Stakeholders of Freeze Drying
What is Freeze Drying and What are its Uses?
Freeze-drying, also known as lyophilization, is a powerful technique used for long-term preservation of labile samples in various fields, from pharmaceuticals, and food industry, to biotechnology, and scientific laboratories. In principle the process involves removing up to 99% of water from a sample, which is a key factor in metabolic activity and subsequently product deterioration.1 The process begins by freezing the material at low temperatures, then applying a vacuum to convert ice directly into vapor by sublimation. This method preserves the structural and chemical integrity of the sensitive compounds without the need for refrigeration.
Though its roots trace back to indigenous practices as far back as the 13th century, modern lyophilization saw a breakthrough during World War II, when it enabled the stable, transportable storage of blood plasma and penicillin to its recipients while preserving the chemical stability of the product.2 Since then, the utilization of lyophilization has advanced significantly and found applications in a wide range of fields, including preservation of food for NASA expeditions, pharmaceutical drugs, vaccines, microorganism, proteins, and even valuable documents and books.
Lyophilization is no longer a niche technique, it’s a cornerstone of our everyday life.
What Happens When You Freeze Dry Your Sample?
Freeze-drying removes water molecules from a sample while preserving its physical structure and chemical integrity, a key advantage when working with sensitive materials. The process starts by freezing the material at a low temperature, converting the water into ice crystals. Next, the sample is placed under a strong vacuum, dramatically lowering the atmospheric pressure, then the temperature is increased. Under these conditions water bypasses the liquid phase and transitions to its gaseous phase, in a process known as sublimation.
This conversion of ice to vapor protects the shape and integrity of fragile samples. By eliminating water, a key component in many chemical reactions, freeze-drying significantly slows down molecular activity. This results in a stable, structurally intact sample with an extended shelf life, ideal for long-term storage or transport.
What are Lyoprotectants and Cryoprotectants?
Simply removing the water is not enough to preserve a sample’s integrity during freeze-drying. The process alone can cause molecules to change shape and lose their function. Therefore, the sample needs to be protected from being damaged by the stress of the freeze-drying process. Special additives, known as excipients, are utilized to shield the sample from damage during the processing cycle.
- Matrix Agents:These serve as a physical scaffold, maintaining the structural shape of the sample during and after freeze-drying. Commonly used agents include skim milk and bovine serum albumin (BSA), which provide mechanical support 3,5.
- Lyoprotectants: These additives protect biomolecules, especially proteins and cells, from stress-induced damage during drying. Widely used lyoprotectants include disaccharide sugars such as sucrose and trehalose, which form a glassy matrix that mimics the hydrogen-bonding role of water molecules1,3.
As water is removed during freeze-drying, those components will form a glassy matrix around the biological material. This matrix also helps maintain the native conformation of proteins, a critical factor for preserving biological activity post-rehydration. Importantly, salt should be avoided in freeze-drying solution formulations, as they become highly concentrated during freezing, and cause local dehydration, damaging biomolecules and cells.
An alternative to freeze-drying is cryopreservation. This process is used to store biological materials such as cells, tissues, and organs at extremely low temperatures, using low temperature freezers or liquid nitrogen. The main goal, like freeze-drying, is to slow or stop biological processes and maintain viability for long periods. Freezing, however, the water inside the sample can form sharp ice crystals that might puncture cell membranes or damage delicate structures. To prevent this, cryoprotectants are added to the sample before freezing.
Although both lyoprotectants and cryoprotectants serve to stabilize biological materials, their mechanisms and compatibilities differ:
| Preservation Method | Type | Function | Examples |
| Freeze-Drying | Lyoprotectants | Prevent structural collapse during drying | Trehalose, Sucrose |
| Cryopreservation | Cryoprotectants | Prevent ice crystal damage during freezing | DMSO, Glycerol, Ethylene Glycol |
While some lyoprotectants may offer mild cryoprotective benefits, cryoprotectants like DMSO and glycerol are unsuitable for lyophilization. They interfere with the freezing and sublimation steps, preventing proper dehydration and potentially destabilizing the product.
Understanding these distinctions is essential when designing a preservation strategy, ensuring the selected excipient matches both the sample type and storage method.
