Freeze-drying proteins and peptides for biopharmaceuticals is a complex process that presents many challenges. In this blog, I discuss the critical factors that influence the freeze-drying of formulations to ensure the preservation of your proteins.

These days, when I’m not tinkering with lab equipment, you’ll find me in my garden, covered in soil, planting flowers and vegetables. Now, you might be wondering what gardening has to do with the freeze-drying of proteins and peptides for biopharmaceuticals, but stick with me; you’ll be surprised.

What are the components of a freeze-drying formulation?

This crazy idea came to me when I was working in the lab with freeze drying specialist Bruno. While cleaning the freeze dryer, we started talking about our hobbies. Bruno loves football and is a die-hard bostero who never misses a Boca Juniors match, even if it means staying awake until 3 or 4 a.m. As you all know, I prefer spending my time with something I can put my soul into, and gardening is one of my favorites.

I was telling Bruno I was unsure what to plant in the shady back part of my garden. Bruno told me his abuelo always said it’s about getting the right conditions and soil mixture to ensure the plants survive. Bruno had learned the hard way; he had once asked his grandfather if he could plant pineapples to make paletas – a traditional Latin American ice pop that contains chunks of fruit enjoyed in Argentina. His wise abuelo told him to focus on other crops, but he was stubborn and planted the pineapples against his advice. After spending his whole summer tending to them, nothing happened. Bruno told me that, in time, he realized that the soil pH on the farm was not appropriate for pineapples that thrive in acidic soil with pH levels of 4.5 – 6.5. The altitude was also not ideal, and although they had plenty of sunshine, pineapples like continual moisture rather than a lot of water.

While reflecting on Bruno’s advice, it occurred to me that freeze drying formulations were very much like the soil in my garden. Freeze drying formulations are carefully designed to preserve the integrity, stability, and efficacy of the material being dried. This is especially critical for sensitive materials like pharmaceuticals, biopharmaceuticals, or certain food products.

There are many reasons for freeze-drying biopharmaceuticals; they may not be stable in a liquid state or have rigorous storage requirements. Freeze drying is great for products that do not require further processing, as they can be dried in vials and sealed immediately after the process to avoid contamination.

A biopharmaceutical formulation consists of an active ingredient that provides the desired effect, such as a protein or peptide. To support and stabilize this primary component, additional substances, known as excipients (constituents), are added, creating a composition ideal for freeze-drying.

The following is a list of excipients for biopharmaceutical formulations:

• Bulking Agents: Materials like mannitol, sucrose, or lactose that add volume and help create a stable matrix.
• Cryoprotectants: Substances like glycerol or dimethyl sulfoxide (DMSO) that protect the active ingredient from freezing stress.
• Lycoprotectants: These protect the active ingredient during the drying phase and include sugars like sucrose or trehalose.
• Stabilizers: Ingredients such as buffers that help maintain the pH and ionic strength of the formulation.
• Surfactants: These are used to stabilize proteins and other sensitive molecules against aggregation.
• Preservatives: To protect the product if it is susceptible to microbial growth.
• Solvents: The choice of solvent can be crucial, and often water is used. In special cases, organic solvents are used.

The choice of excipients depends on multiple factors. Just like certain plants require specific types of compost or soil, the active ingredient for a biopharmaceutical needs the right formulation to thrive. It is important to know as much as possible about the nature of the material to be freeze-dried, including its stability under different conditions and the intended use of the freeze-dried product. Like in my garden, I need to understand the types of plants or seeds I am planting before I prepare the soil. If the wrong choices are made in the beginning, you’re setting yourself up for failure right from the get-go.

For proteins, generally speaking, their long-term stability is associated with the water content of the formulation and their conformational structure. Proteins need water to avoid denaturation, and care should be taken when choosing the protein solvent. Moreover, a protectant such as trehalose should be used to stabilize the molecule to help it retain its functional activity.

What are the Critical Compound Characteristics for Successful Lyophilization?

Before you choose and prepare the type of soil or compost mix, you must first understand the plant you wish to grow and its specific needs. If a plant requires good drainage, you may choose loam or sandy soil made of larger particles. For acid-loving plants like rhododendrons, azaleas, and blueberries, you need an acidic (Ericaceous) soil/compost. The same is true for proteins and peptides. To make the right choices, you need to understand as much as you can about the compound characteristics. The ultimate goal is to stabilize the formulation so that the biological activity of the protein or peptide is preserved during and after lyophilization.

