Gemma Blackwell:
Hello, and thank you for joining today's webinar, What Does the Future Look Like for lyophilized Amplification Assay? I'm Gemma Blackwell, the Associate Director of Lyophilization at NEB, and I lead a team of production and development scientists who specialize in the design, development, and manufacture of lyophilized products.

I'm joined by David Shire, who is an application scientist at NEB, specializing in the lyobead format.

In this webinar, we will start by describing lyophilization, and how and why it is used. We will then introduce lyobeads, how they are produced, packed, and used, and how they compare to other lyophilized formats. We'll also discuss NEB's various other lyophilized and lyo-compatible products and capabilities. We would love to hear your questions, but please send them in during the webinar and we will answer them at the end.

David Shire:
Thanks, Gemma. So we'll start with, what is lyophilization? This is best described as a low temperature dehydration method. Simply put, the aim of lyophilization is to dry your desired product at a low temperature, resulting in the removal of the majority of the ice. H2O being removed, however, is never present in the liquid form, and from here on, we'll refer to it as our state-specific term of ice.
The ice is removed via the process of sublimation where the H2O moves from a solid to gaseous stage, crucially without passing through the liquid phase.

The most common applications of freeze drying in our industry include things like amplification reaction materials, something which became very common during the COVID-19 pandemic. An example of this is the L4001 LyoPrime Luna RT-qPCR kit, which is shown on the right-hand side of the screen. Other materials which are commonly lyophilized include oligonucleotides, proteins such as antibodies or enzymes, vaccines, along with food stuffs.

So, we've discussed what lyophilization is, now to discuss how it's done. The first step is a formulation preparation. This formulation cannot simply be freeze-dried straightaway. The first step is to analyze the components and assess their impact on the physical characteristics of the product. This could be associated with their freezing temperature, viscosity, or chemical characteristics, such as their ability to form a crystalline structure. This may mean that the formulation to be dried is altered to become lyo compatible, something which Gemma will discuss more later on.
The formulation is also developed with the inclusion of excipients to aid in structure, and additives, which may enhance the shelf life of the product, but also its performance. Once the mixture is formulated, it is then frozen by decreasing its temperature as shown in the graph on the left-hand side.

The next step for the product is to reduce the pressure by pulling a vacuum, allowing the ice to transition directly from solid to gas phase. This water vapor is pulled out of the product due to a diffusion gradient, which is generated in the freeze dryer by the condenser. At predetermined points in the cycle, the temperature is raised to drive off residual moisture and add energy into the system. This is why many freeze drying cycles include ramp steps to slowly aid in the removal of moisture and prevent the loss of product integrity through the melting of ice.

So why lyophilization? The main aim is to increase stability of the product. Water molecules which normally bind to biologics in question are removed and replaced by excipients. These excipients, often things like sugars, have high hydrogen bonding capabilities, making them perfect for replacement of water, which you can see in the top right-hand corner here.

Ambient storage and transit are beneficial characteristics of a lyophilized product for a number of different reasons. The first and one that's close to NEB is the improved sustainability of shipments. The reduction of polystyrene and dry ice necessity means that we can utilize more sustainable and recyclable materials instead. Delays are also commonplace for shipments across the world and therefore lyophilization means any delays do not lead to product damage. Lyophilization also opens up new destinations as room temperature storage is much easier to come across, rather than specific requirements such as ultra low temperature freezers, for example. Lyophilization also gives a ready to use solution where the best case scenario is just a simple water rehydration step, removing the need for multiple additions of reagents or complex SOPs.

This also opens up non-lab uses as well. So applications such as infield sampling for conservation efforts, or if there's damaged infrastructure due to a disaster response, humanitarian response can also be used.