Lyophilization Protocol Overview
The process of lyophilization, or freeze-drying, involves three major steps: freezing, primary drying, and secondary drying. Thanks to technological advances, modern freeze-drying equipment is now available from small pilot units to large industrial scale.
- Freezing Phase
The first step involves cooling the product chamber to approximately -40°C. It’s crucial to allow the chamber to reach and stabilize at this temperature before beginning the freeze cycle. The hold time is dictated by sample volume and fill depth2:
- ≤1 cm fill depth: ~1-hour hold.
- 1-2 cm fill depth: ~2-hour hold.
- >2 cm fill depth: Longer hold times may be needed for uniform freezing.
Proper freezing minimizes the formation of large ice crystals, which can damage cellular structures and reduce viability upon rehydration3.
- Primary Drying
Once the sample is completely frozen, primary drying begins. This phase involves sublimating ice under a vacuum, typically at 50-200 mTorr, while the chamber temperature is gradually increased—often to around -15°C2. This step is the longest and most critical, as it poses the greatest risk for product collapse or meltback if the temperature exceeds the formulation’s collapse temperature4.
Drying duration varies significantly depending on sample characteristics, ranging from several hours to multiple days.
- Secondary Drying
During primary drying most of the ice has sublimated, secondary drying removes bound moisture. After primary drying, the temperature is then increased further to around +20°C to remove residual water molecules. This phase typically lasts 3 to 6 hours2. Completing this step ensures better stability and shelf life by minimizing moisture content.
A clear understanding of each step in the process is important for selecting optimal conditions and settings, ensuring successful sample preservation, and consistency across different batches. It also can be a lifesaver when troubleshooting difficult samples.
Why Set Up Lyophilized Quality Control (QC) Tests?
Lyophilization involves multiple steps where minor deviations can significantly affect the final product.
Common QC tests include:
- Visual inspection: Detects signs of collapse, shrinkage, or discoloration.
- Moisture content analysis: Confirms appropriate drying levels.
- Reconstitution testing: Measures how quickly and completely the product dissolves.
- Biological assays: Verify activity and viability in biologics (e.g., enzymes, bacteria).
Implementing QC early helps optimize the process, detect failures, and improve reproducibility across manufacturing runs4.
Where Can Freeze Dried Samples Be Stored?
Once freeze-dried, samples can still be affected by environmental factors such as temperature, humidity, and light. Therefore, proper storage conditions are essential to maintain their stability over time.
- Container integrity: Use airtight, vacuum-sealed, or nitrogen-flushed vials.
- Temperature:
- 2-8°C: Standard for biological samples.
- Room temperature: Acceptable for more stable formulations.
- -20°C to -80°C: Recommended for sensitive or long-term storage.
- Light protection: Amber or opaque containers help protect light-sensitive compounds.
The optimal storage condition depends on the product’s composition, sensitivity, and intended shelf-life5.
Ultimately, selecting the ideal storage condition depends on the type of sample, its composition, and how long it needs to remain stable.
Conclusion
Lyophilization is a powerful method for preserving delicate materials, from pharmaceutical to biological specimens. Mastery of the freezing, drying and QC processes, along with appropriate storage conditions, ensures that freeze-dried products remain stable, effective and fit for purpose. With continuous improvements in technology and process understanding, lyophilization remains a cornerstone of modern preservation science1. Comprehending these stakeholders ensures lyophilized materials retain their integrity, effectiveness, and intended properties.
1https://onlinelibrary.wiley.com/doi/10.1155/2019/9502856
2Corver, Jos. "The Evolution of Freeze Drying". Innovations in Pharmaceutical Technology. Archived from the original on 2018-05-20. Retrieved 2018-05-20.
3https://pmc.ncbi.nlm.nih.gov/articles/PMC10661802/
4Fissore D. (2017). Model-Based PAT for Quality Management in Pharmaceuticals Freeze-Drying: State of the Art. Frontiers in bioengineering and biotechnology, 5, 5. https://doi.org/10.3389/fbioe.2017.00005
5OPS Diagnostics https://opsdiagnostics.com/notes/applicationnotes.html