Although it is considered a relatively gentle drying process, there are still risks and pitfalls when applying lyophilization to biomolecular reagents in diagnostic tests, which are different from the freeze-drying of generic pharmaceuticals.
The reagents generally utilized in PCR and ELISA based diagnostic kits for diseases like COVID-19 usually to contain labile components, like antibodies that are used to test for a patient's response to the virus, which can be difficult to stabilize for commercial use, or enzymes that must be preserved for longevity in the supply chain.
Dr. Kevin Ward, Director of Research and Development at Biopharma Group, UK, recently gave a webinar looking at numerous aspects of formulation and cycle development in the lyophilization of diagnostic reagents. These aspects included those needed for COVID-19 testing, which may be different to regular diagnostics; this article summarizes the webinar.
Lyophilization of biomolecular diagnostic reagents
Different molecules show different sensitivities to storage and processing conditions. In the case of biomolecules, not only must the molecule stay chemically intact during the freeze-drying process, but it is also necessary for the 3D structure to be maintained.
The aim is to minimize the degradation of activity, which can be a result of factors like denaturation and aggregation. There are further challenges with biomolecule-based diagnostics, especially for COVID-19, which are less apparent than when freeze-drying other molecules, and these are summarized below.
Diagnostic kits typically utilize small volumes of reagents, which can evaporate very fast. Samples on plates or in microfluidic channels that are exposed throughout loading are more susceptible to evaporation, while a few microliters of these reagents in tubes may evaporate less.
High throughput is critical due to the urgency of testing for COVID-19 and so the majority of COVID-19 diagnostic kits are performed in 96-well plates, chips, or microfluidic channels. By loading into a cooled freeze dryer, this problem can be partly overcome.
Although most biomolecular components are not sensitive to conditions of freeze-drying (e.g., enzymes and antibodies), there are some that are and it is worth noting what effects these may cause.
Sensitivity to cold denaturation can lead to precipitation or aggregation of proteins; this can be solved by adding a surfactant or cooling slowly. Some components can also be damaged by freeze concentration effects, which can also be accompanied by a pH shift.
A change of buffer or cooling quickly can usually solve this problem as phosphate buffer can be the main culprit of this. Proteins can also be susceptible to interfacial influences, which may be proportional to the surface area of ice crystals. These effects can usually be combated by cooling slowly or using controlled nucleation to decrease the surface area by increasing the size of the crystals.
A protein may be destabilized because of dehydration stresses. In order for the structure to be maintained, structural water is needed in some instances, so adding cryo- or lyo- protectants can combat this by immobilizing proteins and/or mimicking the action of structural water to avoid dehydration stresses.
As seen in Fig 1, the presence of glycerol in PCR reagents can lead to serious problems as it does not freeze under usual freezing conditions. Diluting the glycerol can solve this problem if a high enough stock solution is available; adding an excipient can also help.
Figure 1. Presence of Glycerol in PCR reagents. Image Credit: SP Scientific Products
It is worth noting that the dried cake may still contain glycerol, which can affect the stability and structure. The shelf life may not be as essential as other diagnostic kits in the case of the COVID-19 diagnostic kits because they only need to be stored for a few months at a time. The presence of glycerol will have less effect on the stability of the active components because they can also be stored at low temperatures.
Containers and closures
The format of containers (e.g., wells, vials, tubes, paper, chips, etc.) and closures have a significant long- or short- term influence on stability and thermal transfer. It is worth noting some plates are more permeable than their seals to water, as seen in Fig 2, although there is a challenge to seal a container without letting any moisture in.
Figure 2. Sealing issues. Image Credit: SP Scientific Products
Dr. Ward discusses the utilization of a controlled environment with low humidity conditions, less oxygen, and utilizing a flexible isolator to decrease the moisture levels in the product. Fast throughput and initial performance may be more crucial than long-term stability for COVID-19 testing and so some of these measures may not be needed.
Smaller pilot dryers may be enough for freeze-drying the components of the diagnostic tests, as the majority of companies are maximizing throughput over long term stability for COVID-19 diagnostic kits.
A VirTis Ultra (SP Scientific), which is a typical pilot freeze dryer, is able to freeze dry up to 5,000 x 2 mL vials. That is a high enough output for most diagnostics. Larger freeze dryers that are designed for the pharma industry have additional functions that are not needed for diagnostics, steam sterilization, for instance.
It is crucial to use a freeze-drying strategy for diagnostic biomolecular components according to the particular sensitivity of the molecule in question whilst considering the issues outlined above. It is also worth considering that all the normal rules apply as for the freeze-drying of any other diagnostics.
Reagents will probably be processed in high batch numbers, but small fill volumes in the case of a COVID-19 diagnostic test, and the priority is mainly on throughput, not long-term stability. When designing a lyophilization protocol specifically for a COVID-19 diagnostic test, this must be remembered.
SP Scientific ultra pilot and small production freeze dryer
The low moisture load of diagnostic kits enables the utilization of efficient, compact ‘pilot’ freeze-drying equipment with a large product shelf capacity to floor ratio. The Ultra capacity can handle up to 5,000ea 2mL vials or 144ea 96-well plates with ease.
Image Credit: SP Scientific Products
SP Scientific benchmark production freeze dryer
VirTis Benchmark production freeze dryers are designed for larger batch sizes of diagnostic products. Internal ice condenser designs can help vapor flow for low-Tc diagnostic products being dried at very low shelf temperatures and round product chambers are typical. Shelf capacity ranges from 2 m² up to 20 m²
Image Credit: SP Scientific Products
Produced from materials originally authored by Dr. Kevin Ward, Director of R&D from Biopharma Group.
About SP Scientific Products
SP is a synergistic collection of well-known, well-established, and highly regarded scientific equipment brands — SP VirTis, SP FTS, SP Hotpack, SP Hull, SP Genevac, SP PennTech, and most recently SP i-Dositecno — joined to create one of the largest and most experienced companies in freeze-drying/lyophilization, complete aseptic fill-finish production lines, centrifugal evaporation and concentration, temperature control/thermal management, glassware washers and controlled environments.
SP is part of SP Industries, Inc., a leading designer, and manufacturer of state-of-the-art laboratory equipment, pharmaceutical manufacturing solutions, laboratory supplies and instruments, and specialty glassware. SP's products support research and production across diverse end-user markets including pharmaceutical, scientific research, industrial, aeronautic, semiconductor, and healthcare. In December 2015, SP Industries was acquired by Harbour Group, a private investment firm founded in 1976. Harbour Group is a privately owned, operations focused company based in St. Louis, Missouri. Headquartered in Warminster, Pennsylvania, SP has production facilities in the USA and in Spain and the UK in Europe and offers a world-wide sales and service network with full product support including training and technical assistance.
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