So there are three main formats of lyophilized material. Vials are the main work course of lyophilization and are most commonly seen. This is because they're vastly used in vaccine production. The glass vial itself is a high purity glass to ensure thermal conduction during drying, and often filled with a rubber stopper to prevent water ingress post-lyophilization. Vials are often used for bulk packing. For example, the LyoPrime Luna kit shown here provides 60 reactions per vial.

Plates are another option available at NEB, where the master mix in question is pre-aliquoted into 96 single use cakes. This is perfect for short experimental setup, and our plates also come with a shell frame ensuring compatibility with your thermocycler. The plates are also arranged in tear off strips, perfect for those experiments that don't necessarily need all 96 reactions.
And then finally, the main topic of this webinar are lyobeads. lyobeads are similar to plates as they are most commonly one cake per reaction, but their advantage is that they are easy to move around. This flexible approach allows lyobeads to be placed into plates, vials, or in customer cartridges dependent on the application and stage of product development.

Beads also maximize dry loads. Given that freeze drying is a batch process, this is paramount to ensure a low cost of goods.

So now we'll focus more on the lyobeads production methods and the process that it follows. There are four main processes which I'll go into more detail now.

So the first step is dispensing. The mass mix is dispensed with high precision utilizing a positive displacement piston pump. There are a number of different factors that we need to pay special attention to, which is not limited to the formation of the droplet on the dispensing tip, the speed of the dispense, and of course the volume of the droplet produced. The droplet falls from the tip into a bath of liquid nitrogen, which is sat at minus 196 degrees Celsius. This flash freezes the droplet, which we can now refer to as a bead.

The bead produced must have high precision on the dispense, often lower than 2% CV, as this is the most significant factor upon re-suspension as it will directly impact reaction concentrations. It is also essential that each droplet is separated as it freezes to prevent merger of partially frozen beads, resulting in double dosages.

If the dispensing configuration is not correct, then small volumes of the dispensed droplet can break off and refreeze to the main bead body, producing what we refer to as a satellite. Although the correct volume has been dispensed, these satellites are print breaking off and ultimately result in under dosage.

Finally, it is also important to ensure spherical morphology of the bead. This consistency across the batch is important for drying, but is also essential for high throughput automation methods, as if not fully round, it may lead to difficulty in movement.

So we have frozen beads, what's the next step? The lyobeads are harvested from the liquid nitrogen and they're carefully transported along streamlined workflows in our labs. These processes ensure the safety of the products, but also the operators as we are dealing with liquid nitrogen. Our dedicated lyobead lab spaces allow for the product to remain frozen at all times, preventing any loss of product structure during the sensitive handling period.

As previously discussed, one of the best features of lyobeads, they move very easily. This is true in both the dried format, but also in the frozen state. Therefore, extra care is taken for inline clearances and batch identification as well.

So once the beads are frozen, they then need to be dried. A common misconception is that the drying of a product in a plate is the same as that of a bead due to their similar size and shape.
However, the process of lyophilization is made up of two main processes: freezing and drying. The freezing step majorly impacts the size of the ice crystals. The popularity of science and cooking demonstrated the use of liquid nitrogen to make ice cream, which is a perfect example. The reason for this use is that the rapid freezing produces really small ice crystals resulting in a smooth texture. The rapid freezing of master mix is the same, resulting in small ice crystals. However, this isn't beneficial for lyophilization as these small ice crystals result in a high product resistance caused by the complex path ice has to take in order to leave the product. This can be seen on the right-hand side. As you can see, the small ice crystals have a much more complex path in order to leave, making it more difficult for that ice to safe.

Here at NEB, we have the expertise to develop cycles favored towards this high product resistance by controlling the product sublimation rate, rebalancing of shelf temperature and the vacuum inside the product chamber. The product collapse temperature and resistance are outside of our control, but through methods such as freeze dry microscopy, which you can see in the bottom right-hand corner, we can gain an understanding of how the product will behave and use optimum drying conditions to reduce the live bead which is stable for years.

