Air-dryable master mixes replace lyophilization in the manufacturing of molecular assays

Quantitative PCR (qPCR) is the mainstream molecular technology for in vitro diagnostic assays. The introduction of freeze-drying to create ambient-temperature stable qPCR tests has improved assay stability and consistency and has enabled diagnostic companies to reduce supply chain and shipping costs. However, lyophilization is a complex and expensive process that requires expertise and sophisticated equipment and, consequently, has become a barrier to entry into the ambient-temperature stable assay market. Air-drying is an alternative technology that is less expensive, easier to perform and easily adopted in-house, enabling diagnostic manufacturers to create temperature stable molecular assays.

The first lyophilized panel of qPCR mixes was created in 2013 to provide rapid qPCR detection in a field setting [Sokhna, Cheikh, et al. “Point-of-care laboratory of pathogen diagnosis in rural Senegal.” PLoS Negl. Trop. Dis. 7.1 (2013): e1999]. Since then, freeze-drying molecular diagnostic assays has become increasingly popular because it enhances assay’s stability, enables ambient temperature shipping and storage, extends the product shelf-life and allows for a larger input of patient sample volume. However, there are significant challenges with lyophilization. It is relatively expensive, time-consuming and it generally requires outsourcing to a third-party given the expertise, specialized equipment and humidity-controlled environment required to perform the freeze-drying. These obstacles have limited its uptake by diagnostic manufacturers and have hampered the expansion of the molecular point-of-care market.

An alternative to lyophilization is to use a qPCR mix that could be dried-down using conventional equipment in a standard laboratory.

In the process of developing such a qPCR mix, Meridian’s R&D team explored the possibility of a glycerol-free polymerase that includes a buffer containing specialized excipients to preserve the enzyme as it is exposed to high temperatures and dehydration. After extensive testing, the team found the optimal formulation of excipients and enhancers that stabilized the polymerase (and reverse transcriptase for one-step) in a dried format and allowed for fast rehydration and reactivation of the enzyme preparations. Comparison testing between the wet and dried assay formats of the new air-dryable qPCR/RT-qPCR mixes demonstrated that the mix was able to maintain its high-performance post rehydration.

The technical requirements for developing an assay using air-dryable mixes are minimal and do not require chemistry or physics expertise. Using only a precision oven and precision scale, any laboratory can create an ambient-temperature stable assay. It is important to consider that the mix must be dried sufficiently to remove excess moisture but not overdried as the performance of the enzyme could be impacted. The air-dryable technology is relevant to many applications such as human diagnostics, veterinary testing, food testing, environmental testing and many others. This chemistry is perfectly suited for a wide range of instruments, from automated high-throughput platforms to point-of-care devices to detect DNA and RNA pathogens such as COVID-19.

Air-dryable qPCR mixes have several advantages for the molecular diagnostic industry. They enable manufacturers to save time, reduce costs and control their entire manufacturing workflow while delivering the same or better performance than wet or lyophilized assay-formats. In addition to human diagnostics, other industries such as agriculture, food, environmental, forensics and animal testing can use this mix, simplifying the creation of temperature-stable assays.

Meridian has also expanded its portfolio of enzyme master mixes for ambient temperature-stable assays by developing inhibitor-tolerant mixes suitable for specific specimen types. This enables crude samples to be added directly to the dried assays, eliminating the time-consuming DNA or RNA extraction step. This will reduce the time and cost of processing and analyzing a sample and still provide highly sensitive qPCR analysis.

Steve Hawkins, Ph.D., is the molecular product marketing manager for the life science division of Meridian Bioscience. Hawkins gained his Ph.D. in Biological Sciences from the University of London in 1993 and then focused on molecular biology and biochemistry via postdoctoral work at the University of Cambridge on receptor mediated endocytosis. He has since worked for several companies including Applied Imaging, PerkinElmer and Genetix, responsible for cytogenetics, microarray, qPCR and NGS products.

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Florent Chang-Pi-Hin, Ph.D. leads the global R&D activities for Meridian Life Science. Florent obtained a Ph.D. in 2003 in biochemistry and molecular biology while studying thermostable enzymes with industrial applications at the Institut National de la Recherche Agronomique (INRA) in Reims, France. Florent’s group is focusing on developing new critical reagents such as qPCR enzymes and mixes used by the global diagnostic industry for the development of diagnostic kits.