Thermal Characteristics

There are various analytical methods used to determine compound characteristics, such as Differential Scanning Calorimetry (DSC), Fourier-Transform Infrared Spectroscopy (FTIR), and others. For successful lyophilization, you need to understand the thermal characteristics of the protein or peptide of interest. DSC is a powerful technique for assessing the thermal stability of proteins and peptides. It measures the heat flow associated with phase transitions in the material as a function of temperature. Calorimetry can give you important characteristics of your formulation, such as:

• Glass Transition Temperature, Tg: The temperature at which an amorphous material transitions to a glass (brittle) state. In freeze-drying, it is crucial to keep the product below its Tg during primary drying to maintain structure and stability.
• Melting Point, Tm: The temperature at which a solid substance turns into a liquid. In freeze-drying, it is essential to understand Tm to avoid melting during the process to maintain the integrity of the product.
• Crystallization Temperature, Tc: Temperature at which a solute crystallizes during the freezing process. If crystallization is not desired, this temperature must be avoided.
• Reaction Heat, ΔH: The heat change associated with a chemical reaction. Knowing the ΔH can help in predicting and controlling the heat required or released during phase changes, ensuring a smooth transition between freezing, primary drying, and secondary drying phases.
• Specific Heat Capacity, Cp: The heat required to change the temperature of a unit mass of a substance by one degree Celsius. Cp is vital as it helps determine the amount of heat that needs to be supplied or removed to achieve the desired temperature changes, ensuring efficient and effective drying.

Another analytical technique is Freeze-Dry Microscopy, which helps determine the collapse temperature (confusingly also represented by Tc). This is the temperature at which a product’s structure begins to collapse during the drying phases. Knowing the Tc is crucial for setting appropriate shelf temperatures to avoid Tg and Tm.

Understanding the thermal characteristics influences several factors, including:

• The choice of buffer: This affects thermal stability. A buffer that maintains the pH close to the protein’s isoelectric point enhances stability.
• The concentration of the protein or peptide: Also affects thermal stability; therefore, it is important to perform thermal characterization at concentrations that are representative of the final product.
• Scaling up the process: This may require a re-evaluation of thermal characteristics due to the differing concentration.
• The behavior of the excipients: The influence of excipients on the listed thermal characteristics must also be considered.

Just like you wouldn’t plant a tropical plant in a temperate climate, you need to understand the thermal stability of your biological material, as ignorance here could turn your proteins into an unstable mess.

How do you Optimize the Freezing Phase?

When preparing soil for planting, there is a thing called ‘tilth’ that refers to the physical condition of the soil and describes the stability of aggregated soil particles, the degree of aeration, soil biota, etc. This is similar to what occurs during the initial freezing phase of the lyophilization process. The rate of freezing and the final freezing temperature influence ice crystal formation and size, which in turn affects the sublimation rate and other aspects of the process. The product must be frozen at a temperature that is low enough to ensure that it is completely frozen. This freezing stage creates the structure in which the protein will be embedded. If this is not right, the protein will lose its activity and be locked in the wrong conformation or lose its integrity. For a more detailed description of how to perfect the freeze-drying process, check out the blog I wrote on the topic. With regards to proteins and peptides, they are best stored at -80°C therefore, freezing them slowly at -20°C is not recommended. Rapid freezing, using an ethanol mixture, is preferred because it results in the formation of smaller ice crystals, which are beneficial in maintaining protein stability.

What are the Critical Factors Influencing the Drying Phase?

The drying phases are critical when working with proteins and peptides. Too fast or too slow, and you’ll either kill the protein structure or end up with an inadequately dried product. Primary drying is the longest phase, and we must apply what we have learned to set an appropriate chamber pressure and shelf temperature. The best approach for setting the system pressure is to determine the product temperature using thermocouples or other temperature probes and then consult a ‘vapor pressure of ice’ table to find the corresponding vapor pressure of ice at that temperature. Endpoint determination is also crucial to ensure all the ice has been sublimated from the product, as residual moisture can compromise stability and shelf life. It is important not to prolong the drying phase unnecessarily as it is not cost or energy-efficient and can lead to product collapse. There are various methods for endpoint determination, including the temperature difference test (between the sample and the shelf), the pressure difference test, and the pressure rise test. Modern instruments can determine the endpoint automatically through the implementation of automated endpoint determination. Such process analytical technologies that track the drying progress allow for real-time adjustments, speeding up the optimization process. Automated endpoint determination provides essential tools for monitoring process reproducibility, ensuring consistency across batches. The use of endpoint determination prevents premature transitions to subsequent drying phases, ensuring optimal drying outcomes.

After primary drying, there is usually a residual moisture content of 5 – 10% due to tightly bound water molecules; hence the requirement for secondary drying. The goal is to vaporize the bound water, and this is usually done at lower pressures and higher temperatures. If the temperature is too high, however, it could lead to degradation of the protein or peptide. Secondary drying is important to ensure stability and shelf life. You should also be aware that although a protein can become unstable during the drying process (denaturation), the protein may refold completely (renaturation) and still display pharmaceutical stability after reconstitution as long as the folding mechanism is reversible.

So, there you have it. By understanding your protein or peptide of interest and understanding the critical factors that have been discussed here, you should have no problem preserving your proteins and peptides during the lyophilization process.

Till next time,