How to make your protein formulation last and last and last

Stability is important no matter if you are trying to form a sandcastle, a nuclear reactor, a new relationship, or a new protein formulation! So this is the promised continuation of my protein formulation series, where I go deeper into the spray-drying and freeze-drying methods for protein and peptide applications. In this post, learn more about how to use these techniques to achieve a stable protein formulation.

Recently, I was watching a documentary on stable man-made structures, such as the Hoover Dam and the Torre Mayor. The Torre Mayor apparently not only survived a 7.6 earthquake back in 2003, but the workers inside did not even notice the tremors.

Engineers love stability, but so do scientists. If we’re going to do the work, then at least the product should be made to last, right? I could discuss stability in any research area, but since I touched on protein formulations in my last blog post, how about I stay on topic and give you some more tips on how to use spray drying and freeze drying to optimize your protein formulation?

Whenever we dry a protein formulation, just like those engineers and builders, we are mostly concerned with achieving long-term stability. We can usually formulate small molecules without excipient, as their moisture content can reach a low enough value to keep the formulation stable for longer periods of time.

Proteins, on the other hand, are more labile than peptides. Their stability is closely dependent on their conformational structure and the water content of the protein formulation. Proteins need water to avoid denaturation, so the protein formulation should be optimized and the drying process should be properly controlled to avoid stability issues. In some cases, proteins could even become unstable during the drying process, but if no irreversible reaction, such as aggregation occurs, the protein could refold completely and display proper pharmaceutical stability after reconstitution.

A well-designed formulation considers the specificity of the molecule, the drying stress during the process, the method by which the protein was produced and purified and the molecule’s degradation pathway. And then of course, come the parameters of the technique used for protein formulation:

Protein formulation with spray drying

The results of the spray drying method highly depend on material properties, equipment design and the process parameters. These factors influence the quality of the final product in terms of:

  • Morphology
  • Residual moisture
  • Particle size

To reduce the thermal degradation of the protein or peptide in your protein formulation, you should mainly focus on the temperature and residence time. The outlet temperature is the maximal temperature experienced by the product or the temperature at which the molecule spends the longest time. This parameter is therefore key to preventing thermal degradation.

If the outlet temperature is lower than the degradation temperature of the protein, the material will experience less thermal degradation. But an outlet temperature that is too low can result in shorter shelf life to an increase in residual water content. It is important to find the ideal outlet temperature where you can obtain a product as dry powder, without inducing degradation in your protein formulation.

Generally, spray drying of proteins to be used as biopharmaceuticals, is carried out with an outlet temperature below 100⁰C, with very low feeding rates.

The residence time is important for minimizing the thermal degradation of heat sensitive materials. Fine particles with a high amount of surface moisture tend to experience easy evaporation, so they require a short residence time. Fine to semi-coarse spray that needs a low moisture content requires a medium residence time, whereas coarser spay with even lower residual moisture content requires the longest residence time.

The time required to evaporate a droplet of pure water is reported as 0.03 s for a 10 um droplet and 3 s for a 100 um droplet.

The typical residence time for a lab-scale spray dryer for protein formulation is in the range of 0.2 to 0.35 s. The residence time is given by the drying chamber volume and the drying gas flow rate. Since the drying chamber volume is constant, only the gas flow rate can be adjusted. However, most spray dryers are run at maximal drying gas flow rate to maximize capacity.

Protein formulation with freeze drying

The most critical step of the freeze-drying process is the freezing, as it establishes the microstructure of the dried product. You need to freeze the product at a temperature that is low enough to completely solidify the product. Most liquid products, including protein formulations, freeze by forming ice crystals. Size and shape of the ice crystals depend on the cooling speed and define the freeze-drying ability of the particles.

Rapid cooling results in small ice crystals, whereas slower cooling leads to larger ice crystals. In terms of lyophilization, smaller ice crystals are more challenging to remove from the product than larger ones. Still, the freezing temperature of a protein formulation is defined by its characteristics and composition.

Formulations can generally freeze in two different ways. Eutectic mixtures contain substances that freeze at lower temperatures than the water surrounding them. When cooling a eutectic mixture, water is the first to separate from the substances as it freezes to ice. The protein formulation may appear frozen, but the remaining substances are still liquid. They form concentrated areas that freeze eventually at temperatures below the freezing point of water.

The temperature where all components of the mixture are properly frozen is called eutectic temperature. This is the critical temperature of the protein formulation and the maximal temperature the protein formulation can endure during the freeze-drying process.

Applying a vacuum to an incompletely frozen eutectic mixture could result in the destruction of the product as unfrozen components expand when placed under vacuum.

The other class of mixtures is amorphous and form glassy states when frozen. With decreasing temperature, the protein formulation becomes more viscous and eventually freezes to a vitreous solid at the glass transition point. For amorphous products, the critical point in terms of stability is called the collapse temperature. The collapse temperature is usually slightly lower than the glass transition point. Amorphous products are generally very challenging to freeze-dry.

Proteins are molecules with a relatively high glass transition temperature and are therefore easy to freeze and dry. When freezing, the final structure in which the protein will be embedded is created. If the structure is not right, the protein will be locked in a wrong conformation and can potentially lose potency. It is therefore important to assess the freezing method (slow vs quick) and its impact on protein integrity.

For primary drying, you can usually develop the cycle using information from the glass transition temperature to define shelf temperature and chamber pressure. Phase duration can be determined using tools, such as end point determination (temperature and pressure) or scientific knowledge.

While the primary drying is developed using information from the thermal characteristics of the molecule, the secondary drying cycle is developed using information on the thermal stability of the molecule.

The temperature of the shelf and the duration could impact product potency. Secondary drying also determines the residual moisture level in the product and therefore its stability and shelf life. The remaining humidity can increase degradation reactions during the product shelf-life and should be kept at a low level, typically below 3%.

Tips for your protein formulation

  • You should store proteins and peptides at -80⁰C or on ice, freezing them at -20⁰C is not recommended. Prior to storage, should filter your purified proteins. Rapid freezing using dry ice/ethanol mixture is preferrable than slow freezing at -20⁰C. To increase stability during storage, freezing and thawing cycles, consider adding sucrose or glycerol to your mixture. Take care in choosing your protein solvent and potentially use protectants, such as trehalose to stabilize the molecules and help retain their functional activity.
  • Measure the degradation temperature and of your protein, if possible. Also try to determine the glass transition temperature of your protein formulation (protein + excipients)
  • Use sugar and sugar additives in your protein formulation to protect spray-dried and freeze-dried proteins against dehydration and thermal stress.
  • Consider if your protein formulation design is appropriate for the chosen drying method. It is very difficult to design a freeze-drying cycle around a protein formulation that is not well suited to freeze drying. For example, proteins in phosphate buffered saline are extremely difficult to lyophilize.
  • Develop and optimize your freeze-drying cycle for each product. One cycle does not fit all.
  • Freezing methods used before lyophilization can significantly impact the structure of the ice formed, affecting both the water-vapor flow during primary drying and in the final product. Try to control how a solution freezes to achieve shorter freeze-drying time and more stable protein formulations.

Try to apply the knowledge and tips from this post and let’s see if your protein formulations outlast the Hoover Dam! And if you want further support, reach for a free spray drying guide for more tips on formulation.

Till next time,

The Signature of Bart Denoulet at Bart's Blog