Vaccine Freeze Drying - Challenges in Process Development and Formulation

Millions of deaths from infectious diseases per year are prevented by vaccination, but there are still millions of people who die from these diseases because of long-term stability issues, distribution difficulties, and weak thermostability demanding cold chains.

All of these challenges can be solved by producing a dry product through lyophilization. It is a vital time to discuss the process of freeze-drying vaccines because of the urgency required to develop a coronavirus vaccine.

Ms. Julia Kosan, a Ph.D. candidate from Friedrich Alexander University, Erlangen, Germany, recently held a webinar discussing the suitability of freeze-drying vaccines to heighten their thermostability. This article summarizes the webinar.

Effect of lyophilization on vaccines

There are numerous types of vaccines that trigger certain immune responses. A number of coronavirus vaccines that are currently being developed are based on recombinant vector vaccine, which utilizes coronavirus genomic material packaged into a viral vector.

Freeze-drying a vaccine supplies a significant advantage, but there are a number of challenges that must be overcome. Complex formulations, particularly vaccines which are composed of multiple strains or numerous antigens, can lead to complicated freeze-drying processes and challenging critical formulation temperatures.

Freezing and drying can stress the vaccine, whereby the extent of this process sensitivity will differ between vaccines. Internal ice formation and direct damage to a component of the vaccine, e.g., the lipid membrane, nucleic acids, or proteins, can be stress factors.

Stress Factors During Freeze Drying.

Figure 1. Stress Factors During Freeze Drying. Image Credit: SP Scientific Products

Intraviral ice crystals can form during freezing, which will heighten the volume of the product and can damage the lipid bilayer, as seen in Figure 1. The ice also produces new interfaces between ice and liquid and heightens the risk of surface-induced aggregation.

Drying above the critical formulation temperature leads to heightened mobility of the amorphous phase in the primary drying step of the lyophilization cycle. This enables protein interactions and can improve membrane permeability.

Where the hydration shell of each is removed as part of the secondary drying stage, protein aggregation and inactivation can happen. In the case of phospholipids, the alteration in the thermotropic phase transition can also heighten membrane permeability. Secondary drying influences residual moisture levels directly, which can affect long term stability.

Required characteristics of vaccine formulations

Optimally, vaccines must be stable in both dry states for long term storage and liquid state for at least 24 hours. Vaccines must be developed with appropriate formulation and processes in order to attain this.

Preferential Exclusion Theory.

Figure 2. Preferential Exclusion Theory. Image Credit: SP Scientific Products

Stabilizers (cryo- or lyo-protectants) play a key part in developing a stable vaccine formulation. The amorphous cryoprotectants, like sugar alcohols and saccharides, thermodynamically stabilize during freezing through the preferential exclusion of the cryoprotectant and hydration of the protein, as seen in Figure 2.

They also supply kinetic stabilization through vitrification, which decelerates the aggregation of proteins and lipid membranes. Some cryoprotectants, like dextran, do not penetrate the compound, but by increasing the osmotic gradient they are able to stop internal ice formation.

Water Replacement Theory.

Figure 3. Water Replacement Theory. Image Credit: SP Scientific Products

Lyoprotectants work in the drying phase of the freeze-drying cycle by replacing hydrogen bonds between water and phospholipid or protein, as seen in Figure 3. Kinetic stabilization can be achieved through vitrification, as with cryoprotectants, enabling the mobility of the lipid membrane and proteins and so, stabilization of structure and conformation.

To heighten the stabilization of the vaccine, other excipients such as buffers, surfactants that minimize surface-induced destabilization, and less frequently utilized excipients, like bulking agents, organic co-solvents, and tonicity adjustment agents may be added to the formulation.

Case study – Developing a thermostable lyophilized Polio vaccine with three inactivated serotypes

Different formulations of the polio vaccine were assessed with numerous excipients by utilizing a Design of Experiment (DoE) method and the stability of serotypes was examined.

A stable product was not shown from a basic screening with a limited amount of excipients, so an extensive screening was carried out, which was successful in identifying a stabilization agent.

