A comprehensive picture of the freezing behavior in a Cryowedge 34 (representative of a 300-L cryovessel) has been obtained
during freezing as well as in the frozen state. Broadly, the cryoconcentration effects measurable in the liquid phase as the
solution freezes (presented in Part 1) are preserved in the frozen state. Approximately 3% of the solutes are affected by
the highest level of cryoconcentration (concentration >40 mg/mL). The levels of cryoconcentration observed here compare well
to those seen in previous reports for the Cryowedge 20 (20-L cryovessel), in which the cryoconcentration level of 99.5% of
the total protein was less than 1.2X.4
However, there are some important differences from earlier reported results.4 The main finding is the significant impact of depth on the cryoconcentration effect, whereas earlier results found no effect
of depth. This difference is likely a consequence of the composition of the solution. The solution studied here contains 20
mg/mL MAb in 20 mM histidine buffer as well as 84 mg/mL trehalose dihydrate, whereas the earlier reports studied a BSA solution
at 1 mg/mL in 10 mM citrate buffer only. Disaccharide solutions have significant temperature coefficients for density and
viscosity compared to water or buffer solution alone. In separate measurements, an increase in density of 0.0025 g/mL for
the disaccharide solution (84 mg/mL trehalose in buffer) as opposed to 0.00096 g/mL for buffer solution alone, was observed
on cooling from 25 to 5 °C. As the solution cools, small temperature differences lead to density gradients, which create convection
currents. Material with higher density, i.e., materials containing a higher concentration of solutes, sinks to the bottom.
Thus, in the freezing of solutions containing high concentrations of solutes, there is a combination of convection- and diffusion-driven
cryoconcentration effect, both toward the bottom and toward the last point to freeze, simultaneously. Although it is not known
how this effect translates to large cryovessels with greater available depth of liquid, it is quite likely that a large fraction
of solution in the upper regions is significantly low in disaccharide concentration. If the disaccharide has a critical concentration
to be effective as a cryoprotectant, this function could potentially be compromised. We are currently exploring this question
by placing cores from various positions in the frozen block on long-term stability. Similarly, the high concentration of disaccharides
in the lower regions could lead to the possibility of exceeding solubility limits or crystallization.
Interestingly, in another study of the Cryowedge 20, cryoconcentration effects larger than those seen here were found.6 That study looked at various buffers as well as fully formulated MAbs. Sampling also was performed by coring the ice. Cryoconcetration
effects at the last point to freeze were found to be 12-fold (sodium citrate buffer), 24-fold (histidine buffer), and 20-fold
(NaCl solution). A possible reason for the discrepancy could be the size of the cores as discussed below.
Micro- and Macro-cryoconcentration
The term cryoconcentration as used in the literature and in this paper requires clarification. The method used here to measure cryoconcentration actually
gives a macroscopic value that is somewhat dependent on the sampling method and the core dimensions. An ice core samples a
combination of ice as well as frozen solute matrix (Figure 7). When melted and analyzed, the pure-ice fraction dilutes and
dissolves the solute fraction, giving a lower-than-true cryoconcentration value, as is apparent in the two dashed circles
in Figure 7. The size of the core and its position will therefore determine the value measured, and should be referred to
as a macro-cryoconcentration value. The true or micro-cryoconcentration can be explained as follows: As freezing proceeds, ice formation excludes solutes (including
protein) from the growing ice crystal. Solutes migrate in front of the growing ice front that consist of small finger-like
projections called dendrites.7,8 These dendrites trap some of the solute as they grow. Therefore, solute is found in all regions of the block instead of
only at the last point to freeze. However, as the solution trapped between dendrites freezes, it continues to undergo cryoconcentration
as the water component is removed as ice and the composition changes, following the freezing boundary curve in Figure 8. The
solute potentially can precipitate if its solubility limit is reached at the eutectic point. If precipitation is hindered,
the solutes would be cryoconcentrated to a value close to the theoretical maximal freeze concentration given by the phase
or state diagram (Figure 8). This represents the true conditions experienced by the protein and should be called the microscopic cryoconcentration. Thus, for the system containing trehalose (and most other disaccharides when precipitation is hindered), the maximal freeze
concentration value is approximately 80% w/w.9 This represents cryoconcentration by a factor of ~10 in our system. The protein concentration in this matrix therefore is
200 mg/mL, significantly higher than measured when macroscopic sampling is performed and reported.
From a unit-operations' perspective, process parameters will affect the rate of heat removal, ice formation, and convection,
and thus determine the macro-cryoconcentration distribution in the frozen matrix. However, in the macro-cryoconcentrated matrix,
the protein and solute molecules will be in an environment determined by thermodynamics, i.e., by the phase or state diagram.
Storage temperature in relation to Tg' is key to the stability of this matrix.