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Hybrid Freezing & Thawing: An all-in-one solution

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Biopharmaceutical manufacturers and CMOs typically require separate cold chain management solutions when the drug substance is filled into different single-use primary packagings during bioprocessing: single-use bioprocess containers and bottles.

This requires freezers for cooling single-use bags and separate freezers for cooling rigid containers.

But why not combine both in one unit? The increased number of systems requires a larger footprint on-site, a more sophisticated GMP-compliant infrastructure and therefore leads to incompatibilities, higher costs, but also inconsistent freezing performance. Hybrid solutions for drug freezing and thawing involve the use of both primary packagings.

FIG 1 FTLS_Hybrid

Figure.1: Single Use Support’s Hybrid Freezing & Thawing Platform for Bags and Bottles

Blast vs. plate-based freezing

The challenge in hybridizing freezing of bags and bottles is to combine the proposed freezing methods. The different shapes of bags and bottles require different freezing methods to lower the temperature of drug substance. While 2D single-use bags have a flat design and a low water column, they can be frozen faster than bottles. While plate freezers provide advanced freezing results for single-use bags, bottles are frozen by blast freezing. Both methods are integrated in hybrid freeze-thaw platforms.

Surrogate tests have been conducted to evaluate the freezing results of single-use bags and bottles preventing the effect of cryoconcentration.1​ The results confirm numerous studies that higher freezing rates are preferable to avoid protein chemical and physical instabilities and that fast and homogeneously frozen liquids limit cryoconcentration to a large extent.2​ Homogeneous freezing therefore enables highest product quality after thawing.

Control freezing of single-use bags with cooling plates

It can be argued that the simplest and most intuitive method to refrigerate active pharmaceutical ingredients (API) is simply by placing single-use bags in a conventional static or blast ultra-cold freezer. However, these are designed only to maintain the temperature. Therefore cooling drug substances with conventional upright or chest freezers  takes several hours or days for the entire liquid to reach -80°C. The ice front grows from the outside toward the center of the cryobag. And since water will freeze in its most natural form, it pushes solutes and proteins into its center. Especially during the phase transition from +2°C to -5°C, when liquids become solid.

Solidification in static freezers, for example, takes up to five hours, giving the solutes time to collect in the center, before being homogeneously frozen within the growing ice front. This is the point at which cryoconcentration occurs: the frozen water growing from the surface to the center of the bioprocess container is separated from the collected solutes trapped in the center, the last point of freeze.

Controlled and accelerated freezing prevents protein aggregation, namely cryoconcentration. Plate-based freezing​ does the trick with efficient and homogeneous freezing results. Finally, lower cryoconcentration results in higher product quality of API after the freeze-thaw cycle.3

FIG 2 cryoconcentration_bags

Figure 2: Cryoconcentration in single-use bags frozen in static freezer vs. plate freezer

As shown in Figure 2​, there is a significant difference in protein concentration between conventional and plate-based freezing. In this study, two 10L single-use bags from the same manufacturer were filled with a protein surrogate solution. The liquid contained a mixture of salt and sugar and was specified by a customer of Single Use Support to best mimic the freezing behavior of its product, a monoclonal antibody (mAb) formulation.

A blue dye, which had no effect on the freezing behavior of the surrogate, was added to visualize the effect of cryoconcentration. Furthermore, the frozen single-use bags were sawed and drilled in different sections to evaluate the degree of homogeneity.

The single-use bag frozen in a static freezer showed a high concentration rate in the lower center of the bags. Compared to the homogeneity before freezing, the formulation accumulated up to 212%. In contrast, the single-use bag frozen in the plate-based freezer showed a difference of up to 32% inside the bag, therebyconfirming the more frequent occurrence of cryoconcentration in static and blast freezers. With the plate freezer the effect of cryoconcentration was largely prevented.

Control freezing and thawing of bottles

Due to their physical shape, bottles are not suitable for freezing on plates. However, there are ways to improve freezing and thawing processes for bottles as well. These are usually frozen in freezers by the air flow from integrated fans. The hybrid version of RoSS.pFTU Large Scale​ – a Freeze and Thaw Platform – enables both freezing methods, plate-based and airflow-based, depending on the primary packaging used.

During the freezing process of liquids in bottles, a “volcano cryo-concentration effect” is often observed (Figure 2). The accumulation of formulation in the ice formation due to slow freezing results in an upward pressure bulge and a groove in the center of the bottle.

Single Use Support has conducted a series of test runs to find solutions to achieve homogeneous freezing results in bottles.1​ Two 4-litre bottles from the same manufacturer were filled with a protein surrogate solution. As a result, the combination of the Hybrid RoSS.pFTU Large Scale and the use of insulation bottle caps enabled homogeneity after freezing surrogates in rigid containers to -80°C.


FIG 3 cryoconcentration_bottles

Figure 3: Volcano cryoconcentration in bottles frozen in static freezer without insulation bottle caps (left) vs. frozen in RoSS.pFTU Large Scale Hybrid with insulation bottle caps (right)

The insulating caps are devices that are attached to single-use bags and bottles when they are frozen in static or blast freezers to control homogeneity and pressure at the last point of freeze in cryogenic applications in bottles. These vendor-agnostic insulation devices attached to the bottle cap can be supplied for any type and size of bottle to ensure homogeneous freezing in the hybrid freeze/thaw platform RoSS.pFTU large scale.

Plate-based platforms support both cooling and heating of pharmaceuticals. This means that the same method used for freezing can also be applied for controlled thawing of single-use bags. Bottles, on the other hand, are thawed by a warm air flow. In addition, a shaking function supports the thawing process of drug substances in bottles.  The RoSS.pFTU Large Scale Hybrid shakes with a linear motion which can be adjusted according to the product characteristics, and avoids shaking-induced frothing.  

Kick off for hybrid systems in bioprocessing

Bags are the primary packaging that ensures the most efficient process and the highest quality in liquid and cold chain management. 

Bottles are still widely used due to their overall initial costs and ease of process integration into manual processes. However, manufacturers and CMOs face challenges when using bottles in commercial scales. These include limited scalability and storage capacities due to the bulky shape and lack of seamless integration into automated workflows. Most importantly, the slower freezing time results in longer process times and reduced product quality by higher risk of cryoconcentration.

There is a trend towards single-use bags, however it is unlikely that bottles will be replaced anytime soon. Hybrid solutions support manufacturers to embrace the handling of both single-use bags and bottles. They offer additional process flexibility and an efficient footprint by providing an all-in-one platform for modern freezing and thawing.


1.​ Single Use Support GmbH: Determination of Cryoconcentration of Bottles in a Blast Freezer. 2022. Data on file.
2.​ Minatovicz B. et al.: Freeze-concentration of solutes during bulk freezing and its impact on protein stability. Journal of Drug Delivery Science and Technology 58 (2020) 101703, p.13
3.​ Hauptmann A. et al.: Distribution of Protein Content and Number of Aggregates in Monoclonal Antibody Formulation After Large-Scale Freezing. AAPS PharmSciTech (2019) 20:72

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