Optimising lyophilisation protocols to stabilise diagnostic reagents

In the diagnostic testing industry, stability and shelf life of reagents are crucial for reliability, accessibility, and scalability—especially in resource-limited settings. Lyophilisation, or freeze-drying, is a powerful technique used to preserve biological materials by removing water under low temperature and pressure. When done correctly, it can extend the shelf life of diagnostic reagents from months to years without refrigeration. However, the process requires careful optimisation to balance stability, activity, and manufacturability.

Why lyophilisation?

Lyophilisation preserves biomolecules by converting water directly from a solid (ice) to a gas (sublimation) under reduced pressure, avoiding the high temperatures that can denature sensitive components like enzymes, antibodies, and nucleic acids. This is especially important for point-of-care diagnostics, where cold chain logistics may be impractical or expensive.

The challenges

While lyophilisation offers compelling advantages, it's not a one-size-fits-all solution. Each diagnostic reagent—be it an enzyme for PCR, a colorimetric substrate, or a lateral flow antibody conjugate—responds differently to freeze-drying. Common challenges include:

• Loss of activity: Enzymes and proteins can denature or aggregate during freezing or drying.

• Poor reconstitution: Improperly optimised formulations may not dissolve uniformly or rapidly.

• Structural collapse: Inadequate stabilisers or suboptimal processing can cause cake collapse or shrinkage.

• Batch inconsistency: Even small changes in parameters like cooling rate or residual moisture can impact performance.

  
  

Key factors in protocol optimisation

1. Formulation design

The backbone of any successful lyophilisation protocol is the formulation. Key components include:

1. Cryoprotectants (e.g., trehalose, sucrose): Prevent ice crystal damage during freezing.

2. Lyoprotectants (e.g., mannitol, dextran): Stabilise molecular structure during drying.

3. Buffers (e.g., phosphate, Tris): Maintain pH through freezing and drying cycles.

4. Bulking agents (e.g., glycine): Improve the physical appearance and mechanical strength of the cake.

Formulation screening via DoE (Design of Experiments) can help identify optimal compositions quickly.

2. Freezing stage

Freezing is not just a preliminary step—it dictates ice crystal size and morphology, which influence sublimation efficiency and cake structure. Controlled-rate freezing (e.g., −1°C/min) is often preferred over snap freezing to promote uniformity and reproducibility.

3. Primary drying

   During this stage, sublimation removes ~95% of the water. Key parameters to optimise include:

   • Shelf temperature: Too low, and drying is inefficient; too high, and the product may melt or collapse.

   • Chamber pressure: Typically 100–300 mTorr; must be low enough to drive sublimation but not too low to cause channelling or incomplete drying.

   • Time: Long enough to avoid residual ice but not so long as to damage the product.

     
     

4. Secondary drying

   The goal here is to desorb bound water and reduce residual moisture to <1%. Elevated shelf temperatures (20–40°C) are used carefully to avoid degradation. This phase is critical for long-term stability.

   
   

5. Packaging and storage

Lyophilised products are highly hygroscopic. Immediate sealing in moisture-barrier packaging with desiccants is essential. Stability testing under ICH guidelines (e.g., 25°C/60% RH and 40°C/75% RH) helps verify shelf life.

Conclusion

Optimising lyophilisation protocols is a multidisciplinary process that blends formulation science, thermodynamics, and engineering. When done effectively, it unlocks new possibilities for decentralised diagnostics and global health impact. Whether you're developing a lateral flow assay or a molecular detection kit, thoughtful lyophilisation design can be the key to a robust and scalable product.

Need help designing a lyophilisation protocol or selecting excipients? Let’s talk. https://www.fleetbioprocessing.co.uk/contact-us