Evaluating Freeze/Thaw Processes in Drug Manufacturing

What goes on inside the freezer once we close the door might not be something that occupies our daily thoughts, but the conditions under which bulk drug substances are frozen and  subsequently thawed have a significant effect on the quality and stability of the final drug product.

Drug product manufacturing needs to be tightly controlled at all steps of the production process. As well as the core process parameters, this careful monitoring should extend to storage and transport stages.  

As manufacturing processes become increasingly complex and decentralized, there are likely to be more stages where bulk freezing of the drug substance could benefit product stability, quality, and process efficiency. 

_{This article is posted on our Science Snippets Blog.} 

Why do we Need to Freeze Drug Substances?

Freezing the bulk drug substance during or after production is primarily carried out to slow down chemical/biological degradation and preserve the product’s stability. However, it also has various additional benefits, for example, restricting microbial growth and extending the product shelf-life. Freezing also helps prevent contamination from extractables and leachables by limiting the exposure of the drug substance to the walls of the storage container. Finally, a frozen product undergoes significantly less agitation than a liquid product during transport. Excessive agitation (or “sloshing”) could potentially compromise the structure of shear stress-sensitive molecules in the formulation.

Biopharmaceuticals are expensive and are often manufactured in large volumes to achieve commercial scale. The bulk drug substance can be frozen in smaller batches, allowing the final drug product to be manufactured based on actual market demands. Therefore, freezing can also be operationally beneficial, enabling longer hold times between processing steps. 

Optimizing freeze/thaw conditions is essential to the production of a robust final product.

What Options do I Have For Freezing my Drug Substance?

There are many commercially available and purpose-built solutions for freezing drug substances. The decision for which to use is determined by many factors, including the stability of the formulation, production volume, transport needs, storage times, cost, and process requirements.  

Freeze/thaw technologies fall into two main categories: the traditional method of bottles, carboys, or freeze tanks and freeze/thaw bags. Bottles, carboys, and freeze tanks are usually glass, plastic, or stainless steel and they are stored in conventional or blast freezers. Freeze/thaw bags are a more modern method of frozen storage. They are sterile, single-use, disposable technologies offering a more flexible solution.

What are the Challenges Associated With Freezing Drug Products?

1. Cryo-concentration and Thawing

The most fundamental concern when designing freeze/thaw procedures is the phenomenon of cryo-concentration. During the freezing process, solutes are excluded from the ice-liquid interface by diffusion to specific areas of a container. This means the concentration of solutes in the formulation is non-uniform throughout the vessel. In some regions, where the formulation freezes later, there is a high concentration of proteins or excipients, creating a source of variability when the product is thawed. 

Fluctuations in local concentrations can create new formulation conditions, potentially causing alterations to the structure of macromolecules, aggregation of proteins, and changes in pH, ultimately impacting the stability and activity of the drug substance.  

Ultimately, cryo-concentration represents a barrier to maintaining product quality during freeze-thaw procedures and could create problems for maintaining full regulatory compliance.

2. Freezing Rate

An important consideration for limiting cryo-concentration is the freeze-path length, which is the distance from the freezing vessel’s edge to its center. Longer path lengths restrict heat transfer and result in slower freezing rates, measured by analyzing the last point to freeze. Cryo-concentration usually happens when the freezing process occurs slowly. 

It is not just the freezing process that impacts the degradation of the drug product; thawing can also alter the stability of the drug substance. Poorly controlled thawing (slow speeds and excessive shearing) can also cause damage to proteins.  

Therefore, the speed and conditions of the freezing process are critical factors in the manufacture, storage, and shipping of pharmaceutical products.  

3. Scaling-Up

Freezing procedures can be tricky to scale up. Lab- and pilot-scale models of freezing processes are not necessarily reflective of commercial-scale conditions. This is because the dimensions of containers have a considerable influence on freezing due to the freeze path length; the further away a substance is from the container walls, the greater the effect of cryo-concentration. The last point to freeze is challenging to control between scale size and distance from the container walls. Thus, significant parameter optimization and process simulations could be required during early process development.  

These challenges are mostly applicable to traditional, rigid freezing vessels using uncontrolled freeze/thaw rate technologies. There is a need for more robust and controllable solutions to these challenges, and disposable technologies are gaining attention.  

Bags vs. Bottles

Bottles – The Norm 

Traditionally, drug substances are frozen in stainless steel containers or bottles (carboys). The primary advantage of this method is its long-standing place as the ‘gold-standard’ in the industry. The protocols are well-established, and the procedures are considered simple. However, carboys and bottles are often bulky and offer limited control of the freeze/thaw process, making it tricky to maintain the intended formulation conditions. 

Controllable Freeze/Thaw Solutions in Disposable Bags  

In contrast, freeze/thaw bags offer a much greater capacity for control, enabling the maintenance of homogeneity in the drug substance during the freezing process. Bags provide a shorter freeze-path length than conventional systems, improving the heat transfer, minimizing cryo-concentration, and promoting uniform product distribution.  

Systematic research has demonstrated that the freezing process is an order of magnitude faster in controlled freeze/thaw bags compared to conventional methods. This study also revealed that freeze/thaw bags exhibited minimal cryo-concentration, in contrast to conventional carboys [ref]. 

Bags vs. Bottles – Who wins on Scalability, flexibility, and convenience?

Freeze/thaw bags are often manufactured with an identical freeze path length across container sizes, limiting the effects of variable cryo-concentration. This makes the process more scalable than traditional methods, facilitating a seamless transition into commercial-scale manufacturing, with a reproducible last point to freeze all across the scale-up. 

Bags also offer a logistical advantage, as they save space and provide more convenience in handling. Traditional systems can require a considerable amount of maintenance, cleaning, and testing. In contrast, single-use technologies are fully assembled and sterilized, meaning they are ready to use for closed aseptic processes. This limits the need for operator involvement and reduces the risk of contamination. The flexibility offered by single-use disposable technologies also allows users to quickly respond to changing clinical and commercial environments.

Freeze/Thaw Bags - Necessary For All Drug Products?

Not all freeze/thaw bags are made equal; there are many parameters affecting performance, including the interaction between the product and the container, the mechanical properties, suitability for cold chain temperature, and the permeability of the bag. 

Importantly, controlled freeze/thaw solutions are more expensive than traditional storage systems. Therefore, the decision of whether or not to implement these advanced solutions could be dictated by cost. Additionally, not all drug products are sensitive to denaturation. In cases where the drug product is robust enough to withstand freeze/thaw-mediated perturbation, users may wish to take their chances on uncontrolled freezing. However, newly emerging technologies, such as mRNA vaccines, tend to be very sensitive and need a highly controlled freezing environment. 

Undoubtedly, controlled freeze/thaw bags offer a superior solution for maintaining uniform conditions for the frozen storage of drug substances. They offer enhanced process reproducibility and ensure the final drug product is of consistent and reliable quality.