Controlled Freezing in Biopharmaceuticals

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<h1 class="blog-h1">Controlled Rate Freezing, Thawing, and Biopharmaceutical Viability</h1>

The overarching goal for any bioprocess engineer is to minimize physiological changes in biopharmaceuticals by controlling the production process. Biopharma companies often employ a variety of cGMP procedures to monitor molecular stability throughout product processing. Cryopreservation can extend a drug product’s shelf life at the storage step, sometimes indefinitely. However, continued efficacy relies on ensuring how products are frozen and thawed.

<h2 class="blog-h2">Risks to Biopharmaceutical Product Quality Attributes (PQA) During Freezing and Thawing</h2>

Biological sciences at the forefront of life science, biopharma, and biotech industries are often rooted in the physical attributes of cellular biology, where the recrystallization temperature of water at -120°C is a crucial metric. Here, the viability of a mammalian cell is predicated on whether the cellular wall integrity is maintained during processing, pulldown to temperature, long-term storage, and thawing.

Proper cell preparation is critical to viability at all phases of the process. Suspension agents such as glycerol or dimethyl sulfoxide (DMSO) are used to prevent the formation of microscopic ice crystals, either outside or within the cell. Formation of such crystals can compromise the cell wall, and cell lysis, thereby destroying the cell or the product the cell is engineered to produce.

<h2 class="blog-h2">Uncontrolled Rate Freezing of Biopharma Drugs and Therapeutics</h2>

Drug products or drug substances are typically stored and thawed using either a controlled rate freezing process or an uncontrolled rate freezing process. To ensure product degradation is arrested so shelf life is extended adequately for long-term storage and to prevent destabilization of drug product molecules, samples must reach the glass transition temperature (Tg′) of the protein through a controlled cooling rate.1

Even with standard procedures or limited controls, uncontrolled freezing and thawing of drug substances produce results that are not scalable or reproducible. Uncontrolled rate freezing can also create stress factors like pH shifts and cold denaturation that impact the PQA of biopharmaceuticals. It is only through controlled, slow freezing, and gradual thaw of biopharmaceutical drug products and therapeutics that consistent, optimized results can be assured.

<h2 class="blog-h2">Maintaining Sample Integrity Through Controlled Rate Freezing and Thawing</h2>

The cell itself is at the center of most life science research and development based on mammalian cell culture. While biological attributes are endemic to the cell, either engineered or natural, the cell is the vehicle wherein all activity is focused. When pharmaceutical manufacturing focus originates at the cellular level, the need to protect molecular stability at all production phases remains a common denominator to assure drug product integrity, viability, and efficacy. The speed at which biopharmaceutical products are frozen, the temperature at which they are initially cooled down, the dwell time required to soak the load thoroughly, and the release to the final storage temperature are functions that can be achieved only with a forced-air system. When cGMP criteria are imposed and compliance with FDA 21 CFR pharmaceutical or drug quality-related regulations are mandated, performance and documentation are essential to verify pulldown time and temperature uniformity of ultra low temperature equipment. The selection of an active control ultra low temperature freezer as a part of the overall production process sets the process apart from conventional freezer designs.

Additionally, as pharmaceutical products are moved through manufacturing bulk, the need for high-volume cooling capacity demands a balance between cabinet size and design, refrigeration capacity, BTU removal capability, inventory loading design, and performance tracking and recordkeeping at all stages.

<h2 class="blog-h2">Equipment Considerations for Biopharma Product Pulldown and Storage</h2>

Not all ultra low temperature freezers can safely deliver the conditions necessary to freeze and thaw biopharmaceutical products. Regardless of the intended temperature, a basic understanding of ultra low temperature freezer categories includes knowing the difference between storage freezers and process freezers, cold-wall and forced air cooling, steady-state and process operation, and heat removal capacity and its effect on cellular mechanisms. While the science of biological preservation is extensive and includes permutations specific to what is being frozen, for what purpose, and for how long, the need to freeze a biological product, store it, thaw it, and transport it is central to the biopharma industry.

