Lyophilization may appear like a gentle drying process, but it comes with significant risks when used with biomolecular reagents in diagnostic tests that are not the same, like the freeze-drying of general pharmaceuticals.
The reagents that are mostly used in PCR and ELISA-based diagnostic kits for conditions like Covid-19, are known to have labile components. These may include enzymes that are necessary to preserve longevity in the supply chain. They may also be vital in preserving the longevity of the antibodies used for testing the patients’ response to the virus. These reagents may not be easy to stabilize when considering commercial use.
In a recent webinar by Dr. Kevin Ward – the Director of Research and Development at Biopharma Group UK, explored the various aspects of formulation and cycle development in the lyophilization processes of diagnostic reagents. This included Covid-19 testing, which may be the same as the regular diagnostics. In this technote, you will get a summary of the webinar, as well as an overview of some of the questions raised during the Q & A session.
Lyophilization of Biomolecular Diagnostic Reagents
Different molecules have varying sensitivity when it comes to storage and processing conditions. For biomolecules, it is vital that the molecules remain chemically intact during the dry-freezing process and they must also maintain the necessary 3D structure. The goal is to reduce the chances of degradation of activity that may be experienced as a result of denaturation or aggregation.
Further challenges still exist when it comes to biomolecular-based diagnostics, especially for Covid-19, which is not predictable compared to dry-freezing other molecules. These issues were discussed in the webinar, and then summarized as follows:
Low Volume Diagnostics
The diagnostic kits that are normally used tend to have small volumes of reagents that may sometimes evaporate very quickly. In as much as a few microliters of these reagents in tubes may take slightly longer to evaporate, samples in microfluidic channels or on plates that are already exposed throughout the loading process may be more susceptible to evaporation. Since there is an urgency for Covid-19 testing, high throughput is necessary. Consequently, most of the Covid-19 diagnostic kits happen in 96-well plates, microfluidic channels, or chips. One method that can be used to partly overcome this method is by loading onto a cooled freeze dryer.
Stabilization of biomolecules
Most of the biomolecular components are never sensitive to conditions of freeze-drying. However, some are, and it is vital to take note of the kind of effects that these few minorities might have. When a biomolecular component is sensitive to cold denaturation, it may lead to precipitation or aggregation of proteins. This is, however, a challenge that can be easily overcome through the addition of a surfactant or by cooling slowly. There are also chances that some of the components may get damaged as a result of freeze concentration effects that will normally feature a shift in the pH. Phosphate buffer is usually the main cause of this.
As such, some of the potential remedies to this is a change of buffer or cooling quickly. It is also possible for proteins to be susceptible to interfacial effects, which may measure proportionately to the surface area of the ice crystal. However, these effects can be overcome by cooling slowly or through controlled nucleation that may increase the size of the crystal while at the same time reducing its surface area. It is also possible for the protein to be destabilized through the dehydration process. In some instances, it may be necessary to have structural water to help maintain the structure of the protein.
Having glycerol in a PCR reagent may come with some serious problems. This is because glycerol will not freeze under normal freezing conditions. However, this problem can be overcome by simply diluting the glycerol out with a high enough stock solution or by adding an excipient. But it is still possible that you will get some glycerol in the dried cake.
This has the potential of affecting the stability and the structure. For Covid-19 diagnostic kits, there might not be as much emphasis on shelf life, as there is with other types of diagnostic kits. The reason for this, is because Covid-19 diagnostic kits don’t require lengthy storage like the other types of diagnostic kits. It is also possible to store them at low temperatures, and as such, the presence of glycerol will not be pronounced too much regarding the stability of the active components.
The nature of containers used such as vials, wells, tubes, chips, paper, etc, and their respective closures, may impact the long or short-term effects of stability, as well as the thermal transfer. In as much as it is challenging to seal a container without allowing some moisture to get in, it should be remembered that some plates may have higher permeability to water compared to the permeability of their seals to water.
During the webinar, Dr. Ward touched on the use of a controlled environment with low humidity, and reduced oxygen, as well as the use of a flexible isolator, to reduce the amount of moisture present in the product. In the case of Covid-19 testing, a lot of emphasis may be placed on initial performance and rapid throughput, compared to long-term stability. As such, some of these measures may not be applicable.
Freeze Drying Equipment
While most companies are making frantic efforts to maximize throughput over long-term stability for Covid-19 diagnostic kits, some companies are leveraging the power and speed of contract lyophilization.
Canada Peptide is equipped with multiple freeze dryers of various sizes. Capabilities to lyophilize up to 6000 vials per machine, per run. Our ISO5 fill & finish lyophilization suite is well suited for the production of diagnostic kits.
It is important to consider dry-freezing strategies for diagnostic biomolecular components. But this should be in accordance with the component’s sensitivity, while at the same time considering all the issues discussed above. For Covid-19 diagnostic tests, there is a very high possibility that the reagents will be processed in small fill volumes, but at high batch numbers with the main focus being on the throughput, as opposed to long-term stability. Therefore, this has to be considered in the design of a lyophilization protocol for the Covid-19 diagnostic test.
How does spray drying compare to freeze-drying?
Several interesting studies have been done on this topic. This has been the main focus area for Dr. Sune Klint Aderson in Denmark. We strongly recommend reviewing his articles for further reading.
When you compare a normal lyophilized vial product and a diagnostic biological product, you may get at least a 3% difference in the moisture content. What is the normal range for the moisture content and how long is its shelf life?
The range in the target moisture level may be wide, and this depends on the specific formulation, as well as how long it took the lyophilized form to become stable at the desired temperatures. This is a factor of classic kinetics, such as Arrhenius or Williams-Landel-Ferry.
However, any presence of moisture may plasticize the product, leading to a reduction of its dry state glass transition temperature. Also, the presence of a high number of residual water molecules may result in an increase in hydrolytic-based reactions.
Are there any advantages for multi-steps of first drying?
With the greatest challenge in most cases being an increase in the product resistance to vapor flow, sometimes it may be possible to develop more aggressive conditions during the early stages in primary drying. This will increase the efficiency and it may also help in reducing the heat input, which in turn, will help to maintain the product within its critical temperatures until all ice is removed.
What is the relevance of stoppering in small vial works?
Stoppering in small vials should work just as well with large vials. However, you should get more low-profile frames or trays whose heights are shorter than the heights of fully stoppered vials. You may also find the force applied by the stoppering ram necessary. Also, you need to ensure that the stroke length is enough to allow for stoppering throughout all levels.
Why would one Covid-19 diagnostic test yield faster results than another?
Some of the tests are DNA or RNA-based, while others are antibody-based. Also, some tests may be designed for use with different types of patients, containing the molecule of interest at different concentrations. The dynamics of the reaction will vary according to these parameters.
Does high cake resistance lead to higher moisture levels after primary or secondary drying?
High cake resistance at the initial start of primary drying can prolong the overall process. If other factors remain constant, primary drying may be faster with larger ice channels. But, this may imply that the material has a lower specific surface area. Hence, secondary drying may be slower. Most people will prefer more efficient primary drying, since it is usually the longest part of the process, and it comes with the highest risks if skipped.