An assessment of the fraction of total protein mass affected by cryoconcentration effect was performed. Figure 5a shows the
data as a percentage of affected protein mass for the top half of the Cryowedge. The top half shows lower concentrations than
the nominal for most of the fractions. Approximately 14% of the total protein was at concentrations between 0 and 9 mg/mL
whereas the majority of the mass (~77%) was at concentrations between 9 and 12.5 mg/mL. The most concentrated region (19.5
to 23 mg/mL) accounted for 20% of the total protein in the initial solution load. Along similar lines, the osmolality distribution
had three distinct regions of 90–140 mOsm/kg, 140–190 mOsm/kg, and 190–240 mOsm/kg which accounted for 25, 44, and 17% respectively,
followed by a small region of 14% where osmolality values were between 240 to 340 mOsm/kg (Figure 5b). Analysis of the bottom
half shows that ~48% of the total protein was at a concentration range of 10–20 mg/mL, whereas 32, 14, 5, and 11% lay in concentration
ranges between 20 and 30 mg/mL, 30 and 40 mg/mL, 40 and 50 mg/mL, and 60 and 70 mg/mL, respectively (Figure 5a). Osmolality
trended in a similar fashion: 150–315 mOsm/kg and 315–480 mOsm/kg accounted for 52 and 27%, respectively. The highest osmolality
fraction (975–1,140 mOsm/kg) accounted for ~3% of the solution (Figure 5b).
Material was collected from around each sample port position (Figure 1 in Part 1) for the top and bottom half of the MAb solution
block, and density was measured on the resulting thawed solution. Figure 6 shows the contour map for the solution density
gradient trapped in the frozen state as a function of cryowedge depth and sample position. The highest solution density is
seen around position 3 and is higher in the bottom halves. It is likely that the concentration and osmolality differences
between the top and bottom halves (in a relatively small total depth of 7.5 cm) is a consequence of a convective effect created
by these density gradients in solution during cooling.
Several studies have been published investigating cryoconcentration effects in various freezing systems, as reviewed in the
introduction. The reported results from these studies are in general agreement with the results observed in our study. With
all the current results and previous studies taken together, it is clear that cryoconcentration is inevitable for all practical-scale
freezing systems. Additionally, the extent of freeze concentration may be proportional to the solution depth because of density
gradient-driven convection during the freezing process.