RESULTS
Mapping the protein and solute in the frozen state allows us to understand the stress that protein is subject to during frozen
storage. Another objective of mapping the solution properties during the freezing process and after freezing was to determine
if the changes occurring in the liquid phase during freezing are representative of the final solid phase. Ice cores were taken
from the frozen block and osmolality and protein concentrations were measured. A core sample from the frozen block represents
an average concentration of solutes and protein at that position because the ice sampled in the core dilutes the sample after
it is melted for analysis. The implication of this on the measurement of cryoconcentration is discussed later.
Mapping of Solute and Protein in Frozen State
Figure 3
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Cryowedge mapping during freezing as a function of freezing rate and time provided information on the development of solute
and protein distribution.5 A key question is whether the solute distribution observed during freezing is sustained in the frozen state. This was addressed
by coring blocks of formulation buffer and of MAb solution that were frozen using the intermediate cycle described in Part
1.5 The coring configuration for the frozen block is shown in Figure 1. Cores also were obtained from upper and lower halves
of the ice block to assess the convective effects on cryoconcentration.
Formulation Buffer
The cores obtained from the cryowedge corresponding to positions 0–8 (Figure 1 in Part 1)5 for the upper and lower half of the frozen block were tested for osmolality. Data are shown in Figure 2 and a clear difference
is seen between the two halves. In the top half, the osmolality ranged from 203 to 395 mOsm/kg, with the highest value at
position 3. The osmolality values for the bottom half ranged from 200 to 1,291 mOsm/kg, with the highest value also at position
3. These data show that depth has a large effect on the outcome of the freezing process. This observation is significant,
considering that the solution depth in the cryowedge was only ~7.5 cm, whereas 200- and 300-L cryovessels can be as deep as
107 and 130 cm, respectively (Sartorius-Stedim Cryovessel specification).
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