And then finally onto packaging. We spent a lot of time and effort into making these lyobeads and making them dry. So the last thing we want to do is introduce a load of moisture.
The packaging can be done in a number of different ways, one of which is in our dedicated dry room spaces. These have a relative humidity of less than 10%, but we also have the capability here at NEB to pack in 100% dry nitrogen if moisture and oxygen sensitivity is important. This is dependent on the application.

The packaging can also vary. This can be in large bulk vials, ranging from 30 to 200 mil in size, but also precise aliquoting into small three mil or five mil vials for lab bench or for precise re-suspension.

Our beads can also be singulated into strips or tubes, as you can see on the top right-hand corner. This is done in a static free environment to ensure that the correct dosage of material is present in each container.

And then finally, if we're utilizing plastic consumables over glass, then it's favorable to sell these in foil pouches with desiccant or oxygen scavenging pouches. This ensures moisture and oxygen protection before shipping to customers.

So in summary, some advantages and applications of lyobeads, one of the most recent applications of beads is they're using cartridge-based systems for point of care and point of use diagnostics. This became commonplace during the COVID-19 pandemic, whereby centralized testing labs utilized lyophilized material, but also places like doctor's offices and pop-up testing sites. lyobeads are seen to have a stability matching that of other formats, but allowing that flexibility in where they're placed.

Glass vials, as we've discussed previously, are used for bulk format, but they also leave you with a slightly difficult to dispose of consumable. A cartridge-based system can be very easy to use, and also disposed of much more easily as well.

The high precision dispensing methods used to any bead for bead production allow for a repeatable and reliable Lyo bead. This high precision means that loading multiple beads has very little impact and no buildup of error.

For example, two reagents which interact can be separated into two different beads, and prevent unwanted reactions during formulation and dispense, which can often also lead to larger batch sizes as well.

Another benefit of beads is that their dispense volume and concentration can be changed very easily, allowing optimization to any reaction. A reaction which may be sensitive to excipients may benefit from a more concentrated bead to reduce this excipient carryover.

Finally, as mentioned previously, lyophilization is a batch-based process and therefore freeze dry real estate is an important parameter. This allows the maintaining of high throughputs and low costs. As you can see on the right hand side, if we take Lyo Luna as an example, the number of beads you can fit in a dryer is almost double that of the number of reactions you get from a vial-based product, and over 25 times that of a plate-based product.

Gemma Blackwell:
Thank you, David. Next I would like to introduce New England Biolabs localization sites, and this is us. We're based just south of Oxford in the UK, and around a year ago we moved to a 30,000 square foot purpose-built facility, which tripled our space compared to our previous site, and which includes custom-built spaces for material preparation, drying and packing and ultra low humidity, as well as separate self-contained spaces for QC testing and R&D activities.

These are some of our labs. Actually, these photos were taken shortly after we moved in, so the benches are a lot fuller now, but they give you a feel for the facility. We have various sizes of freeze dryers. You can see some of our smallest on the bottom left and two of our mid-size dryers on the top right.

We offer several lyophilized products and formats in the NEB catalog, including QPCR and LAMP products. These materials have the same high performance qualities as their liquid equivalents with the simplified transport and storage that you would expect from freeze-dried material. They're ideal for applications, as Dave discussed earlier, where ambient shipping and storage are important. They're also useful as a step towards developing a custom lyophilized material. Primers can be added wet during initial testing, then assuming that material is suitable for the application, primes and probes can be added to the formulation and dried in. We typically find that dropping in primers and probes to an existing live-ready formulation is very straightforward. That makes these lyophilized products available from the catalog also an ideal turnkey solution for RT-qPCR, LAMP and RT-LAMP product development.

In addition, NEB offers an ever expanding range of glycerol-free and biocompatible molecular biology reagents. These include RT-qPCR and RT - LAMP and LAMP mixes as we've already discussed, but also a range of BST enzymes for isothermal amplification, RPA enzymes, several DNA polymerases including taq, hot start taq, and Q5, restriction enzymes, ligases, as well as enzymes proteomic applications.

