The advent of point-of-care (POC) and point-of-need (PON) testing allows users to rapidly receive in vitro diagnostic (IVD) test results without a centralized laboratory testing location. A challenge facing this industry is that the reagents necessary for diagnostic testing frequently have short shelf lives when stored at ambient, or even refrigerated, temperatures.
Additionally, these reagents would typically require cold-chain shipping and then storage at low, and, in some cases, ultra-low temperatures. Because POC and PON testing are intended to be used in non-lab environments, such as at home, in an emergency or operating room, and in the field, these cold storage conditions are likely unavailable. To solve this problem, lyophilization can be used to stabilize the reagents at room temperature. Lyophilization removes water from the reagent, decreasing the rate of physical and chemical degradation of key molecules.
Lyobeads and lyocakes simplify daily clinical tasks
Lyophilized singleplex and multiplex assay reagent beads are increasingly popular in IVD laboratory testing, particularly in the POC and PON markets. Lyophilized reagent beads, or lyobeads, are formed by using precision equipment to dispense a specified volume of reagent into liquid nitrogen. The frozen beads are then freeze-dried en masse to remove the frozen water.
Another option is lyocakes, which can be made in a few different ways, such as bulk lyophilization of reagents in vials. In this case, the dry lyophilized cake would then have to be measured into required individual doses. Lyocakes can also be made by directly dispensing the required volume of reagent into test or assay cartridges, microcentrifuge tubes, or PCR tubes, and then freeze-drying in place.
These shelf-stable lyobeads and lyocakes can then replace traditional reagents that would have required cold storage and precise dispensing for their designated experiments.
Strategizing bulk lyophilization can reduce QC-related losses
POC and PON testing often involve an assay reaction taking place in a microfluidic cartridge or lab-on-a-chip device. These devices are sophisticated and limited in space, with the lyophilized reagent required to fit into chambers that have millimeter-sized dimensions or smaller.
For reaction chambers that are particularly small or are difficult to access, lyocakes may be useful. Filling the cartridge or device chamber with diagnostic reagent liquid and lyophilizing by placing the entire cartridge into the freeze-dryer can be the best way to get lyophilized reagent into the reaction chamber. However, it is important to note that lyophilizing entire cartridges does not make efficient use of space inside a freeze-dryer, will reduce throughput, and increase costs. This can also lead to unforeseen expenses if a batch fails quality control (QC). If there is a problem with the reagent or cartridge, both are unusable, leading to a substantial loss of funds.
Lyobeads are lyophilized in bulk and placed into cartridges during assembly. Entire batches of cartridges are not lost in the case of reagent QC failure. Lyobeads are also a good choice for assays that require multiple components. Two or more distinct types of lyobeads can be placed together in the reaction chamber, if necessary.
Maximizing lyophilization scalability requires multiple considerations
Lyobeads are freeze-dried in monolayers or bulk layers making them highly space-efficient. A 12” x 24” tray can fit over 10,000 regular-sized lyobeads. Lyobeads make direct contact with freeze-drying trays, meaning heat is transferred efficiently and sublimation occurs quickly. Their small diameter means lyophilization cycles are also short.
Lyocakes can be less space-efficient. If lyophilized in a cartridge, PCR tube, or microcentrifuge tube, their packing density will be less efficient—meaning, fewer doses can be lyophilized per unit area of a freeze-dryer. In such cases, the cartridge or tube becomes a barrier between the reagent and the shelf of the freeze-dryer. This barrier can provide thermal resistance that could slow down the sublimation.
Space efficiency, good heat transfer, and small bead size make lyobeads a better choice for high throughput work compared to lyocakes. One exception to this trend is the preparation of lyocakes in glass vials. Glass vials may be filled with sufficient volume to offer good scalability. However, there is an additional requirement of achieving the desired dose of the dried reagent post-lyophilization, which is never required for lyobeads used in IVDs as each bead contains the exact amount of reagent required for one reaction. When dosing lyophilized reagents, reproducibility is generally diminished compared to that of precision microfluidic volumetric dispensers.
POC diagnostics demand rapid reagent reconstitution
Rapid reconstitution is a key requirement for lyophilized POC reagents, as POC diagnostics typically warrant fast-paced procedures and homogeneously mixed reagents. Moreover, in many cartridge or device designs, reconstitution must occur without aids like mixing, shaking, or heating. Ensuring consistent and efficient reconstitution is crucial to maintain the reliability and accuracy of POC testing.
Lyobeads maximize surface area-to-volume ratio, which can help speed up the rate of reconstitution as the entire surface of the bead is available to absorb a solution. For some lyocakes, like those lyophilized in tubes, only one face of the lyocake surface will contact the solution, which may decrease the rate of dissolution of the solid.
Choosing excipients that cut down reconstitution time is another factor to consider when designing POC tests involving lyobeads and lyocakes.
Optimally designing and implementing lyophilization
Stabilizing diagnostic reagents to extend shelf life and reduce cold chain requirements through lyophilization is crucial to the IVD and POC diagnostics market. Lyobeads and lyocakes each offer distinct advantages for particular use cases.
When designing a POC test, it is critical to consider the space available for lyophilized reagent, the reconstitution time, and the scalability of the lyophilization step(s).
Evik Diagnostics manufactures rapid diagnostic assays and immunological/molecular diagnostic assays with dry reagent components that are ambient temperature-stable through lyophilization. Being ISO 13485:2016 certified for this process, we can assist with lyophilizing proprietary reagents, customizing reagents, and determining the best lyophilization practice for a specific POC or PON testing assay.