Cryogenic freezing has become a critical process step in advanced therapeutics

Cryogenic freezing is widely known for its ability to preserve organisms and their viability. For that reason, it is an important process not only in medicine but in the biopharma industry, example, for cell and gene therapy. Nevertheless, safe long-term storage at ultra-low temperatures is highly complex and requires precise methods and protocols, to ensure safety and functionality. Consequently, it comes as no surprise that there are multiple challenges that can occur in the field of cryopreservation.

We had the opportunity to talk to cryogenic freezing expert Michael Wieland, Senior Director and Head of Global Applications Services at Single Use Support and ask him all about cryopreservation in biopharma, its challenges and solutions.

What significance does cryogenic freezing have for the pharma industry?

Michael Wieland: It is no secret that freezing process intermediates, e.g. bulk drug substance, offers tremendous advantages to drug manufacturers, such as providing added process flexibility, shelf life, and ease of transportation. However, to some processes cryogenic freezing is strictly necessary, as for example with advanced therapeutics, where cryogenic freezing has become a critical process step.

What are your experiences with cryogenic freezing in gene therapy? What substances are being frozen in the manufacturing process?

Wieland: Freezing a process intermediate can be of value for most of the intermediates that are far downstream in the process, as early steps often involve too much volume to be frozen efficiently and first need to be concentrated down. Cryogenic freezing can be of most value when it allows bulk drug substance to be stored and shipped freely, without fearing for loss of stability or scheduling issues.

Which difficulties can occur with cryogenic freezing?

Wieland: Traditionally not much attention has been paid to the process of cryogenic freezing and the potential challenges that can be associated with it, such as cryo-concentration, where changes in pH and ionic strength, caused by the expanding ice, can lead to protein denaturation and degradation. Also, phenomena like turteling, or the effect of low temperatures on plastics, can lead to damage and loss of product.

Have you ever had a freezing process “backfire” and impact the cells and their viability negatively?

Wieland: Yes, of course. Freezing cells is always tricky if done in large volumes, because so -called cryo-protectants have to be added to protect cells from the effects of freezing. However, these cryo-protectants are cytotoxic, unfortunately. This means, unless the cells are frozen rapidly, it will result in loss of cell viability. To avoid that, it’s smart to use a plate freezer as using a plate freezer can really take the process of freezing cells in large volumes to the next level, since cells will freeze faster.

What cryopreservation equipment is needed to ensure safe freezing and thawing?

Wieland: This question is not easy to answer because it highly depends on process specifics. But in general terms, as with other process steps, the biopharmaceutical industry is aiming at a linear scale-up of processes from development to large scale. In freezing, the relevant parameter to keep constant during scale up is the speed the ice-front moves through the liquid. Using equipment that can control the rate of freezing, for example a plate freezer, this parameter can be scaled up in a controlled way.

Have you ever experienced that cells froze onto cp bags? How do you prevent that from happening?

Wieland: That is an excellent question! Although, I have never experienced this myself, I am aware that this is a painful subject in the industry. I believe the key here is speed; freezing the suspended cells before they have time to congregate at the bottom of the bag. For small volumes a traditional freezer might be sufficient, but for full scale volumes only a plate freezer will provide adequate freezing speed.

What do you think the future holds for cryopreservation?

Wieland: As it stands now, a critical bottleneck in the production of advanced therapies, especially cell therapies, is the time it takes from filling to freezing, as keeping this time as short as possible is crucial for the quality of the product. In the future, I see more automation to speed up filling of bags and moving into a freezer. Ideally, this would be a semi continuous step, where bags are frozen immediately after they are filled.