Our third offering is custom Lyophilization. So, when there is a preexisting formulation that works well in a customer's application, or when the Lyo-Ready products available via the catalog aren't suitable, or in different formats or product product packaging is needed, we can develop a custom product. A large part of the work that we do with NEB Lyophilization Sciences involves producing a custom product specific to a customer's requirements. Often these are built from our available glycerol-free lyocompatible reagents, but if these aren't available, it's often possible to remove glycerol or to reformulate to make a material compatible with lyophilization.

We're also able to accommodate a wide variety of packaging requirements, obviously vials, tubes and plates, but lyophilized lyobeads also allow pick and place into custom cartridges and containers, either not suitable for in-situ drying or which are prohibitively expensive to dry into because of their size.

Previously, I mentioned the lyocompatible reagents available from NEB, and that while we often build custom products from these materials, we also regularly develop lyophilized products based on other NEB materials which aren't immediately compatible with lyophilization. This is possible because we have this fantastic resource available in NEB's 50 years of entomology experience. This means not only do we know the exact formulation of the materials that we're working with, we can also easily speak to the scientists who developed and who produced these enzymes and reagents, to understand the impact of any changes we need to make to the formulation to make them compatible. It not only makes troubleshooting easier, but means that we're less likely to need to troubleshoot because between our own knowledge of lyophilization and our colleagues' knowledge of the materials we're working with, we can design the formulation to be suitable for freeze drying from the beginning.

Now, I would like to talk through the stages of lyophilized product development. We already discussed propelling formulation for lyophilization by making it lyocompatible. This can involve removing glycerol or other incompatible components, which we do alongside our colleagues in enzyme development and production. We then select suitable excipients in collaboration with either our customers if it's a custom product, or our colleagues if it's a product developed destined for the NEB catalog.

Excipients fundamentally need to be compatible with the reaction that they're added to. The screening initially focuses on assay performance alone. Once we have identified excipients that at least don't negatively impact assay performance, as it's sometimes possible for excipients to have a positive impact on assay function, we can move on to identifying excipient combinations that produce a cake or a bead with the desired characteristics in terms of robustness, appearance and reconstitution. That produces a lyo-ready formulation.

Once we have that, we can move on to refine the drying cycle suitable for the lyo-ready formulation in its container. The cycle needs to produce material that's dry enough to ensure an appropriate shelf life, with the right characteristics for reconstitution. The container needs to protect the material from moisture, but also to facilitate the use of the product, which is where lyobeads are so useful as they can be placed into so many different containers. Some products can leave the process there, such as materials for use in research and development. Others such as those destined to go into the NEB catalog need additional verification steps.

Lyophilized products in the NEB catalog are typically called LyoPrime. So, you'll see LyoPrime Luna, which is a freeze-dried version of our existing RT-qPCR mix. This slide shows the lyophilized products currently available by the NEB catalog and some of those under development, including bead versions of the existing qPCR and LAMP and RT-LAMP products. If you would like to discuss a lyophilization project or a Lyo compatible reagent, please contact our customized solutions team via the form shown here available from the NEB website.

Thank you for attending our webinar. If you have any questions, David and I are looking forward to answering them.

Kari:
Thank you so much, David and Gemma. It was a wonderful presentation, so we can now open that up for questions from the audience.

Gemma Blackwell:
We have one question just to start us off, and that's how long would you expect lyobeads of a nucleic acid amplification test to remain stable in proper storage conditions, and would you expect a reduction in amplification efficiency?

I'll take the first part first. So, for customer products, we typically aim for a shelf life of at least two years, and that's generally readily achievable, we tend to find, two years or longer. I've seen a bead product with a shelf life of longer than four years, and that's only topped out at four years because we stopped testing at that point.

