Intermediate Freezing Process
Freezing by the intermediate process comprises an equilibration step for the solution at 0 °C with the mixer turned on for
2 h to ensure homogeneous solution composition and temperature. After 2 h, the process temperature was decreased in steps
of 5 °C after every 30 min with a final temperature of –50 °C. The freezing time was approximately 8.2 h (Figure 2b). Figure
4a shows the osmolality values obtained for the formulation buffer solution with this freezing profile. A maximum osmolality
value of 839 mOsm/kg was observed for position 3 followed by 522 and 500 mOsm/kg at positions 1 and 5 respectively. The observed
osmolality value at position 3 was just marginally greater than that obtained from the fast freezing cycle (Figure 3a). Except
for the last stages of freezing, the osmolality ranged from 227 to 283 for all positions. When the osmolality values for fast
freeze and intermediate freezing cycles are compared, it can be concluded that the solute distribution during freezing varied
little between the two cycles even though the freezing rates were significantly different. The values of pH and Tg' ranged
from 5.61 to 5.68 and –30.9 to –31.3 °C, respectively, for all positions (not shown).
Figure 4. 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 intermediate freezing process.
Figure 4b shows the osmolality values obtained when the MAb solution was subjected to the intermediate freezing process. The
highest osmolality values were observed for position 3, with the maximum measured as 889 mOsm/kg. The similarity between osmolality
values for formulation buffer and the MAb solution for the intermediate freezing profile suggests that the presence or absence
of protein during the freezing process had little effect on solute distribution. Protein concentration values provided the
same trend as osmolality values, with the highest protein concentration being 48.1 mg/mL, an approximately 2.6-fold cryoconcentration
(Figure 4c). Except for the last stages of freezing, the protein concentration ranged from 17.4 to 18.2 mg/mL for all positions.
The changes in pH and Tg' values were minimal for all the samples (data not shown). In addition, soluble aggregate levels
for all the samples, including the high osmolality and concentration samples, did not show any apparent increase (data not
Additionally, a thaw process profile was run on the frozen MAb solution. After 1,020 minutes, the temperature was increased
to –25 °C and thereafter every 15 minutes to 2 and 25 °C and held at 25 °C with the mixer on during thawing. Analysis of the
samples taken from the thawed solution showed that mixing during thawing had redistributed the protein and solutes uniformly
with no observable impact on soluble aggregate levels.