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PCR is a common and often indispensable technique used in medical and biological research labs for applications including functional analysis of genes, the diagnosis of hereditary diseases and the detection and diagnosis of infectious diseases.
Storage of PCR Materials by Freeze Drying
PCR based diagnostic kits will generally include mastermix, primers, probes, as well as other components such as enzyme and buffer solutions.
Freeze drying PCR components can increase product lifetime from just a few hours/days in liquid form to several months/years in the dried format. Achieving a successful freeze dried product reduces cold chain storage requirements and product wastage (due to product expiry), therefore improving the environmental sustainability of the product. This has clear advantages: reducing the importance and criticality of forecasting product demand, storage facility requirements, transport logistics and associated costs of these.
A successful freeze drying process produces products with a high surface area, which enables them to be reconstituted quickly and easily with the re-introduction of the solvent (usually water). This aspect is valuable for freeze dried components which need to be solubilised quickly whether in vial or as part of an enclosed sample processing unit. PCR testing kits can involve a number of steps in order to prepare and analyse the sample. Ideally units need to be able to be stored on-site ready for use, therefore they need to have sufficient shelf life to ensure that a reasonable number of tests can be kept in stock and that manufacturing campaigns are economic.
In addition to the advantages outlined above, having pre-prepared testing kits with reagents already installed can reduce the chance of contamination and operator error. In short, freeze drying offers a range of advantages to using liquid only components.
Reformulating for Freeze Drying
Many PCR materials are stored frozen or chilled as standard and and therefore in many cases the formulations have been designed with cold storage in mind. This can cause problems as many materials used to ensure stability in chilled or frozen state storage are not required for freeze drying and indeed can be a hindrance to the freeze drying process. Often re-formulation is required where certain components need to be reduced or replaced; for example glycerol is a good cryo-protectant for frozen state storage, but is not suitable for freeze drying and long term stability due to its glass transition in both the frozen and dried state. Other components can also cause problems and it is important to balance and establish what is required for a successful PCR based diagnostic versus what can be removed, replaced or reduced to enable successful freeze drying and long term storage.
Challenges of Small Volumes
Freeze drying of PCR components can require processing of much lower volumes per individual aliquot, for example 20µl compared to 2ml in glass vials. An example of a commonly used tube for small volumes in PCR is given in the picture above.
One of the risks of processing such small volumes is that solvents may start to evaporate off during loading, before the product has been frozen. Uncontrolled drying can lead to post-process issues for example with reconstitution. Care should therefore be taken with loading procedures, and pre-freezing may be required in some cases.
Containers are available in glass and plastic. but glass is not always considered suitable. These small tubes are often not flat-bottomed and will therefore need to be supported within a rack to hold them upright. This introduces different challenges, for example the differences in thermal heat transfer for plastic materials with minimal shelf contact and container closure options.
BTL provides a complete freeze drying consultancy service from characterisation, formulation and cycle development through to, scale-up, optimisation and small-scale processing. BTL is experienced in freeze drying of Polymerase Chain Reaction (PCR) components, having been involved in a number of commercial and research projects. Currently BTL is a member of an EU FP7 project called AngeLab, http://cordis.europa.eu/projects/rcn/105552_en.html and prior to this was involved in freeze drying of PCR components for the development of a fully-automated, real-time PCR system for rapid diagnosis of influenza in a point-of-care environment. BTL has also assisted many other companies to develop their freeze dried PCR products; these often need to be designed for a lab-on-chip format.collaborative research, diagnostics, PCR