Lyophilization refers to the process of removing water molecules from a product after it is frozen and kept in a vacuum. The process is also known as freeze-drying, and is usually a long and complicated process that comes with a plethora of opportunities for potential applications. Peptides are an area of application of lyophilization, since there are a variety of pharmaceutical products that are usually subjected to the process. Apart from peptides, some of the usual targets for lyophilization include chemical API, collagen, proteins, enzymes, and oligonucleotide among others.
It is not always mandatory for the above-named substances to undergo lyophilization. The process becomes necessary and highly recommended when the bulk of the ingredients of the compound display signs of high instability when they are in liquid or frozen forms. There are a variety of reasons why the bulk of a compounds’ ingredients may be unstable in liquid or frozen forms, with the most notable culprits being sensitivity to heat, oxidation, chemical reactions, biological growth, aggregation and degradation among others.
The process of dry-freezing makes it possible for the substances to have a longer shelf life, and to make the transportation process of the substances easier. When the water molecule has been removed and the substance sealed in a vial, it’s not only easy to ship the substance but it can also be easily reconstituted to its original form for research, or any other application. Additionally, it is easy to store lyophilized products under room temperature conditions because they don’t contain any water molecules that might affect their formulation to lead to their degradation or a change in their chemical composition.
The Process of Lyophilization
As noted earlier, the process of lyophilization is to remove water molecules without damage so that they have an increased shelf life, can be transported and stored easily and also be reconstituted to its original form without damaging them in any way by simply adding back the removed water molecules. The entire process is usually an elaborate process beginning by freezing the product to be lyophilized under atmospheric pressure.
The very first drying phase is known as primary drying, and in this process, the water which is now in the form of ice is eliminated through another process known as sublimation. In the sublimation process, the frozen water molecules are converted into vapor without turning into a liquid. The second phase of lyophilization is known as secondary drying. At this stage, water is removed through a process known as desorption. When it comes to the final quality of the lyophilized substance, it will be determined by the specific conditions under which each of the processes took place.
The Freezing Process
The freezing process aims to transform the product through the extraction of heat to create a state with the right conditions for sublimation drying – the evaporation of ice without turning it into a liquid. When the product to be frozen is cooled down, a crystal nuclei is formed. All the water surrounding this crystal nuclei are absorbed by the crystal, causing the crystal to increase in size and form a different shape. The shape and size of the crystal formed, however, will depend on factors such as the composition of the basic product, the speed of the freezing process, the amount of water in the substance, presence of crystallizing and non-crystallizing substances in the product, and the viscosity of the liquid. These factors will also have a direct influence on the overall process of sublimation. Large crystals will result in a relatively wide and open lattice after the process of sublimation is over, while the smaller crystals will result in relatively narrower spaces once sublimation is over and all the water vapor is removed.
The Primary Drying Process
The sublimation of the ice of the substance’s surface is the genesis of the primary drying phase. As the process sets in, the surfaces where sublimation takes place begin to withdraw inside the product, causing the ensuing vapor to escape through the surface that had previously been dried. The implication of this is that the drying process will mainly depend on the speed at which the vapor transfers as well as the speed at which the sublimation heat is removed.
The sublimation heat is conducted through convection and thermal conduction, whereas thermal radiation is responsible for transferring very minimal amounts of the sublimation heat. It should be noted that following the reduction of pressure in the drying chamber when the pressure falls below 10mbar, the convection process will practically stop. Due to this, it is necessary for the pressure in the drying chamber to be adjusted to the highest possible levels during the primary drying process because of its direct impact on the sublimation temperature.
There is no amount of heat needed on the surface of the substance for the sublimation process to take place. Heat is only needed at the ice core boundary that is receding into the center of the product as the drying process proceeds. Though the water vapor will be flowing from within the substance towards the outside, the flow of heat must be in the opposite direction. Since the dried product layers usually have low thermal conductivity, there has to be a temperature gradient so that the transfer of heat can steadily rise during the process. This makes sure that the substance is not damaged and that the maximum allowable temperature for the substance being dried is not exceeded.
Also, great care needs to be taken to ensure that the required sublimation temperature is maintained throughout the drying process. This means that the supply of heat to the ice core must be maintained at a steady rate, and that there are no instances of overheating anywhere near the sublimation zone. The primary drying phase will proceed until all the ice present in the product has been removed through the process of sublimation. Only then will the substance be ready to proceed to the secondary drying phase.
