Democratizing Molecular Diagnostics with Custom Lyophilized Reagents

Lyophilization as an elegant solution to challenges in molecular diagnostics.

Q: WHAT IS LYOPHILIZATION AND HOW IS IT DIFFERENT FROM THE CURRENT APPROACH FOR MOLECULAR DIAGNOSTIC ASSAYS SUCH AS QUANTITATIVE PCR (RT-QPCR)?

A: Lyophilization is a process that immobilizes enzymes and reagents in a solid matrix. Virtually all reagents in molecular biology—irrespective of their use—are readily lyophilizable, if you know how.

Today, most molecular biology laboratories mix reagents at the time of use. This can lead to inefficiencies in the workflow, and contamination or errors in mixing reagents. Many reagents cannot be premixed because they are unstable at room temperatures or could interact with each other, reducing their efficacy. As a result, these reagents need to be manufactured and handled in a “wet” format. Wet reagents are dispensed as liquids in separate tubes and frozen. Thus, they require a cold chain supply system, with expedited shipping on dry ice or frozen gel packs in styrofoam containers and storage in freezers at the customer site.

Compared to wet reagents, lyophilized products are solid and stable until they are resuspended and used. Lyophilization offers an elegant solution for many different challenges in the molecular diagnostics applications, including cold chain logistics, product storage and shelf life, laboratory workflow efficiencies, economics, and environmental impact.

Des O’Farrell is the president of the genomics division at Fortis Life Sciences. He has more than 30 years of industrial experience and owns more than 50 patents. Des became CEO of Empirical Biosciences (now a Fortis Life Sciences company) in 2014, and led all aspects of the business thereafter. Before Empirical, Des has been an investor, founder, and executive in various medical device and life science companies, including DCN Dx and a co-founder of Shoulder Innovations LLC & Transcorp Spine Inc.

Q: WHAT ARE THE ADVANTAGES OF LYOPHILIZED OR LYOPHILIZATION-READY POLYMERASES, ENZYMES, AND REAGENTS COMPARED TO THE WET CHEMISTRY APPROACH?

A: Lyophilization allows us to create a complete, ready-to-go assay—whether it is in a single reaction or a bulk reagent format—enabling easy use, reducing errors, sample loss from workflow inefficiencies, and decreased turnaround time to obtain test results.

Lyophilization eliminates the need for a cold chain from the point of manufacturing to the point of use, which offers dramatic economic and environmental advantages. The stability of lyophilized products translates to increased shelf life and greater temperature tolerance (from frozen up to 45–50°C), compared to wet reagents that require a cold chain during transportation and freezer storage. You save significant amounts of money by no longer paying for cold chain handling, shipping, and delivery, nor for freezer space. Environmentally, you reduce contamination from toxic refrigerants and coolants, eliminate dry ice use, minimize packaging waste, and decrease energy consumption.

Lastly, lyophilization enables molecular diagnostics to be used in settings where you do not have the capital equipment, logistics infrastructure, or facilities available to support cold chain supply or to perform complex laboratory procedures. This includes settings such as low-income countries, in the field, or in remote locations. Lyophilized products enable molecular testing to be performed at the point of use instead of in a laboratory and democratize molecular diagnostics and testing.

Q: WHAT STEPS NEED TO BE TAKEN TO PREVENT THE POSSIBLE LOSS OF ACTIVITY DURING LYOPHILIZATION AND ENSURE A ROBUST AND RELIABLE ASSAY?

A: During lyophilization, phase transitions from frozen liquids to gases occur, leaving behind solidified reagents, so the physics and the biochemistry associated with that reaction are pretty involved. A fundamental understanding of the biology and chemistry of the reagents, of the physics underlying the lyophilization process, and how these can impact reagent performance, is crucial to creating lyophilized products without loss of performance.

We work with our customers to gain a thorough understanding of the customer’s end performance requirements or of their platform, because every assay and platform behaves differently. The success of our applications comes from using our expertise to provide an integrated solution for our customers.

It is much more efficient to make lyo-compatible products from the start, rather than trying to make wet reagents lyophilizable. For example, many wet reagents are mixed with glycerol, salts, and detergents that impede lyophilization. That is why we developed our processes to eliminate lyo-incompatibilities at every step, from the raw materials used to manufacture enzymes, to the secondary components in buffers, and all the way to the end product.

Q: WHAT ARE THE TECHNICAL REQUIREMENTS FOR DEVELOPING LYOPHILIZATION-READY ASSAYS AND REAGENTS, AND WHY SHOULD LABORATORIES SEEK EXTERNAL EXPERTISE?

A: Most developers of molecular diagnostics products are experts at identifying the functional requirements of their assay or the performance of their platform. While lyophilized assays are incredibly easy-to-use for the end-user, developing those assays from scratch can be a complex process with many scientific and engineering considerations. Our experience shows that there is no magical “one size fits all” approach for creating a reliable and robust lyophilized assay for a particular application. You need the knowledge and expertise at every step.

We have a thorough understanding of lyophilization mechanisms and of virtually all the processes used by various designated contract lyophilizers. Our technical expertise goes beyond just providing a reagent. We have been developing lyophilization-ready reagents for almost ten years. The extensive firsthand knowledge and experience we have gained along the way enables us to support our customers in creating the optimized solution for their project—whether it is working with them directly during development and scale up or working with their preferred lyophilization partners to ensure that the customer gets a finished product that works.

Q: HOW DO YOU THINK LYOPHILIZED ASSAYS AND REAGENTS WILL IMPACT MOLECULAR DIAGNOSTICS IN THE FUTURE?

A: Lyophilization allows most molecular diagnostic tests that previously required a high-complexity laboratory to be efficiently run on a single sample device, without the need for highly skilled staff or complex equipment, and to have the results be quickly reported to patients or provider at the point of care. Lyophilization—and in general, solid-phase chemistries—will democratize molecular biology. An analogy would be how immunodiagnostics was democratized by lateral flow, because lateral flow can deliver immunodiagnostic products in an ambient-stable format, is easily transported, easily used, reliable, and repeatable.

As a result of the COVID-19 pandemic, there is an emerging trend toward having molecular tests in prefabricated, preassembled reaction cassettes. People have seen the importance and the value of molecular diagnostics, particularly when used at the point of care. The applications of lyophilization are endless, from human clinical diagnostics to water quality testing to species-of-origin testing in food markets. Lyophilization of molecular biology tests will have important implications for future health care and life sciences overall.