There's no good reason, I think, to expect that the stability should be shorter than any other lyo product. Storage conditions though are absolutely key. They have to be protected from moisture and if anything is sensitive to oxygen, they also have to be packed under an inert gas. So, there's no good reason to think that a well formulated bead with a good freeze drying cycle that's appropriately packed would have a shelf life stability shorter than any other lyo product.

In terms of performance and whether we would expect a reduction in amplification efficiency, compared to another life life product, again, with the same caveat of proper development, proper cycle and formulation, we wouldn't expect a drop in efficiency just for the bead format. Compared to a wet mix, we tend not to see it, but again, the caveats are our proper formulation, proper storage, proper cycle. It can happen though, and we tend to find... Well, there can be various causes. I'd say the number one, if I had to pick one, would probably be excipient compatibility. So, if you can get that excipient screening done and make sure that your assay's happy with the excipients you're adding to it, you're giving yourself the best chance of not seeing an amplification efficiency problem. Hopefully that answers the question.

Let me just find another... Sorry, another question. So whether we can get catalog items customized with a customer's own primers, certainly we can. So I mentioned that we have on the catalog RT-qPCR mixes, RT-LAMP and LAMP products, and actually that's a very straightforward thing to do, to drop customers' own primers and probes into one of those mixes, either in the vial, plate, or bead format. Typically, we don't find that adding oligos really affects how a material freeze dries at all, so we would expect it to be a very straightforward and quite a quick project to drop a primer or a probe... Sorry, primers and probes into either a QPC or a LAMP mix. So yes, that's definitely possible.

David Shire:
So, we also got a question about lyobeads being aliquoted into 96 well plates. There are a number of different methods that we use in-house, some with higher throughput than others, but a lot of customers aliquot their own lyobeads into plates. So if they get them in a bulk format, you can utilize compressed air to move them around.

And a similar question was whether they fall out of the 96 well plate. Yes, they can do. The reason for the use of lyobeads in a plate format is either to use them during screening processes, which you would then eventually move to a lyobead in a cartridge based system so that you're not having a design change between your early stage work and your later stage work, or simply because in the long term, you can fit more in a dryer, which is one of the major advantages, as I mentioned earlier.

Freeze drying is a batch process, so freeze dryer real estate is the phrase we use, is very important for some customers.

Gemma Blackwell:
Another similar question as well, which was how are lye beads aliquoted into wells of a 96 well plate, and how much water is used to hydrate a bead? If I take the second part first this time, that depends on the size of the reaction that you want to run. And so, we typically make beads at two times the final concentration of the reaction that's to be run. Of course, we can vary that as needed, and I think we've got a project that we're working on at the moment that has a five or six times concentrated bead. We can also make them at the same concentration as the reaction. So it depends on how big the bead is, how concentrated it is, and the final size of the reaction. Typically, we make beads at between 10 and 40 microliters. There are sort of standard sizes, but we can go outside of that for particular requirements.

How are lyobeads aliquoted into the wells of a 96 well plate? There are a few different ways of doing that, and it sort of depends on the complexity of the aliquoting as to whether you're having the same thing or just one thing in each of the 96 wells of a plate, or the eight or 12 wells of a strip. If it's just one thing, then we have a machine that can do that for us.
If it's a little bit more complicated, and you're putting either different things into different wells, or multiple things or multiple different things into different wells, then we do have equipment that can do that, but you can also use vacuum tweezers to move beads around.

If you're just working with beads in your own lab, you don't necessarily need to have vacuum tweezers. If you have a pair of long nosed metal tweezers, that's absolutely fine. Obviously the main issue there is as soon as lyophilized material is opened, it's then drawing in moisture from the air and that will affect over time function and stability. So if you are working with lyophilized material in standard room air rather than dry air, it helps to work quickly, and then if you're setting up a reaction to set it up and run it straight away as soon as you open the container.

