How to freeze dry PCR diagnostic kits for COVID-19

PCR diagnostic kits and vaccines for COVID-19 are essential for battling the pandemic. One of the biggest challenges associated with their storage, transport and use is the need for refrigeration or freezing of the material. This cold chain makes diagnostics and vaccination particularly difficult in locations without suitable infrastructure and storage possibilities. In this post, I examine using the freeze-drying technique as a possible solution to the cold chain challenge.

A good friend of mine lives in Vancouver, Canada and they have had a scorching heat wave this past week. She told me their schools are closed, cooling centers are full, and cables are melting from the high temperatures. It is strange for them to experience such extraordinary weather, but there are places on Earth that are regularly exposed to extreme heat. And they must deal with a lot of consequences, much worse than ice cream that comes out already melted from its packaging.

Take the current Covid-19 pandemic. PCR diagnostic kits and vaccines must often be refrigerated or even frozen during transportation and storage. In hot climates and locations that do not have the necessary infrastructure in place to maintain cool temperatures of these medical stocks, it can be very challenging to vaccinate or test the population.

One way to approach this problem is to avoid refrigerating or freezing the diagnostic kits and vaccines. Overcoming the cold chain requirement and improving stability of vaccines and PCR diagnostic kits can be achieved by using freeze drying or lyophilization . If you’ve been following the blog, you know freeze drying is one of my favourite processes , so I am grabbing the opportunity to discuss with you how we can use freeze drying for PCR diagnostic kits for COVID-19 and other diseases.

But before I get into the freeze-drying part, let me just quickly give you a rundown of how PCR diagnostic kits work. Polymerase chain reaction (PCR) is an in-vitro technique for rapidly synthesizing large quantities of a given DNA segment. During the process, DNA is copied and becomes a substrate for further replication. By identifying the replicated material, the target DNA can be ascribed. In the case of PCR diagnostic kits, if the sample contains the DNA of interest, say from virus or bacteria, then replication occurs, and the PCR result is positive.

Now let me point out that lyophilization brings more benefits than avoiding the cold chain requirement for PCR diagnostic kits and vaccines. Because the process removes water as a reaction partner, freeze drying can increase the stability and shelf life of products. Lyophilization is suitable for processing heat-sensitive products, as well as products sensitive to oxygen, as these can be sealed in an inert atmosphere. Since freeze-drying creates a highly porous dry product, you can count on fast reconstitution times as an additional benefit.

Regarding PCR diagnostic kits, freeze-drying can help you improve the following:

  • Whereas liquid PCR diagnostic kits expire within 6 months in the fridge or freezer, freeze-dried kits can remain stable at 30°C for more than a year
  • Since no freeze-thaw cycles are required, there is no activity loss of components
  • The technique reduces hassles related to cold chain management, as well as packaging challenges
  • Lyophilized PCR kit master mix is preformulated – Not only does the kit contain all needed components, you improve your efficiency and consistency, avoid contamination and eliminate pipetting errors

Freeze drying of pharmaceutical products, including PCR diagnostic kits, is a multi-step process:

PCR diagnostic kits, lyophilization, freeze-drying

Let me walk through these five steps and give you a few tips on how to optimize them for pharmaceutical applications.

1. Sample preparation – Choose the right container for your sample size. Consider the thermal and chemical properties of the active component in your sample. Pay attention to what freezing temperature is needed and if your API is sensitive to pH changes. Be certain that any solvent present in your sample can experience proper freezing at that particular concentration. Potentially add excipients such as a bulking agent to stabilize the cake structure in cases of solutions with low solid contents or collapse temperature modifiers to increase the shelf temperature for safer and faster sublimation of your PCR diagnostic kits.

2. Freezing – Here, sample solidification is a must. Ice crystal formation during this step depends on speed and freezing temperature. The size of the forming crystals impacts freeze-drying speed and final product porosity.

As a rule of thumb, the smaller the ice crystal, the longer the process time.

Importantly, freeze concentration due to ice formation can lead to changes in pH and subsequent conformation and structural changes. Ice crystals can exert mechanical stress on the active components, including damaging cell or protein membranes. To alleviate this issue, you could try using cryoprotectant.

3. Primary drying – Once you have your frozen samples, you can go on to primary drying. Here, you need to maintain low sample temperature even during batch loading into the freeze dryer . This is to avoid collapse due to high water content in frozen samples. To optimize the process time, you should also track your sublimation processing using endpoint determination techniques . You could either check the change of sample temperature or monitor the change in measured pressure, which is dependent on the amount of sublimated vapor in the drying chamber.

At the completion of the primary drying step, you can achieve a residual moisture content of about 5-10% in your PCR diagnostic kits.

I just wand to add that you can achieve precise and effective energy input for a controlled sublimation via:

  • Heating the freeze-dryer shelf (conductive heat transfer)
  • Convective heat transfer
  • Higher vacuum setting, depending on melting/collapse temperatures
  • Minimizing inactive energy input via radiation, a random energy, to reduce effects on process reproducibility
freeze-drying, sublimation, lyophilization, energy input, convection, conduction, radiation

During primary drying, it is important for you to find a good balance between efficient sublimation and stable cake structure by applying necessary energy for the sublimation front.

For PCR diagnostic kits in 96-well formats, there is only a small contact surface between the sample and freeze dryer shelf. To try to solve this problem, you could try using aluminum blocks to maximize the heat transfer from the shelf.

4. Secondary drying – You can start with secondary drying once all the ice has sublimated in primary drying. This phase relies on desorption of residual ice, such as crystal water in the sample. You are free to set up the temperature of the shelf to as much as 50°C. Consider thermal characteristics, such as degradation temperature of the “dry” cake when setting up your shelf temperature.

At the end of secondary drying, the final moisture content in your sample should be in the range of 1-3% in your PCR diagnostic kits.

5) Further processing – After you go through the drying steps, you need to seal your product in a moisture-proof container for product storage and shipment. If you want to quality control your product first, you could measure final moisture via Karl-Fisher titration.

I hope you have not overheated from all this information and have managed to keep a cool head. It is an interesting topic and I could write for ages on it, but I will stop here and invite you to watch a webinar with a panelist from a company who is manufacturing freeze-dried PCR kits for more information.

I really enjoyed re-visiting lyophilization with you today. I might try to do this more often. Stick around and see if I do.

Till next time,

The Signature of Bart Denoulet at Bart's Blog