August 11, 2022

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Vaccine freeze-drying: Challenges in process development and formulation

Vaccine freeze-drying: Challenges in process development and formulation


Vaccine freeze-drying: Challenges in process development and formulation.  Millions of people die from infectious diseases each year, and vaccination can prevent these infectious diseases.

However, due to long-term vaccine stability problems, difficult delivery, cold chain requirements, and poor thermal stability of certain vaccines, millions of people still die. Because of these infectious diseases.

All these challenges can be solved by freeze-drying to produce relatively drier products. Due to the urgency of developing a coronavirus vaccine, now is a critical moment to discuss the vaccine freeze-drying process.


The effect of lyophilization on vaccines

There are many types of vaccines that can trigger certain immune responses. Many coronavirus vaccines currently under development are based on recombinant vector vaccines, using coronavirus genome materials packaged into viral vectors.


Freeze-dried vaccines have significant advantages, but many challenges must be overcome. Complex formulations, especially vaccines composed of multiple strains or multiple antigens, may lead to complex freeze-drying processes and challenging formulation temperatures.


Freezing and drying will have a certain impact on the vaccine, and the sensitivity of the vaccine freeze-drying process varies from vaccine to vaccine. Internal icing and direct damage to vaccine components (such as lipid membranes, nucleic acids, or proteins) may be stress factors.

Vaccine freeze-drying: Challenges in process development and formulation

Figure 1. Stress factors during freeze-drying.


During the freezing process, ice crystals in the virus are formed, which increases the volume of the product and may damage the lipid bilayer, as shown in Figure 1 above. Ice also creates a new interface between ice and liquid and increases the risk of surface-induced aggregation.


Drying above the critical formulation temperature will result in improved fluidity of the amorphous phase in the primary drying step of the freeze-drying cycle. This allows proteins to interact and can increase membrane permeability.


In the case of removing each hydrated shell as part of the secondary drying stage, protein aggregation and inactivation may occur. In the presence of phospholipids, thermally induced phase transition changes can also increase membrane permeability. The secondary drying directly affects the residual moisture content, thereby affecting the long-term stability.


Characteristics required for vaccine formulations

In the best case, the vaccine must be stored for a long time in a dry state and stable for at least 24 hours in a liquid state. In order to achieve this goal, vaccines must be developed with appropriate formulations and processes.

Vaccine freeze-drying: Challenges in process development and formulation

Figure 2. Priority exclusion theory.


Stabilizers (cryoprotectants or cryoprotectants) play a key role in the development of stable vaccine formulations. Amorphous cryoprotectants, such as sugar alcohols and sugars, are thermodynamically stable by preferentially eliminating the hydration of cryoprotectants and proteins during the freezing process, as shown in Figure 2 above.


They also provide dynamic stability through vitrification, thereby slowing the aggregation of protein and lipid membranes. Some cryoprotectants, such as dextran, cannot penetrate compounds, but by increasing the osmotic gradient, they can also prevent internal ice formation.

Vaccine freeze-drying: Challenges in process development and formulation

Figure 3. Water substitution theory.


As shown in Figure 3 above, the lyoprotectant plays a role in the drying phase of the freeze-drying cycle by replacing the hydrogen bonds between water and phospholipids or proteins. Like cryoprotectants, kinetic stability can be achieved through vitrification, allowing lipid membranes and proteins to flow, thereby achieving structural and conformational stability.


In order to improve the stability of the vaccine, other excipients can be added to the formulation, such as buffers, surfactants that minimize surface-induced instability, and less commonly used excipients, such as fillers, organic co-solvents, and Tonicity modifier.


Case study-development of a heat-resistant freeze-dried polio vaccine with three inactivated serotypes

By using a design of experiment (DoE) method, different formulations of polio vaccine were evaluated with multiple excipients, and the stability of the serotype was checked.

Basic screening with a limited amount of excipients did not show a stable product, so extensive screening was carried out and the stabilizer was successfully identified.

Compared with liquid formulations and other commercially available polio vaccine formulations, the optimization of the best candidate produces a final formulation with high thermal stability.


Process development

As shown in Figure 4 below, freezing has a huge impact on product characteristics, which in turn affects product stability. Slow freezing will result in the formation of a small number of large crystals, which may be harmful to the film. Quick freezing reduces the time for the release of permeate water, but creates a greater risk of internal ice formation.

Figure 4. The effect of freezing rate.


The choice between fast or slow freezing is difficult, but it is affected by vaccine sensitivity and formulation. Therefore, it is very important to consider the effect of freezing rate on stability during the development of the freeze-drying cycle.


Product temperature is very important in the entire primary drying step, it will affect the drying time, sublimation rate and stability. When optimizing the main drying parameters of vaccines, it is worth considering the cost efficiency of reducing drying time and product stability.


Removal of the hydrated shell during the secondary drying will reduce product stability. Increased residual moisture can also cause collapse, aggregation and degradation. Therefore, the optimal residual moisture content and secondary drying conditions should also be part of the development phase.


Case study-the importance of product temperature to long-term stability during primary drying

In the example of the bacterial vaccine examined, three different cycles were tested based on the product temperature (Tp), and the product characteristics were checked in terms of stability. The stability was quantified by comparing the live cell count of the live bacterial vaccine after freeze-drying.


There is no difference between being conservative immediately after freeze-drying (Tp is much lower than the collapse temperature (Tc) but higher than the glass transition temperature (Tg’)) and aggressive cycles (Tp higher than Tc).


The aggressive cycle performs poorly after a few days and one month, and the intermediate (Tp at Tc) and aggressive cycles are not as good as the conservative cycle, as shown in Figure 5 below.

Figure 5. Correlation of critical formula temperature (CFT) and freeze-drying conditions on vaccine stability.


It is recommended to use conservative conditions to start the drying cycle, but for some vaccine formulations, preliminary drying above Tc may not result in loss of stability.


Sum up:

It has been proven that freeze-drying is an ideal technology to improve the thermal stability of vaccines. The development of vaccine formulations should explore the choice of freeze-drying protectants and cryoprotectants, other stabilizing excipients, and the influence of freeze-drying schemes in the freeze-drying process to prevent any damage to the vaccine.


In development projects, the impact on process conditions and how it affects product quality attributes should be considered based on formulation and process issues, but by understanding these potential mechanisms, reasonable development can be achieved to obtain long-term stability.



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