Slow Freezing Process
Temperature profiles for the slow freezing profile are shown in Figure 2c. Supercooling was recorded in this case at all thermocouple
positions in the cryowedge. A remarkable nucleation phenomenon was observed visually. Ice nucleation began at the heat transfer
surfaces and rolled across the cryowedge in about 1 min, leaving the whole solution opaque with ice crystals or nuclei. Some
of these nuclei melted back, as evidenced by a slight clearing of the solution over time. Complete solidification followed
the time course per Figure 2c. The time required for freezing was 598 minutes (defined as before). Osmolality values obtained
for formulation buffer are shown in Figure 5a. The maximum osmolality value of 1391 mOsm/kg was measured for position 3. This
maximum osmolality value for the slow freezing of formulation buffer was significantly greater than in the other processes.
The slow rate of freezing allowed a greater time for diffusion or convective movement and thus produced a greater degree of
cryoconcentration. Except for the last stages of freezing the osmolality ranged from 221 to 305 mOsm/kg for all positions.
As expected, these values were lower than those seen in Figures 3a, 4a, and 5a. Again, no apparent changes in either pH or
Tg' values were observed.
Figure 5. Changes in (a) solution osmolarity of formulation buffer, (b) solution osmolarity of MAb solution and (c) protein
concentration as a function of position in the cryowedge and time during the slow freezing process.
Osmolality values for the MAb solution frozen by this slow process are shown in Figure 5b. The maximum osmolality observed
was at position 3 followed by position 1, with values of 778 and 770 respectively. The maximum in this case was significantly
lower than that in Figure 5a. We attribute this difference to a possible sampling discrepancy. The protein concentration reached
41.8 mg/mL, approximately 2.33-fold higher than the initial value of 17.9 mg/mL (Figure 5c). The soluble aggregates level
did not change, nor did the pH or Tg' (data not shown).
A comprehensive picture of freezing behavior in a Cryowedge 34 (representative of a 300-L cryovessel) has been obtained for
a number of freeze profiles. The concentration changes in the liquid phase as the solution freezes were monitored, based on
protein concentration and osmolality measurements. The last point to freeze shows the highest concentrations, whereas the
regions close to the heat-transfer surfaces are depleted of solutes. Our results with various rates of freezing demonstrate
that a slower freezing rate allows a higher degree of concentration polarization to occur. Slower freezing enables pure ice
crystals to grow slowly into the solution without trapping solutes, hence increasing the solute concentration in the unfrozen
liquid phase. Diffusion and convection rates of the solutes in competition with the overall freezing (ice formation) rate
determines the distribution of the solutes.
We have systematically analyzed solute distribution and solution property changes during freezing for various process profiles
in a cryowedge, representative of a 300-L cryovessel. Our results suggest that during freezing, as water from solution is
converted into ice, it pushes the solute toward the center resulting in a macroscopic cryoconcentration (discussed further
in Part 2). A >2-fold increase in protein concentration was observed for all the freeze profiles. A >4-fold increase in osmolality
was observed for the slow freeze cycle, whereas the increase in osmolality for the intermediate and fast freeze cycles was
>2-fold. No significant differences in pH, Tg', or soluble aggregates were observed.
Parag Kolhe is a principal scientist, Alanta Lary is a senior scientist, Steven Chico is an associate scientist, Elisabeth Holding was an associate scientist, and Satish K. Singh is a research fellow all at Pfizer, Inc., Chesterfield, MO, 636.247.9979, firstname.lastname@example.org