How to Evaluate Lyophilization for Molecular Reagents and Assay Products?

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Lyophilization, also known as freeze-drying, is a process commonly used in the biotech and food industry to minimize the moisture content and water activity of products.

Our teams here at DKSH have found that our biotech customers in the Asia Pacific region are becoming increasingly aware of the choice to lyophilize their products as a value-added process. Aside from the obvious reason to reduce transport costs, it is also popular for applied biotech applications such as food or veterinary where access to ultralow refrigeration can be limited and certain point-of-care IVD applications where simple preparation is important.

An investment into lyophilization for pharmaceutical or food manufacturers is a relatively straightforward consideration as the freeze dryer is very similar to other manufacturing process equipment. But for those working with polymerase chain reaction (PCR) or other molecular reagents, it can be quite challenging to evaluate this type of product.

In this short article, I will explain the typical considerations for molecular reagents and assay manufacturers evaluating lyophilization.

Lyophilization is a physical process that removes water from a sample or product by freezing it in a vacuum and sublimating the ice.

A product or sample is first brought to a low temperature inside a dedicated freeze dryer equipment. For molecular reagents, this is typically done at -85 degrees Celsius or lower to accommodate for enzymes. Gradual freezing is controlled by the freeze dryer in a pre-programmed protocol of freezing and drying stages. Drying is conducted in two phases: a primary drying phase and a secondary drying phase.

The primary drying phase involves reducing the pressure of the surrounding environment below that of the triple point of water. The application of a small amount of heat then energizes the free ice crystals to undergo sublimation, transitioning directly from a solid to a gaseous phase. This step removes approximately 95 percent of the water and results in the product or sample transforming from a glassy frozen state to a largely dry powder.

The secondary drying phase removes residual water molecules adsorbed to the product or sample, leaving the product or sample at one to two percent water content. The result is a lyophilized powder that is much lighter in terms of mass and smaller in volume than the original product or sample. Readying this lyophilized powder for use then simply involves adding sterile, distilled water or an appropriate buffer to form a solution in a process called reconstitution.

1. Increase shelf-life

By removing the need to dehydrate by heating, lyophilization provides a convenient and safe method for the long-term storage of molecular reagents and assay products while preserving their activity. The first to degrade in your assays are usually enzymes and RNA, which will degrade even in low-temperature storage when in solution.

After reconstitution, all reagents will become saturated with water again in solution. Some users find that reconstituted probes can have slightly less fluorescence activity, but not to a degree that will affect the detection. It is important to test your freeze-dried formulations and lyophilization protocol to obtain the best results.

2. Lower storage and shipping costs

Lyophilized molecular reagents can be stored at ambient temperature for up to two years, minimizing cost, packaging and environmental impact. Lyophilization will also reduce the weight and volume of your reagents or samples.

From a logistical point of view, eliminating dry ice from the equation can be the factor that pushes those on the fence over the edge. Not having to deal with IATA and other regional or local regulations can help speed up access to new markets.

3. Added value to laboratory customers

Freeze-thawing and assembly of multiple PCR reagents create the potential for pipetting errors, batch-to-batch inconsistency and poor result reproducibility due to the labor-intensive nature. Using pre-formulated lyophilized reagents that contain all the components needed for PCR offers simplicity and minimizes hands-on time. This is essential for improved laboratory throughput and time-sensitive results.

For food, veterinary and remote COVID-19 screening applications where the primary barrier to low-temperature shipping are infrastructure restrictions and the point-of-use, it is very important to have room temperature stable reagents.

The first step into evaluating lyophilization is considering whether it is the right solution for you. You should consider the typical storage and shipping conditions of your top customers. If your customers are based internationally or test in remote and outdoor locations, freeze-dried kits can be a big cost-saving option.

The second step is understanding the investment in time and equipment. Depending on your scale, you will want to start with a laboratory or pilot scale freeze dryer. The purpose is to work on your formulation, which typically takes six to twelve months.

Each kit is different, and you will have to optimize your kits with lyophilization-compatible reagents and freeze-drying excipients. If you use glycerol, you will have to find a substitute as glycerol does not pair well with lyophilization. There are commercial products available so you will not have to reinvent the wheel.

Due to the nature of freeze-drying nucleic acids and proteins, the freeze dryer will need to have an operating temperature of at least -80 degrees Celsius with fully programmable drying steps. This will require you to invest in a pharmaceutical-grade freeze dryer.

We find that most small and medium-sized enterprises can satisfy their manufacturing output with a pilot-scale freeze dryer. If you require a scale beyond that, production freeze dryers will involve HVAC engineering and manufacturer installation.

In our experience, we have found that lyophilized reagents or assays are already a standard offering for larger multinational companies/manufacturers. These companies will typically offer both standard solutions and lyophilized products as separate offerings. For the manufacturer, lyophilized products are not meant to be a replacement to their existing product line. Lyophilization is also compatible with reagents for loop-mediated isothermal amplification, next-generation sequencing and other molecular techniques.

Lyophilization has a steep initial investment and it will require your time and effort to optimize for your assays, but the costs are typically paid back through added-value products and reduced costs in storage and transportation within two years.

DKSH can provide you with the right freeze-drying equipment and expertise. We also offer related testing instruments such as climate chambers and moisture testing equipment.

About the Author

James Hsu joined DKSH in 2019 as Business Development, DKSH Technology. In this role, he is responsible for growing the life sciences and scientific solutions business. His previous experience was accumulated in the bustling Asian genomics and proteomics sector, where he worked on bringing a digital PCR startup to market. James graduated from the University of California, San Diego.

James Hsu

Life Science