The normal freezing point of pure water is usually zero degrees Celsius. If there are any other substances dissolved in the water, then it will no longer be considered pure, and it's freezing point is likely to go down. In the case of inorganic salts dissolved in water, if the solution is frozen, the freezing of pure ice will lead to an increase in the concentration of the inorganic salts in the solution, and this will further lower the freezing point of the solution. What has been observed with this type of freezing is that products will behave differently and the ultimate results will depend on the specific freezing technique used.
Before a freezing technique is preferred for any particular substance, its parameters must be ascertained before the process of sublimation drying. The freezing behavior of the product must be researched so that it is known in advance what should be expected once the process starts. In most cases, and the majority of pharmaceutical applications, the two main freezing techniques used include freezing by contact with cooled surfaces and dynamic or rotation freezing with the help of a coolant bath.
The secondary drying is the final drying phase in the lyophilization process. Its purpose is to reduce any residual moisture content that may be present in the substance to the lowest levels possible so that the product can be in a state where it can be stored permanently. At this stage, all the water molecules that might have been bound through the adsorption process at the product’s internal surfaces have to be removed. For this to happen, the capillary forces of the water must be overcome.
As such, the lyophilization service provider or the freeze-drying plant should be designed in such a way that it can give a higher pressure gradient during the second phase of the drying process because it is not always possible to increase the temperature of the product without causing any damage to it. This secondary drying process must be controlled precisely to avoid any instances of over-drying the product.
After the drying process
The after-treatment of the lyophilized substance is majorly aimed at protecting the substance once the drying process is over. At this stage, the substances are usually hygroscopic because of their large internal surfaces, and if adequate protection is not offered, they may end up absorbing water and undoing all the work done. If the drying process took place in a vial, flask or a bottle, it is a good idea to close the container immediately once the drying process is completed. There are specially designed ribbed rubber stoppers that can be fitted to the necks of the flasks, vials or bottles to act as stoppers and to stop the substances from absorbing any moisture. If possible, the sealing of the containers should be under vacuum or they can also be sealed under a protective gas atmosphere. The sealing method is chosen and the other after-treatments will vary from one substance to another.
How Lyophilization Impacts Long Term Sample Storage
In most cases, samples that should be stored for an extended period in pharmaceutical and biotechnology are usually highly labile. This means that heat can be used in the desiccation process and can also damage them or have a significant impact on their stability and quality. Substances such as enzymes and peptides, for example, can be used in a variety of assay kits, and when they are desiccated, it becomes possible to extend their shelf life and also easy to transport.
These proteins display very precise micro molecular structures and they are used for precise biological applications. When they are exposed to heat, their noncovalent bonds are likely to be broken and their overall structure may also be disrupted. This is why lyophilization is the highly recommended process to facilitate the long term storage of such substances.
Lyophilization and Long Term Storage Challenges
Due to the advance effects of water and heat, the removal of water molecules through lyophilization has presented a myriad of advantages, ranging from improved shelf life to reduced costs, increased purity to improved stability. When dehydration by heating is eliminated, dry freezing provides a safe and convenient way to store lab samples and pharma products for the long term without compromising their activity in any way. In addition to increasing the shelf life, the lyophilization process can also reduce the volume and the weight of the substance. As a result, there will be a reduction in the shipping costs and the overall impact on the environment will also be reduced. With such, it is not necessary to have shipping procedures in place for maintaining the sample stability such as the use of dry ice, which further helps reduce the logistics costs.
These benefits of lyophilization are of immense importance when it comes to the transportation of assays and samples to remote destinations and developing countries known to have budget deficits and resource constraints. Sometimes the samples and the assays need to be transported over long distances during which the transportation conditions cannot be controlled. For example, the assays may have to be transported to hot and humid environments which may create substantial risks to the stability and purity of the samples and assays. With lyophilization, however, it is possible to have a temperature-controlled chain when necessary during transportation over long distances and storage over long periods.
Choosing the Right Lyophilization Service Provider
Lyophilization is a delicate and complex process, and not every peptide synthesis company is suited to offer this service. Consequently, it is imperative to take your time to research and choose a reputable company with a track record of offering the best lyophilization services. Don’t just choose the first company that comes your way, lest you end up wasting your time and money.