The objective of mapping the solution properties during the freezing process and after freezing (described in detail in Part
2) was to determine how solute and protein concentration changes evolve during the freezing of the liquid phase and to compare
these concentrations to the final distribution in the frozen solid phase. The freezing time of the process is defined as the
time interval between the first probe to reach 0 °C and the probe at position 3 to read –20 °C.
Mapping of Solute and Protein During Freezing
Solution samples removed during freezing were analyzed for pH, osmolality, Tg', protein concentration, and soluble aggregates.
Fast Freezing Process
The fast freezing process profile is shown in Figure 2a. In this cycle, the process temperature is reduced to –50 °C and held
for an extended time to achieve the fastest freezing rate possible in the wedge. Positions 0 and 7 are the first to freeze,
followed by positions 2, 4, 8, and 6. The longest time to freeze is observed at position 3, preceded by positions 1 and 5
(Figure 2a). Slight (approx. 1 °C) supercooling is seen at positions 2 and 4. The last point to freeze (position 3) is a factor
of the geometry of the wedge and (heat-transfer capacity weighted average) distance from the actively cooled surfaces as shown
in Figure 1. The time taken to complete the freezing process (defined as above) for this run was 391 minutes (Figure 2a).
Figure 3a shows the osmolality values obtained for formulation buffer solution when the solution was subjected to the fast
freezing process. A significant osmolality increase is observed at position 3, followed by smaller increases at positions
1 and 5. The initial osmolality level for the formulation buffer was 256 mOsm/kg. The highest osmolality value measured was
667 mOsm/kg, representing cryoconcentration by a factor of approximately 2.6 compared to the initial. The solutes in the formulation
buffer progressively migrate (by diffusion or convection) toward position 3 as the freezing progresses. The osmolality values
at the other positions (before completion of freezing at that position) ranged from 242 to 302 mOsm/kg with a minimum observed
at position 7. In general, except for the last stages of freezing at positions 1, 3, and 5, the osmolality values range between
228 and 274 mOsm/kg (compared to initial of 256 mOsm/kg). No significant changes in the pH of the solution were observed during
freezing (in liquid state), with pH ranging from 5.55 to 5.63 (not shown). The corresponding Tg' of the solutions removed
from these positions also showed no significant changes, ranging from –30.6 °C to –31.5 °C (data not shown). The pH and Tg'
results show that there is no significant partitioning or separation of the components of the solution during the freezing
Figure 3. Changes in (a) solution osmolality of formulation buffer, (b) solution osmolality of MAb solution, and (c) protein
concentration as a function of position in the cryowedge and time during the fast freezing process.
Figure 3b shows the osmolality values obtained at different positions of the cryowedge as a function of time with the MAb
solution undergoing the fast freezing process. A maximum osmolality value of 705 mOsm/Kg was observed at position 3. The lower
osmolality value observed at the later time point is likely an artifact of sampling, because at this point in the cycle, a
significant amount of ice crystals are present at this position. It is possible that some ice crystals were drawn into the
syringe along with the unfrozen solution, resulting in dilution. The observed maximum osmolality value is not significantly
different compared to that seen with the formulation buffer (Figure 3a), suggesting that the formulation buffer and the MAb
solution behave similarly. The time taken to complete the freezing process (–20 °C at position 3) for this run was 442 minutes.
The protein concentration trend was similar to the osmolality trend, showing a peak concentration of 41.9 mg/mL (a 2.2-fold
increase compared to initial concentration of 18.8 mg/mL) (Figure 3c). Again, except for the last stages of freezing in positions
1, 3, and 5, the measured MAb concentration ranges from 17.6 to 17.9 mg/mL. Soluble aggregate levels, measured by size exclusion
chromatography, did not show any difference between the samples, indicating that an increase in local protein concentration
did not have any impact on soluble aggregate levels for this MAb solution in the experimental time frame (SEC was performed
within 15 days, and all aliquots were stored at 2–8 °C; data not shown). No substantial variations were observed for pH (range
5.48 to 5.58) or Tg' (range –28.3 to –30.4 °C; data not shown).