
maximum osmolality of 1,132 mOsm/kg (core 2, position 3) corresponds to a >4-fold increase from the initial osmolality value
of 270 mOsm/kg.