Debunking the myths: clearing up six common misconceptions about reagent lyophilization
Posted on Friday, March 3, 2023
Topic: What is Trending in Science
Lyophilization is a powerful technique for preserving and storing sensitive reagents at ambient temperatures. It offers several advantages in a range of scientific fields, from biotechnology to pharmaceuticals. Diagnostic testing in point-of-care and low-resource settings is made possible with lyophilized reagents because their stability at room temperature eliminates the need for refrigeration, reducing costs and logistical challenges associated with cold-chain shipping and storage. This enables diagnostic testing to be conducted in remote areas where it might otherwise be unavailable.
Lyophilization involves removing the water from a reagent mixture by freezing it and subjecting it to low pressure, which causes the ice to sublimate into vapor. This leaves a dry, stable product. It requires a dedicated freeze dryer that automates the drying process and optimization of step changes in temperature and pressure to ensure a thoroughly dried product while maintaining reagent activity. Using the right cryo-protectants (i.e., excipients) can protect active materials (i.e., enzymes), through the drying process and when these materials are stored at ambient temperatures.
Our technical teams at NEB Lyophilization Sciences™ Ltd., find that some questions are commonly raised regarding the process, robustness, shelf-life and customization of reagent lyophilization. Let's address some of the most common misconceptions.
Myth #1: Lyophilizing the enzyme is the most challenging part of the process to perfect
Typically, it is not the enzyme that requires the most optimization; it is the buffer composition. High concentrations of salts in the buffer can be challenging – as can some specific salts, for example, ammonium sulfate. Organic solvents like Dimethyl Sulfoxide (DMSO) can also be problematic to the instrumentation used in the process. There are often simple workflow solutions to overcome these obstacles, such as substitution for lyo-friendly alternatives, and using a rehydration solution to add back any incompatible components after the lyophilization process.
Myth #2: The enzyme will lose activity when lyophilized
Some researchers have observed the need to supplement their lyophilized reactions with additional enzyme to achieve the same activity level as the liquid form. This leads to the misconception that the drying process will require more enzyme. However, a lyophilized assay with optimized composition and drying parameters should consume the same amount of enzyme as the liquid form and perform nearly identically. There should be no need to compensate for activity loss in a freeze-drying process since the process is equivalent to a single reagent freezing step. If lower activity is observed, it is likely due to issues in formulation or process stabilizing the enzyme during lyophilization that may affect the stability at room temperature, suggesting further optimization is required.
Myth #3: Once glycerol is removed, the process becomes easy and straightforward
One way to make a product lyo-compatible is to remove glycerol from the reagent, as glycerol concentrations above 0.2% are generally incompatible with lyophilization. But other components pose challenges for lyophilization besides glycerol. As mentioned above, the overall composition needs to be considered because other components, such as salts or solvents, might be incompatible with the process. In some cases, changes to the formulation may be necessary. Finally, primary packaging and product format decisions play a significant role in the lyophilization process and are often not trivial as specific tools may be required for dispensing, drying and packing.
Left: A typical freeze-dryer; Right: Glass vials are ideal for reagent manufacture: glass is impervious to oxygen and moisture
Myth #4: Lyophilized reagents have a shorter shelf-life
Sometimes there is an assumption that the shelf-life of a lyophilized product at ambient temperatures will be shorter than its liquid counterpart stored at -20°C. If a lyophilized product has a shorter-than-expected shelf-life, this may suggest suboptimal fine-tuning of the composition or process. But, if the reagent and the process are optimized, robust stability and long-term storage at room temperature can be achieved. Products may attain stability for many years with only marginal reductions in performance. The composition of the formulation should be designed to maintain the stability of the product during lyophilization and storage.
Myth # 5: Lyophilized products are fragile and not stable
Some lyophilized products appear powdery or crystalline-like, leading to the perception that they are delicate. However, a well-made lyophilized "cake" remains intact even when agitated. A high-quality lyophilized product will have a glassy-poriferous structure that maintains its shape and form. The main challenge with all lyophilized reagents is susceptibility to moisture upon storage, even from atmospheric humidity. Lyophilized reagents readily want to take on water as they are hygroscopic, which is desired during rehydration because intended water addition generally results in complete, quick dissolution. On the other hand, prolonged and unwanted exposure to moisture will cause it to collapse, often impacting the ease of rehydration and overall reagent performance. That said, lyophilized products have a robust, stable structure if shielded from moisture and atmospheric exposure, and this is accomplished through product packaging.
Myth #6: Customizing with primers, probes and templates complicates the lyophilization process
Researchers interested in customizing target-specific assays may wish to include primers, probes, or control templates to enable whole workflows, though there is often concern that this may affect the lyophilization process. On the contrary, adding primers, probes and templates into an optimized reagent composition is typically low-risk and straightforward. Such incorporation rarely jeopardizes product robustness or stability. Typically, during product development, buffer optimization is the first step, then the enzyme is added, and finally, primers, probes, and templates. Synthetic sequences are required for templates if they are to be assembled into reagents in a clean room environment.
Learn more about Key Considerations for Optimal Lyophilized Reagent Development
If you have more questions about lyophilization, don't hesitate to call our customized solutions team.
NEB Lyo Sciences offers custom lyophilization of several NEB reagents, including but not limited to Luna qPCR and LAMP products. Reagents from other NEB portfolios or researchers' home-brewed mixes and reagents from other vendors can also be lyophilized if the opportunity is right.
NEB Lyo Sciences can incorporate custom primers, and provide dried reagents in custom formats, including custom or standard vials, tubes and plates. We also provide contract R&D to help guide you in developing specific manufacturing processes and offer recommendations for optimal drying processes and packaging for your product.
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