The challenge of viral vaccines

Successful production of a viral vaccine means managing a range of challenges. The complexity of the molecule structure, the need to scale production to meet market demand, and the high requirements for purity and speed all play into the end result.

Viral vaccines need to be produced fast, in large quantities. Routine immunizations and preventive campaigns, like stockpiling in response to emergencies, involve millions of doses, and speed is essential. Shortening the time to market is beneficial for both the manufacturer and the patient.

Working with flavivirus

The flavivirus molecular structure is complex, incorporating nucleic acids, lipids, carbohydrates and more than one molecule of protein. The structure itself is delicate, and can be sensitive to commonly-used process conditions. On top of these considerations, there is the need to maintain high purity levels and keep the virus particle intact. This is required to optimize the production process and keep up the high production volumes required.

Flavivirus vaccines can be based on live, attenuated or inactivated virus (whole virus vaccines), recombinant virus subunits, virus-like particles (VLP) or plasmid DNA, and viral vectors.

Whole virus vaccines use a modified virus to trigger an immune response. Vaccines based on a viral subunit can be a viable alternative when viruses can’t easily be propagated in cell culture.

Design of experiment (DoE) is an important part of the work. The approach helps determine which components in the media will have the most beneficial effect on cell growth. Surface Plasmon Resonance (SPR) analysis during process development provides data about virus behavior and host interactions. This is valuable both for regulatory compliance and future drug development.

Cell-based versus egg-based

Egg-based vaccine production can yield 100 to 300 doses per fertilized hen egg. But the eggs must come from pathogen-free chickens, which limits availability and makes scaling difficult. Another drawback is that patients with severe egg allergies might have a reaction to the finished vaccine. An alternative is cell-based production, which allows more flexibility and scalability.

Cells for vaccine production are traditionally cultured in T-flasks or roller bottles. Although the standard has moved towards stainless steel bioreactors. These improve reproducibility and take up less space, but still require extensive cleaning and preparation time before and after use.

Enhancing capabilities with single use

Single-use equipment represents a next-step from stainless steel in viral vaccine production. It cuts down on operator time and effort by eliminating cleaning and preparation stages. This is possible as all surfaces in contact with the process material are disposed of after use. By the same token, there’s less risk of contamination as no open handling of the material is required. The process is more flexible, as start-up and changeover can be affected quickly. And there’s less cleanroom space needed, allowing a smaller production footprint.

Where stirred-tank bioreactors are used, moving to single-use versions is a simple transition. The scale-up and scale-down principles are the same as in stainless steel equipment.

Managing adherent cells

Cell-based virus production often uses anchorage-dependent cells, such as Vero cells, which require a surface to adhere to. Using a bioreactor with dextran bead microcarriers meets this need better than traditional shake flask and roller bottle systems, as it creates a greater surface-area-to-volume ratio. This allows higher titers with a smaller physical footprint. The dextran bead structures are translucent, making manual inspection straightforward.

Adherent cells are also sensitive to shear stress. This introduces a challenge for process designers looking to maintain molecular integrity while creating enough movement to aerate the culture. Rocking bioreactors, such as GE’s WAVE range, reduce shear by agitating gently with no physical stirring of the culture. They can give a representative reflection of the process performed in a stirred-tank bioreactor.

Downstream considerations

High upstream titers create greater demand on downstream processes and equipment. Fortunately, there are a range of modern technologies and solutions available to mitigate these pressures. Ultracentrifugation and precipitation can be substituted with chromatography, which is both highly selective and scalable.

Modern chromatography resins are designed to offer improved pressure-flow properties and increased productivity compared to their legacy predecessors. As a result, they are more suitable for manufacturing applications that require larger volumes to be produced in a short time.

Cation exchange, anion exchange, and affinity chromatography resins are all used in virus vaccine purification. Multimodal resins, which operate in more than one mode, can be used for more challenging separations. A novel class of multi-modal resin uses dual layers. Larger molecules are excluded from its surface, while a ligand-activated core captures smaller particles.

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Our Fast Trak team developed a process for production of inactive whole-virus yellow fever vaccine.

  • Aim: To improve productivity and scalability and meet the concerns with live, attenuated vaccines
  • Cells: adherent Vero cells
  • Medium: Cytodex 1 microcarriers in animal-derived component-free medium supplemented with recombinant human albumin
  • Bioreactor: Xcellerex XDR-50 bioreactor system. Its impeller design and adjustable agitation speed range address the requirements of the shear-sensitive culture.

Downstream purification was initially based on sulphate Cellufine™ affinity chromatography, with a sucrose gradient ultracentrifugation step added to increase purity. However, the ultracentrifugation step was costly and cumbersome, and required equipment that would not be available at some manufacturing facilities.

We replaced the sulphate Cellufine and the ultracentrifugation steps with two simple chromatography steps. Capto DeVirS was used for the initial viral capture step, and Capto Core 700 for the final polishing step. The optimized process could be conducted at room temperature, whereas the legacy process was performed at 2°C to 8°C.

Host cell protein (HCP) was significantly reduced at a similar or slightly higher recovery in half the process time. Manufacturability was significantly improved.

The new process is more convenient to carry out and is easily scaled. It’s compatible with both single-use and conventional technologies and meets stringent regulatory requirements.