Safe Freeze–Thaw of Protein Drug Products: A QbD Approach

Apply a DoE strategy to test several formulations in parallel.
Dec 01, 2010
Volume 23, Issue 12


Commercializing therapeutic proteins involves a series of processes aimed at maintaining safe and efficient protein drug solutions before final patient administration. Common operations include important steps such as pre-formulation, drug product formulation, sterile filtration, freezing, thawing, and freeze-drying intended to stabilize the protein drug before fill-and-finish, and during storage and transportation. Freeze–thaw operations used in the biotechnology industry still are generating debates regarding safety problems because methods to freeze and thaw samples can affect the purity, activity, safety, and efficacy of the final product. This article presents a Quality by Design (QbD) approach to define a safe freeze–thaw space where a protein's quality is not affected by the freezing or thawing method used.

Sartorius Stedim Biotech
Freezing is a process step used by most biopharmaceutical companies to store proteins. Yet little attention is generally given to this important step, even though reports suggest that processes for freezing protein solutions require detailed precautions.1–8 During freezing, the physical environment changes dramatically, which can significantly affect the protein quality. Moreover, freeze–thaw variations can exist in or between batches, and heterogeneous processes raise serious validation concerns. Cryoconcentration has been recognized as the most likely and important stress for biopharmaceuticals during freezing, and occurs at two levels.1–9 At the microscopic scale, when water molecules of the bulk buffer crystallize, an unavoidable dehydration of the amorphous phase occurs, called amorphous phase cryoconcentration.8,9 Although highly concentrated, the amorphous phase quickly reaches its frozen glass transition temperature (Tg'), below which the high viscosity prevents molecular interactions, leading to product loss.

At the macroscopic scale, a bulk-scale or progressive freeze concentration may result from back-diffusion of solutes from the solidification front to the remaining unfrozen solution.6 This redistribution of solutes in front of the advancing surface of ice can lead to product and solute concentration in the liquid phase for an extended period of time and may be detrimental for product stability (e.g., oxidation, aggregation, denaturation).

To limit the cryoconcentration effect, Sartorius Stedim Biotech has developed the Celsius Control Freeze Thaw (CFT) technology: a single–use system for controlling the freezing and thawing rate at manufacturing- and laboratory-scale.7 With this system, the ice crystal growth rate in the direction of the heat flux is sufficient to prevent back-diffusion of solutes from inter-crystalline space into the liquid bulk, thus minimizing the bulk-scale cryoconcentration.9

In this article, we evaluate the suitability of the laboratory-scale Celsius S3 system as a screening tool for determining a safe freeze–thaw space where a protein's quality is not affected by the freezing or thawing method used.