Optimization with the best candidates resulted in a final formulation that attained high thermal stability when compared to the liquid formulations and other marketed formulations of the polio vaccines.

Process development

As seen in Figure 4, freezing has a huge effect on product characteristics, which influence product stability. Slow freezing results in the formation of a small number of large crystals, which can be detrimental to the membrane. Fast freezing decreases the time for osmotic water release, which generates a bigger risk of internal ice formation.

Impact of Freezing Rate.

Figure 4. Impact of Freezing Rate. Image Credit: SP Scientific Products

The choice between fast or slow freezing is difficult, but it is affected by vaccine sensitivity and formulation. So it is vital to consider the effect of the freezing rate on stability during lyophilization cycle development.

The product temperature is crucial throughout the primary drying step and affects the drying time, sublimation rate, and stability. When optimizing the primary drying parameters for vaccines, it is worth considering the cost efficiency of decreasing the drying time versus product stability.

Removal of the hydration shell during secondary drying can decrease product stability. Increased residual moisture can also lead to collapse, aggregation, and degradation. Optimal residual moisture levels and secondary drying conditions should also be part of the development phase because of this.

Case study - The importance of product temperature during primary drying for long term stability

Three different cycles were tested based on product temperature (Tp) in an example of a bacterial vaccine examined and product characteristics were examined with respect to stability. Stability was quantified by comparing the live cell count of a live bacterial vaccine after freeze-drying.

There is no difference between conservative (Tp well below the collapse temperature (Tc) but above the glass transition temperature (Tg’)) and aggressive cycles (Tp above Tc) Immediately after freeze-drying.

The aggressive cycle did not perform as well after a few days and after a month, the intermediate (Tp at Tc) and aggressive cycles were not as good as the conservative cycle, as seen in Figure 5.

Relevance of Critical Formulation Temperature (CFT) and Impact of Freeze Drying Conditions on Vaccine Stability.

Figure 5. Relevance of Critical Formulation Temperature (CFT) and Impact of Freeze Drying Conditions on Vaccine Stability. Image Credit: SP Scientific Products

It is recommended to begin the drying cycle using conservative conditions, but for some vaccine formulations, primary drying above Tc may not cause the loss of stability.

Summary

It has been demonstrated that freeze-drying is an ideal technique to heighten the thermostability of a vaccine. Vaccine formulation development should explore the effect of the choice of lyo- and cryoprotectants, other stabilizing excipients, and the freeze drying protocols during lyophilization to stop any damage to the vaccines.

During the development projects, the effect on process conditions and how it can influence product quality attributes should be considered based on formulation and process issues, but through knowledge of the underlying mechanisms of these, rational development can be realized to attain long term stability.

Acknowledgments

Produced from materials originally authored by Julia Kosan, a Ph.D. candidate from Friedrich Alexander University, Erlangen, Germany.

About SP Scientific Products

SP is a synergistic collection of well-known, well-established, and highly regarded scientific equipment brands — SP VirTis, SP FTS, SP Hotpack, SP Hull, SP Genevac, SP PennTech, and most recently SP i-Dositecno — joined to create one of the largest and most experienced companies in freeze-drying/lyophilization, complete aseptic fill-finish production lines, centrifugal evaporation and concentration, temperature control/thermal management, glassware washers and controlled environments.

SP is part of SP Industries, Inc., a leading designer, and manufacturer of state-of-the-art laboratory equipment, pharmaceutical manufacturing solutions, laboratory supplies and instruments, and specialty glassware. SP's products support research and production across diverse end-user markets including pharmaceutical, scientific research, industrial, aeronautic, semiconductor, and healthcare. In December 2015, SP Industries was acquired by Harbour Group, a private investment firm founded in 1976. Harbour Group is a privately owned, operations focused company based in St. Louis, Missouri. Headquartered in Warminster, Pennsylvania, SP has production facilities in the USA and in Spain and the UK in Europe and offers a world-wide sales and service network with full product support including training and technical assistance.


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Last updated: Dec 15, 2020 at 10:49 AM

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