Still frozen in fear of freeze-drying of PCR diagnostic kits?

My last blog post touched on an important topic, mainly how to improve the stability of PCR diagnostic kits for COVID-19 using the freeze-drying method. I think the subject is of such interest that it never really dried out of my thoughts. Instead, I’d like to invest some more time into the matter but dig even deeper and get you even more comfortable with this approach. Read this post to gain valuable insights, and tips and tricks into how to best perform freeze-drying of PCR diagnostic kits for various diseases.

I have a small group of friends who I meet with a few times a year and we visit a local bar to do some catching up. This time around, there was a new bartender that was doing some very fancy drink pouring techniques. We chatted with him briefly and he explained how he went from trying to simply pour without spilling to throwing bottles up in the air and juggling them around without a wasted drop. Sure, practice makes perfect, but he admitted that he also had a great mentor who taught him a few tricks of the trade.

I thought to myself, that we also have these lab masters, who have performed certain methods for so long that their knowledge is an absolute treasure chest for those starting out with the same technique. In my previous post for example, I discussed how we can perform freeze-drying of PCR diagnostic kits . I gave you the bare basics, so you could pour drinks without spilling. But in this post, I’d like to show you how to throw a bottle 5 m in the air, spin around and catch it with 1 finger. Or in other words, I’d like to give you a few tips and tricks on how to optimize your freeze-drying of PCR diagnostic kits.

Considerations for your equipment for freeze-drying of PCR diagnostic kits

It is rather obvious that nearly all you need to consider regarding your freeze-drying equipment and process for PCR diagnostic kits is governed by the small size of the reagent batches. These tend to be in microliters, so you can dispose of the typical 1:1 shelf surface to ice condenser surface area ratio. I can also recommend the following to you:

  • Use heatable shelves – This is because the surface of the tubes is not ideal for uniform heat supply. When freeze drying a 96-well plate, you run into a problem of working with plastic tubes with minimal surface area that is in contact with the freeze dryer shelf. To try to reduce the negative impact of the reduced surface area, you could try using aluminum cooling-blocks to increase conductive heat transfer. I would recommend using 4-6 shelves for this application. For more information, check out my previous post on the topic of shelf freeze drying .
  • Avoid large condensers – Since not much water needs to be sublimed, consider using a smaller condenser, such as one with 6 L capacity. Avoiding oversized units reduces the energy and environmental cost of the lyophilization process, and it eliminates the need for cooling water utilities.
  • Stopper your freeze-dried PCR reagents and diagnostic kits – Since these are highly hygroscopic samples, you must seal them at the end to avoid exposure to ambient moisture. Try to work in a humidity-controlled room to slow moisture re-absorption. For storage and transport, seal off your products in moisture-proof containers, such as an aluminum bag.
  • Use pressure for endpoint determination – Since the product sample size is in the microliter range, it is very challenging to use product probes for endpoint determination. Instead, consider using pressure difference test as an indicator. To find out more on using pressure as an endpoint determination criterion , check out my previous blog post on the subject.

Ok, if I have to be honest, I might have mixed up a bit of methodology with the freeze-drying equipment part but let me try to give you a few focused pointers only on the technique of freeze-drying of PCR diagnostic kits.

Considerations for your method of freeze-drying of PCR diagnostic kits

  • Do not skip the secondary drying step – During this part of the process, you remove as much water as possible, which is the goal of you performing this technique in the first place.
  • Optimize stability of formulation with additional reagents — Consider using excipients, such as bulking agents, to stabilize solutions with low solid contents. To increase the shelf temperature for safer and faster freeze-drying of PCR diagnostic kits, try adding collapse temperature modifiers to your sample.
  • Make higher vacuum settings work for you – Convective heat transfer is important in lyophilization, particularly for achieving uniform heat distribution across the shelf and for mitigating the edge effect caused by differences in radiant heat transfer. Normally, I would recommend using slightly higher vacuum settings, such as 100 mTorrs instead of 60 mTorr, to achieve a more homogeneous batch. However, many PCR reagents have very low melting/collapse temperatures, so a higher vacuum setting during freeze-drying of PCR diagnostic kits may not always be feasible. But if you are committed to the idea, you could add excipients to the PCR mix, which increase the collapse temperature and allow the use of higher vacuum settings. I’d like to point out one additional benefit, mainly that higher vacuum settings also reduce the chance of choked vapor flow to the condenser.

We are getting closer to last call, but I do want to mention one last thing.

If you want to scale up your process, you could think about using lyophilised bead-format assays, which bring several advantages to the table. These formulations offer homogeneous granules with controlled density and small particle size distribution. In fact, each bead contains a specific volume of reagent. With the freeze-dried mixtures, you eliminate aliquoting, freeze-thawing and pipetting, all of which reduce errors and improve efficiency of use. Freeze-dried products can also be quickly reconstituted, and large amounts of beads can be freeze dried simultaneously to enable scale-up production in a R&D laboratory unit.

lyophilization; freeze-drying, lyophilized bead-format assay

Now, if I didn’t manage to thaw your fear of freeze drying yet, consider flipping through some more of our free resources on lyophilization. Check out a guide on basic theory and applications of freeze drying , as well as an illustrated tool kit for general users of lyophilization . Or another brilliant idea, just skim through some of my past posts on freeze drying to enrich your knowledge.

The method is cutting-edge, the application is exciting, but all good things, including my lovely get-together with my friends, must come to an end. As must this blog post. But another meeting and another post is sure to come. Cheers.

Till next time,