Most ultra low temperature freezers used for routine storage and long-term preservation are designed as cold-wall cabinets with passive temperature control. Cold air surrounds the freezer load, which naturally achieves a steady state at the desired setpoint, typically -80°C, without added air circulation. Once frozen, cells can be stored indefinitely under these conditions, although it is impossible to know the effects of the freezing process until the cells are thawed, perhaps months or years later. While traditional ultra low freezers work well for finalizing frozen drug products, only a forced-air freezer designed for high throughput freezing can eliminate uncertainty about whether a product load is completely frozen at the proper temperature after an approved pulldown.


<h2 class="blog-h2">Forced Air Convection Technology</h2>

While passive cold wall storage has its place in the overall equation, the need for controlled rate freezing remains a consideration in how to get biopharma drug products to setpoint in the first place. Here, a forced-air freezer, often called a forced-draft or “blast” freezer, provides active control over pulldown and soak temperature, release to warmer storage temperature, and the capability to initiate controlled rate thawing on demand. Critical to this process is control accuracy, usually established as many degrees per minute, temperature accuracy to within a degree, and temperature uniformity throughout the stored product. Without close temperature uniformity, the expectations for reproducibility and optimum drug product PQA cannot be assured.

Despite the biochemistry and physics of biopharma production, temperature control throughout the process is absolute, from production to storage to cold chain protection. This was widely proven during the COVID-19 pandemic when pharmaceutical companies faced transportation and last-mile storage challenges created by ultra low temperature storage requirements of mRNA vaccines. When transit conditions could not be documented, vaccine lots were rejected as their efficacy could no longer be assured.

The use of ultra low temperature freezers in biopharma production is a convergence of refrigeration technology, the chemistry and physics of blended refrigerants, the science of cellular biology, the logistics of production and storage, and an integrated quality assurance commitment that offers end-to-end documentation. Anecdotal evidence suggests that pharmaceutical products frozen for storage are not always frozen throughout. More than appearance is required to ascertain target temperature compliance. For this reason, a forced-air freezer can be programmed to freeze a product from ambient at a controlled rate. This allows bioprocess engineers to continue the freezing function through the target storage temperature and hold the product load at a deeper temperature dwell to thoroughly soak the bulk materials at a dwell below the long-term storage target. Once the dwell period is completed, the load can be released to a warmer storage temperature, typically -80°C.

Once pharmaceutical products are properly frozen, they can be transferred to walk-in rooms for inventory storage before shipment. Here, the freezer function is maintenance instead of a pulldown process. Walk-in rooms should be used for storage only, not for pulldown. Unlike storage freezers, including walk-in rooms, forced-air freezers are performance-based for cell protection, a deliverable that emphasizes getting the job done quickly and with a primary focus on quality. Because forced-air ultra low temperature freezers can cool faster and with better control over the pulldown process, they are an essential component in the production process.

While most pharmaceutical processes remain proprietary, additional material on controlled rate freezing and thawing is available from industry resources such as the International Society for Biological and Environmental Repositories (ISBER) and manufacturers such as FARRAR™, powered by Trane Technologies.

Purpose-built for bioprocessing applications, the 4000 Series Controlled Rate Chamber offers uniformity and repeatability in rapid, controlled freezing and thawing applications. Unique forced-air convection cooling technology significantly reduces +40°C to -80°C freeze/thaw times from up to days or weeks to hours. Programmable freeze/thaw profiles and universal container acceptance mean that one rate chamber meets multiple drug product/drug substance needs.




  1. William J. Rayfield, Sunitha Kandula, Heera Khan, Nihal Tugcu, “Impact of Freeze/Thaw Process on Drug Substance Storage of Therapeutics,” Journal of Pharmaceutical Sciences, Volume 106, Issue 8, 2017, Pages 1944-1951,
  2. Nikola Radmanovic, Tim Serno, Susanne Joerg, Oliver Germershaus, “Understanding the Freezing of Biopharmaceuticals: First-Principle Modeling of the Process and Evaluation of Its Effect on Product Quality,” Journal of Pharmaceutical Sciences, Volume 102, Issue 8, 2013, Pages 2495-2507,

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