This two-part article series presents an in-depth look at the process of freezing and solute distribution in cryovessels by using a scale-down model called the cryowedge. Part 1, published in the June issue of BioPharm International, discussed the evolution of solute distribution in the cryowedge during the freezing process. Here in Part 2, we discuss protein and solute cryoconcentration changes in the cryovessel. For the study, we prepared a comprehensive map of frozen state solute distribution in terms of protein concentration and osmolality. Solution properties were measured on core samples removed from the frozen mass and compared with data obtained from solution samples removed during the freezing process (presented in Part 1). The results are in general agreement. An approximately three-fold cryoconcentration was observed. The properties of the frozen mass as a function of depth in the cryowedge were assessed and the results suggest a significant effect of convection on protein concentration, osmolality, and density distribution during freezing. Active freezing systems can affect the overall distribution of ice and solute in the matrix, i.e., the macro-cryoconcentration, but not the micro-cryoconcentration experienced by the solute and protein in the matrix.
Freeze-thaw operations represent a critical step in protein drug product manufacturing. Maintaining the protein quality during freezing, frozen storage, and thawing operations is challenging, especially at large scales. One factor that can affect protein quality is the protein and solute distribution that results from a freezing operation. A few studies have been reported on solute distribution in various freeze systems. A study that mapped the performance concentration and osmolality of 2-L polyethylene terephthalate glycol (PETG) bottles frozen at –20 °C or –80 °C found that the maximal concentration is centered in the lower half of the bottle.1 The protein concentration and osmolality were increased by a factor of ~2. Another study of the solute distribution for a monoclonal antibody (MAb) solution in 4-L high density polyethylene (HDPE) bottles, after thawing but before mixing, found that an ~3-fold concentration factor was preserved in the lower portions of the bottle.2 Similar results were seen in the frozen state in 1-L HDPE bottles in the lower and center portion of the bottle when the protein solution was frozen from 25 °C to –20 °C. In a 20-L carboy filled with 16 L of MAb solution and frozen at –70 °C, an ~9.3-fold increase in protein concentration and an osmolality increase of ~7-fold were reported at the last point to freeze.3 On the other hand, for Celsius bags (30-mL, Sartorius-Stedim Biotech, Aubagne, France), an ~2-fold increase in protein concentration and osmolality were observed. Freeze concentration effects also were evaluated for various bottle systems and the cryowedge system for 1 mg/mL bovine serum albumin (BSA) in buffer.4
The geometry of the cryowedge represents a scaled-down model of commercially available controlled-rate freeze-thaw systems called cryovessels. The heat and mass transfer surfaces and distances in the wedge are identical to those in a cryovessel of corresponding size, allowing the freeze and thaw behavior of a full-scale vessel to be studied at laboratory scale. An ~1.3-fold increase in protein concentration in the Cryowedge 20 system (Sartorius-Stedim) was reported, representing a 20-L cryovessel.4 The highest concentration was recorded near the last point to freeze in the wedge, and no significant depth variation was seen at this point. On the other hand, freezing in 1-L bottles increased BSA concentration ~4.6-fold (when frozen at –20 °C) and a >8-fold (when frozen at –80 °C). These concentration hot spots were found near the bottom center of the bottles. For small tubes and vials, the extent of cryoconcentration was lower (1.1 to 1.5-fold). It was concluded that ice-front velocity was the critical factor in determining the extent of bulk-scale freeze concentration;4 faster freezing led to a smaller overall degree of cryoconcentration. The objective of the studies described in this article series was to examine the behavior of proteins and solutes in the cryovessels, because this behavior ultimately determines the outcome of the frozen state storage. In Part 1, solute distribution and solution property changes during freezing were mapped for various processing rates as a function of position in the cryowedge.5 This Part 2 presents an exhaustive and comprehensive protein concentration and osmolality map in the frozen state as a function of cryowedge solution depth. We found that convective effects become important and are likely unavoidable for the distribution of solutes in practical systems.