MATERIALS AND METHODS
Solute distribution and solution property changes in the frozen state were monitored in a Cryowedge 34 (Sartorius-Stedim).
Processing was carried out by a Cryoproccessor B using silicone oil as the heat transfer fluid (HTF, Syltherm, Dow). An in-house
IgG2 MAb solution at 20 mg/mL in a 20 mM histidine buffer, pH 5.5 with 0.2 mg/mL polysorbate 80, 84 mg/mL trehalose dihydrate
was used as a model protein for this study.
Polytetrafluoroethylene (PTFE) dry lubricant (Loctite Krytox RFE) was purchased from DuPont, Inc. (Johnston, IA). A bench-top
band saw (Delta SM400 Shopmaster 3 Amp 9-inch band-saw) was used to cut the frozen cryowedge piece, and a Dewalt drill with
a custom-made drill bit (diameter: 1.5 cm) was used to take cores (~2.5 cm long) from the ice.
Cryowedge Mapping in the Frozen State
The intermediate freezing process profile (defined in Part 1) was used for mapping the solute distribution in the frozen state
for the formulation buffer solution and the MAb solution.5 Removing the frozen block of solution from the cryowedge posed a significant challenge because the bottom surface of the
frozen ice block adhered strongly to the cryowedge surface. Running the thaw cycle for a short period to melt the ice from
the edges did not solve the problem. In the case of the formulation buffer, a thin coat of PTFE dry lubricant, when sprayed
and allowed to dry on the bottom of the cryowedge, enabled the frozen block to be removed. Approximately 4 L of formulation
buffer were added to the cryowedge and frozen. After the solution went through the freeze cycle, the edges of the frozen block
inside the cryowedge were thawed slightly to loosen and remove the block from the Cryowedge. The block was stored at –40 °C
until further processing. This method of removing the frozen block did not work for the MAb solution, however. Instead, an
aluminum foil interior mold of the cryowedge was prepared. The MAb solution was poured into the aluminum foil and then subjected
to the freezing cycle. After thawing the sides, the frozen block could be removed easily and stored at –40 °C until further
processing. To ensure that the heat transfer properties of the system were not significantly altered by the aluminum foil,
a control experiment was performed by freezing water with and without aluminum foil. A comparison of the temperature profiles
recorded by the five thermocouples showed identical thermal behavior between the two (data not shown).
The frozen block was cut according to a predetermined template around positions 0 through 8 (Figure 1). These portions were
further cut into two pieces each to represent the top and bottom halves of the frozen block. These smaller pieces were then
drilled to obtain ~2.5 cm long cores. For the block made with formulation buffer solution, cores were taken only at the sample
port positions (Figure 1 in Part 1).5 The frozen block of MAb solution was extensively cored in both the top and bottom halves to develop a detailed map of the
solute and protein distribution. These samples were analyzed for pH, glass transition temperature (Tg'), osmolality, density,
and high molecular mass species (soluble aggregates) by size exclusion chromatography (SEC) and protein concentration. No
changes were seen in pH, Tg', or aggregation levels, and therefore, these data are not discussed here.