What are Lyophilized beads?
Lyophilized beads are durable spheres of freeze-dried material formed from accurately measured volumes of customizable formulation.
Like all lyophilized formats, these single-dose beads retain long term stability at ambient temperature and consequently do not need expensive cold chain shipping or refrigerated storage.
However Lyophilized beads also help to improve assay accuracy as well as delivering a number of efficiency benefits over traditional lyophilized formats.
Key Benefits of Lyophilized beads
Lyophilized reagent beads deliver the following performance, efficiency and cost benefits over traditional lyophilized formats:
Assay accuracy
Lyophilized beads improve assay accuracy because the single dose consistency of beads minimizes the potential for variance.
Reduced risk of contamination
There is no central stock to corrupt.
Increased speed to result
Beads reconstitute faster because of their volume to surface area ratio.
Reduced risk of error
Beads do not need aliquoting after reconstruction, significantly lowering the risk of dosing error.
Ease of handling
Each all-in-one bead is pre-packaged and ready to use.
Reduced wastage
Because each bead contains an individual dose there is no unnecessary reconstitution or exposure to moisture of any excess stock.
Simpler quality control
Quality control is quicker, easier, and more cost effective with beads.
More efficient manufacturing
Lyophilized beads offer a much more efficient route to large scale manufacturing, especially for Point of Care devices.
How are Lyophilized beads made?
The process of manufacturing lyophilized (lyo) beads requires a high level of expertise and equipment. Partnering with a modern, experienced contract manufacturer will allow you to deliver the benefits of lyophilized beads without prohibitive start-up costs, wasteful trial-and-error testing, or risk to your capital.
Stage 1 – Formulation optimization
To create a custom lyophilized bead which is stable and suitable for large scale manufacture, the ideal freeze-drying conditions of the assay must be established before the lyophilization process begins. This is usually achieved by the combination of Freeze-Drying Microscopy (FDM) and Differential Scanning Calorimetry (DSC).
FDM uses a miniscule amount of a complete formulation to freeze the sample and put the chamber under vacuum. These conditions are accurately controlled, reproducing those of a manufacturing freeze drier. Observing the crystallisation, freezing and collapse temperature of the sample can help identify optimal temperatures, challenging reagents and the effect varying concentrations of the excipients can have on the final conditions.
DSC can calculate how the Glass Transition Temperature (Tg’) will be affected under different heating rates of the product. This provides an indication of the duration of the lyophilization cycle. This helps to calculate the benefit that annealing (Thermal Treatment) of the sample could have on the final conditions.
Both FDM and DSC will be performed with a range of varying formulations identifying challenging components and the most promising excipient combinations in varying concentrations. This provides a good understanding of the conditions to be applied on a larger scale with a minimum reagent cost to the client.
Stage 2 – Automated dispensing
Once the ideal freeze-drying conditions of the assay are established, accurately measured droplets of this formulation are dispensed into liquid nitrogen. This accuracy will ensure that each lyo bead contains a precise dose for its final application.
The beads are transferred to lyophilization freezers to begin the next stage.
Stage 3 – Lyophilization
Lyophilization takes place in two cycles – primary and secondary drying.
Primary drying cycle (sublimation)
During sublimation, the pressure in the environment is lowered while a vacuum is applied. This causes the ice to sublime, removing 95% of the water.
Secondary drying cycle (desorption)
Desorption will remove the remaining, unfrozen water molecules from the formulation. An inert gas is then used to break the vacuum in the environment, ready for the product to be sealed.
Stage 4 – Quality control
At Biofortuna, we always ensure the highest level of quality in each product before the process is complete. This happens in a controlled humidity environment, where the formulation is tested to meet the required standards for appearance, size and successful drying.
First, the Karl Fischer titration technique is applied to detect the levels of residual moisture.
Successful freeze-drying will have removed most of the water from the formulation and the Karl Fischer test allows us to measure the percentage of moisture left in the dried product. However, more is not always better. There is a fine balance between a product that has been optimally dried and one that has been overdried. Depending on the formulation, excessive dryness could have a negative impact on the assay efficiency and/or the bead’s physical characteristics. It takes a certain expertise to fine-tune this balance.
The beads are then evaluated on their physical properties. This includes ultimate tensile strength (maximum strength a bead can withstand before breaking) and friability (how easily the beads can break into smaller parts under duress).
These are important properties that need to be well defined, especially if there is further manipulation after the manufacturing of the beads, such as bulk transport, or placement into cartridges/microfluidic devices.
Beads are also checked for their dissolution rate. This is a critical property, especially when the beads are to be used in a POC (Point of Care) device, where timing from the sample/buffer injection to the final result are very stringent.
Finally, beads are scored on visual appearance, such as their texture, roundness and consistency. This is usually done for aesthetic reasons, but the presence of irregularities could provide indications on the robustness of the freeze-drying process.
The final part of the Quality Control process is, of course, to test the final product for assay sensitivity. This can be done either at the bead manufacturer’s site or the customer’s site. When the sensitivity of the assay is proven to be within the detection limits defined, then the product can be released.