David Shire:
So, there was also a question of NEBL4401 versus NEB L4402. L4401 is the LyoPrime warm start fluorescent LAMP and RT-LAMP mix with UDG, which contains the lyophilized dye. This is available in both the vial and the plate format. L4402 doesn't contain the dye.

Gemma Blackwell:
Okay. We have a couple more. The average turnaround time for custom beads. So, we say six to eight weeks from the receipt of all the materials that we need to make those beads. So, if primers are involved in the receipt of those primers, either from the primer supplier or from the customer, and if there are customer supplied reagents, if a customer wants to add their own enzyme or a particular material to those beads, it's six to eight weeks from the receipt of those materials.

Also, a comment that we don't see any lyobeads in the US NEB catalog. No, unfortunately don't just yet. They aren't quite ready for launch. We do have them through the customized solution service, so they are available. They just haven't quite hit the catalog yet and we're working on that part.

Sorry, we have another question that is, if somebody wants to dry something that is not available glycerol free yet, would this be a very long development cycle?

No, not necessarily. Particularly where the barrier to lyophilizing something is glycerol. There are various methods that we can use to remove the glycerol. It's very straightforward to do it at a sort of development scale. It's also possible to do at much larger production scales as well. So, it wouldn't necessarily have very much, or to be honest, it wouldn't have to add any time to it to a development project. And I don't expect that it would need to add much time to a full production scale project either.

Of course, there's always, as I said, the opportunity that we have this fantastic bank of enzymology knowledge that we have, being NEB, to work on developing a glycerol-free format. So that can always happen in the background, but that doesn't have to happen first.

We can do the lyo part first. If it works well and we need a glycerol free formulation for some reason, that can run along in the background, but we can certainly get started even if the material that you're looking to work with has glycerol in it. That really isn't a problem.

Kari:
We are coming up on time, so let's take one more question from the audience.

David Shire:
So one other question that we got is about re-suspension and reconstitution of the Fireflys product. We would assume in most cases that this would just be a water rehydration step. If we can do it makes life easier for everyone. One thing that does have to be taken into account is obviously if there's something in one of the buffers or in the reaction, which is particularly difficult to freeze dry, we could work our way around this by changing our excipient formulation, which is what we would work with you on, or by changing the concentration in the bead. So things like very, very high salt concentrations might be a little bit more difficult. But the aim would just be water, yeah.

Gemma Blackwell:
Okay. And then I think I get the honor of taking the last question, which is, it's a good one actually, this one. How do we select excipients, or do we just use trehalose? We definitely don't just use trehalose. Trehalose certainly is obviously a really popular freeze drying excipient and we do test it, but we select freeze drying excipients for freeze drying. As we referred to earlier in the presentation, the focus is on selecting excipients that work for the purpose for which the material is going to be put. So if it's a PCR or a LAMP reaction, the focus is on testing a broad panel of excipients, and making sure that we select those that best work for the assay. We at least want something that doesn't inhibit the assay in any way. And sometimes you can find something that actually improves performance.

Once we've done that, we're then looking to find excipients that perform material with the right characteristics. It needs to be appropriately robust, but also re-suspend well. If it's a bead that's just going into a 96 well plate, it perhaps doesn't need to be as robust as a bead that's going into a cartridge for a pick and place process. So we need to bear all of those things in mind.
Having done that, we have an idea of which excipients work well on which freeze drying cycle. So that factors in as well. But the absolute fundamental thing and where we always start, is making sure that the excipients work well in the function to which the material is being put. And I think that wraps it up for questions. I'll hand over to you, Carrie.

Kari:
Great. Thank you again, David and Gemma for today's presentation and for all of the questions that we received. If we didn't get to your question here live, we will be sure to follow up with you offline. And if you do have any more questions, feel free to email us at webinars@neb.com.
The webinar will also be available on demand after today's event. So feel free to pass it along to any colleagues that might be interested or watch it again. And we thank you so much for joining. Take care and have a wonderful day.