Lyophilization Can Increase Accessibility to Point-of-Care Testing
Lyophilized product advantages come from a specialized upfront development process
Historically, reagents used in molecular testing have required cold-chain shipping and storage. The logistical complexity and cost of maintaining molecular reagents at refrigerated temperatures has contributed to limiting molecular tests to specialized laboratories.
However, lyophilization, or freeze drying, of these sensitive reagents now enables molecular assays to be shipped and stored at room temperature, making point-of-care (POC) testing more accessible.
What is lyophilization?
Lyophilization removes water from liquid reagents by first freezing the mixture and then subsequently reducing pressure such that ice is sublimated into vapor. The resulting lyophilized product is dry, temperature-stable, and compact, making it easier, cheaper, and more sustainable to ship and store.
As a result, lyophilization has proved essential to the development of POC diagnostics. In addition, lyophilization of many reagents together can help to simplify and automate workflows, making them higher-throughput and more accessible to nonspecialists.
However, while lyophilization of molecular assays offers several advantages, transitioning from liquid to lyophilized reagents requires upfront development time and investment. Vendors specializing in lyophilization can help ensure that reformulation, product design, and packaging are optimized for assay performance, compatible with the workflow, and intuitive for the end user.
Improving assay performance and workflow
Developers reluctant to transition to lyophilized products often express concern that a lyophilized version of a formulation may have suboptimal assay performance. This concern is logical, because commonly used liquid reagent formulations contain components—such as glycerol, co-solvents, or other key additives—which help with enzyme performance and assay sensitivity but are not compatible with lyophilization.
Additionally, excipients and cryoprotectants are required to enable successful lyophilization and may impact the assay’s performance. Substitution testing and excipient screening can identify alternative components that can restore assay performance, while protecting the macromolecules during freezing and preserving the dried material.
Further, the use of lyophilized products can improve assay performance by streamlining workflows, improving automation compatibility and reducing opportunities for contamination. A lyophilized POC diagnostic may considerably reduce pipetting steps by combining the reagents used in a workflow into a single lyophilized product.
For example, a lyophilized target-specific assay can include primers, probes, active enzymes, and assay buffer requiring only addition of sample. Such incorporation rarely jeopardizes product robustness or stability.
However, as additional components will need to be assessed and optimized for lyophilization, incorporation of all components in the formulation can extend the development timeline. Typically, during lyophilized product development, buffer optimization is the first step, then the enzyme is added, and finally, primers and probes go in.
A POC diagnostic workflow can be further streamlined through the strategic prepackaging of lyophilized products into instrument-compatible containers (such as 96 well plates), which reduce hands-on time during the workflow. For example, at its most condensed, a workflow might only require rehydrating lyophilized beads, preplaced in a device or cartridge with the sample of interest.
Maintaining long-term shelf life and stability
Lyophilized products have a robust and stable structure, even when agitated and stored at ambient temperatures. As long as the reagent formulation and the lyophilization process are optimized and proper storage conditions are maintained, products may display stability for many years. This makes lyophilized assays ideal for POC tests.
Storage conditions are indeed critical, as lyophilized products are sensitive to moisture and atmospheric exposure because they are extremely hygroscopic and will readily absorb water. When purposefully rehydrated, the addition of water generally results in complete, quick dissolution. However, prolonged and unwanted exposure to moisture, such as exposure to the atmosphere, will impact the ease of rehydration and overall reagent performance.
As a result, the strategic design of primary and secondary packaging for lyophilized materials is as critical to stability and assay performance as the development of the reagent formula and the lyophilization process conditions.
The optimal format and composition of the packaging is dependent on the workflow compatibility with the required integrated instruments. Most workflows and instruments will have an assay housed in a molded plastic vessel designed to work with the associated instrumentation.
However, many plastics designed to work with laboratory instruments are not resistant to moisture and oxygen ingress. When sealed in this type of container, a lyophilized assay will start to absorb moisture and be exposed to oxygen.
Therefore, secondary packaging, which is resistant to moisture and oxygen, is often required. This packaging should also be designed to minimize exposure to light if fluorescence components for applications such as quantitative PCR are included.
Typical secondary packaging includes metalized polymer bags, which are sealed under an inert, dry atmosphere—typically nitrogen gas—and include a desiccant material to adsorb residual moisture.
Beyond the clinic, the future is freeze-dried
Assay developers, aware of lyophilization’s advantages, often approach the process with questions about the performance, robustness, shelf life, and customization of reagent lyophilization. The process can be involved, and is best guided by vendors specializing in lyophilization. This is particularly true for developers who must obtain regulatory approval for human in vitro diagnostic tests.
However, once a lyophilization process is developed, it comes with extensive benefits: improving the cost, sustainability, accessibility, and even the performance of POC testing. In fact, we expect to see the impact of lyophilized molecular reagents extend far beyond POC diagnostic testing, improving the accessibility of complex molecular assays across a wide range of scientific disciplines and applications.