Whether you are lyophilising or air drying your assay, your ultimate goal is to stabilise it for transport and storage at ambient temperature without loss of functional performance.
Both processes involve the removal of water to achieve this and enable you to:
- eliminate the need for cold chain storage
- extend shelf life at ambient temperature
- enable single assay protocols.
However, the most suitable route will depend on the composition of your formulation.
Lyophilising removes more water and is much more controlled and predictable, so you can experiment with conditions to achieve the optimum end product for your application. However, lyophilising requires additional, specialist expertise and equipment.
Air drying is simpler, quicker, and generally lower cost so it’s ideal for formulations that will not be adversely affected by the heat of the process. But it is only truly lower cost if it is ultimately successful – not if you get halfway through the process and discover that lyophilising would have delivered an assay with superior performance.
Air drying also offers the advantages of lower equipment costs and faster turnaround times, taking just 10 to 15 minutes on average at low assay volumes. But for difficult-to-dry formulations, which are typical in molecular diagnostics applications, such a short drying cycle will not be sufficient. So, often, lyophilising is the better choice for these applications.
How do the lyophilisation and air-drying processes differ?
Lyophilising and air drying both involve removing water from the product but there is a principal difference.
Air drying removes the water through evaporation; it uses heat (to around 40°C). Methods vary but usually the samples are either passed through a hot, dry air chamber or subjected to hot air fans.
Lyophilisation involves freezing the product at extremely low temperatures, significantly reducing the pressure in the chamber then removing any frozen water by sublimation. Trays of products are loaded into a refrigerated freeze-drying chamber. A vacuum system removes ice and converts it directly into water vapour.
Formulations containing glycerol: lyophilisation or air drying?
Glycerol is found in many PCR reaction products for diagnostic kits and for allergen immunotherapies. It is used as a protein/enzyme stabiliser and also as a component in electrophoresis loading buffers because of its density. In addition, glycerol gradients can be used in the purification of bacteriophage or proteins.
Lyophilising is the best option if your formulation contains glycerol due to the challenges involved in drying it. Successful lyophilisation of formulations containing glycerol takes time. The short duration of the air-drying process is not suitable for these applications.
Glycerol tends to persist as an excluded liquid layer during lyophilisation – it does not freeze under normal lyophilisation conditions. One answer is to remove the glycerol by dilution or dialysis.
It is possible to remove glycerol by air drying by increasing the duration of the process. However, to do so would kill the enzyme in the formulation; they are very sensitive to high temperatures.
But when lyophilising, you remove water while your formulation is frozen. The enzyme remains viable in its frozen state so you can take as long as you need until you are happy with the end result.
For the same reason, that enzymes remain viable when frozen, a successful lyophilisation process will provide a longer shelf-life when compared with a successful air drying process.
Which process is quicker?
Air drying is always a much quicker process, which is why it should be considered for formulations that will not be compromised by the heat required.
Lyophilisation is a more involved process. There are four stages:
- Pre-treatment – any treatment before freezing. Typically, this involves freeze drying microscopy, to determine the collapse temperature, differential scanning calorimetry and analysis of the performance of various excipients to establish which will be most suitable for the lyophilisation process.
- Freezing and annealing – the product is cooled below its triple point (the lowest temperature at which the material’s solid, liquid and gas phases can co-exist).Freezing slowly or annealing (raising and lowering the temperature in a cycle) produces larger ice crystals for faster sublimation and water removal. But large crystals can also break cell walls, affecting the structure of the material being frozen. For this reason, structurally sensitive materials are frozen more quickly – avoiding the formation of large ice crystals.
- Primary drying – pressure is lowered, heat is applied – causing the ice to sublimate, removing 95% of the water. This phase is slow; it can take several days. Rushing it by adding too much heat can detrimentally alter the structure of the material. Applying a partial vacuum speeds up sublimation and drying.
- Secondary drying – to remove unfrozen water molecules. This occurs at a higher temperature to break any physio-chemical interactions between the water molecules and the frozen material.
Which process is more cost-effective?
Lyophilising is more expensive than air drying because of the specialist equipment costs and the energy costs (not least because the lyophilising takes longer).
But that is a simplistic summation. There are other critical factors to consider:
- Shelf life – air-dried products may not last as long as lyophilised products, increasing the risk of wastage.
- Assay damage – attempting to air dry a complex formulation that cannot dry within a short period of time is likely to damage it
- Innovation – lyophilisation is a more versatile process that allows you to experiment. Without it, you may not be able to create the next generation of products and be first to market.
Lyophilised beads offer significant advantages for molecular diagnostics because they:
- are much more reliable – single-assay beads reduce pipetting errors, contamination and wastage
- are easier to handle – they are quicker and more convenient to use, reducing set-up times
- offer faster reconstitution
- are highly scalable
- enable direct dispensing into point-of-care testing (POCT) devices, removing the challenging and often-impossible task of lyophilising the assay within the device.
How outsourcing lyophilisation and air drying benefits new product development
Stabilising your assay by either lyophilising or air drying helps you to create additional value by reducing storage and transportation costs for the end user. This in turn increases access to routine diagnostic testing.
It can be the difference between you hitting or missing demanding targets, enabling you to challenge accepted failure tolerances within your sector or market.
Outsourcing to a stabilisation specialist can:
- prove concepts when your in-house teams are struggling due to lack of specialist resources
- overcome barriers to scaling up, particularly if the concept is not proven or validated
- reduce development costs and minimise risk.
Still carrying out proof-of-project concept research? Always planned to outsource at key stages? Need help with a stalling project? Whatever the circumstances, it pays to involve a specialist early on in the development process.
Assay developers outsource their projects to Biofortuna as a trusted partner for custom development, contract manufacturing and genomic services. We can develop PCR assays in lyophilised, air-dried and liquid formats, using a range of different technologies.
We support diagnostic product and platform developers, research groups and not-for-profit organisations providing custom services and turnkey solutions.
Working with a knowledgeable and experienced lyophilisation partner can:
- accelerate formulation development
- ensure a cost-effective air-drying or lyophilisation process
- support you through a regulatory approval process
- reduce the overall time and cost of bringing an assay to market or deploying an assay in the field.
Our support is tailored around your exact requirements and defined by a problem-solving culture. We listen to precisely what you need and tailor a solution to meet those needs.
A combination of technical expertise, ISO13485 and to ISO17025 accreditation, FDA registration and state-of-the-art facilities mean that developers can rely on the highest scientific standards when bringing their product to market.
Like to know more? Our team would be delighted to have an informal exploratory discussion.
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