Large-Scale Freezing of Biologics: Understanding Protein and Solute Concentration Changes in a Cryovessel—Part I - What really happens to protein and solute concentrations during bulk freezing

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Large-Scale Freezing of Biologics: Understanding Protein and Solute Concentration Changes in a Cryovessel—Part I
What really happens to protein and solute concentrations during bulk freezing at different rates.


BioPharm International
Volume 23, Issue 6

MATERIAL AND METHODS

Materials


Figure 1. Sample ports and positions on the Cryowedge showing the placement of thermocouples that monitor the temperature changes at the respective positions during the freezing and thawing process. The active heat transfer surfaces are identified; these comprise the full top and bottom edges, as well as the dotted regions on the left and right hand edges.
Solute distribution and solution property changes during freezing (at three different freezing rates) and in the frozen state were monitored in a Cryowedge 34. Processing was carried out by a Cryoprocessor B using silicone oil as the heat transfer fluid (HTF) (Syltherm, Dow). Figure 1 shows the Cryowedge when the solution is completely frozen, along with the custom-made sample ports that are marked as positions 0 through 8. Thermocouples used to monitor the process were inserted at positions 1 through 5.

The model protein for this study uses an in-house IgG2 monoclonal antibody (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.

Cryowedge Solution Mapping During Freezing


Figure 2. Temperature profiles for (a) fast freezing, (b) intermediate freezing, and (c) slow freezing processes. The actual temperatures recorded by five thermocouples (labelled as TC 1–5) at the various positions indicated in Figure 1 also are shown.
Cryowedge solution mapping was performed for three freezing process profiles, designated as slow, intermediate, and fast. The intermediate profile was suggested by the manufacturer to best represent freezing in a 300-L tank. The profiles are illustrated in Figure 2 along with the solution temperature recorded by the thermocouples at positions 1 through 5 during a typical freezing run. Approximately 4 L of either the formulation buffer solution or MAb solution was added to the cryowedge and covered with a plexiglass lid. The freezing process was initiated per the identified profile, and at predetermined intervals, liquid samples of ~500 L were withdrawn from all the sample ports, until the liquid underneath the sample port became completely frozen. These liquid samples were withdrawn by attaching a BD spinal needle (18G, 6 inch length) to a 3-mL BD syringe and passing the needle through the sample using the thermocouple port of the Cryowedge. Using a narrow needle for the liquid sample, we attempted to obtain the "mother liquor" without dilution by any ice crystals that may have formed at that position. These samples were analyzed for pH, glass transition temperature (Tg'), osmolality, high molecular mass species (soluble aggregates) by size exclusion chromatography (SEC), and protein concentration.


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