Vaccination remains one of the most effective methods to combat infectious viral diseases such as influenza. The recent COVID-19 crisis has placed unprecedented pressure on the vaccine industry to develop and deploy vaccines against emergent diseases faster than ever before.

An eBook, Solutions for Vaccine Development, released by Eppendorf has been created to provide the reader with an in-depth understanding of how the COVID-19 pandemic has irreversibly altered the landscape of global vaccine development. The content includes application notes, product guides, and expert insights to accelerate your vaccine research and development.

1. Learnings from a pandemic – Efficient vaccine development using parallel control and powerful software solutions

The ebook starts with a Q&A about vaccine development in light of the COVID-19 pandemic. We learn about the typical development process for vaccines, how new technologies such the mRNA-based platforms used for the COVID-19 vaccine have opened new production possibilities, and positive influencers for the future including the development of more efficient downstream processing techniques to keep pace with the upstream development work.

The speed to market, safety and flexibility play important roles in vaccine development. Eppendorf’s expertise in upstream bioprocessing, , paired with a range of devices and single-use technologies can expedite process scale-up from R&D labs to pilot and manufacturing facilities to accelerate vaccine development timelines.

2. Eppendorf Protein LoBind® Tubes – Your Excellent Choice for Handling and Storage of Viral Samples (in Vaccine Production Workflows)

In downstream processing of virus particles produced for vaccine production and gene therapies, the goal is to achieve high-purity virus preparations. A variety of protocols are used across the industry including filtration, ion-exchange chromatography, and gradient centrifugation. All these techniques rely on high quality plastic labware for handling and storage of viral samples. However, non-specific adsorption of virus particles to plastic labware can result in undesirable product loss. The use of Eppendorf Protein LoBind® tubes can alleviate this problem.

In a peer-review publication, the Protein LoBind tubes were tested alongside eight others from various manufacturers in an ion-exchange virus purification protocol. The author found that the virus particle concentration in the LoBind tubes remained stable during extended storage in contrast to the other tubes tested, which showed a significant decline in concentration. The data clearly demonstrates that the LoBind tubes did not bind viral particles and is recommended for collection and storage of viral samples.

3. Vero Cell-based Vaccine Production: Cell lines, Media and Bioreactor Options

In addition to the novel RNA-based vaccine strategy to develop COVID-19 vaccines, traditional vaccine platforms also saw an increase in demand, with many developers using tried and true cell culture methods, including Vero cells, to produce COVID-19 virus fragments and/or spike protein for vaccine development purpose. The Vero cell line has been used for years in various virus vaccine development, and its performance in quality and quantity of viral yield is well-established. Vero cells are widely recognized in the vaccine industry as a safe and efficient production tool and the need for this host production platform may continue to grow.

In this white paper, Eppendorf expert Ma Sha reviews several key concepts including:

• The history of vaccine development to combat infectious diseases
• The shift away from egg-based vaccine production towards mammalian cell culture systems
• Successful optimization strategies for Vero cell-based vaccine production using rabies and influenza as examples featuring Eppendorf products including the Fibra-Cel® disks, which provide support to enable high-density expansion of anchorage-dependent Vero cells in large-scale bioreactors

The continued interest has driven the scientific community towards growing Vero cells in suspension bioreactor cultures and serum-free cell culture media formulations to further expand the capability of the Vero cells in the vaccine development and production market. Additionally, improvements in bioreactor design combined with these various technological advances result in a greatly improved and more functional production train.

Eppendorf’s technical expertise and specialized equipment, ranging from microcarrier spin filters, cell-lift impellers, packed-bed bioreactors, and single-use solutions, offers clients a wide range of technologies well-suited for vaccine research and manufacturing using mammalian cell culture systems like Vero. These technologies are highlighted in several applications notes also included in this eBook, which are briefly summaried below.

4. HEK293 Suspension Cell Culture Using the BioFlo® 320 Bioprocess Controller with BioBLU® 3c Single-Use Bioreactors

The Eppendorf Bioprocess Applications Lab has developed numerous bioreactor application notes for large-scale suspension cultures of mammalian cells for vaccine and gene therapy development. This application note features human embryonic kidney 293 (HEK293) cells, which is a widely used biotherapeutic platform because they are easy to transfect and can produce large quantities of recombinant proteins and virus particles. To achieve high-density cell growth, suspension-adapted HEK293 cell lines such as ThermoFisher’s Expi293F™ are often utilized in bioproduction workflows.

In the study presented in this article, the Expi293F is evaluated in batch culture using a BioFlo® 320 bioprocess control system and a BioBLU® 3c Single-Use Bioreactor. The cultures were monitored and analyzed for cell growth, viability, and metabolic activity (levels of glucose, ammonia and lactate in the medium) during the culture period. The efficient and straightforward configuration of the BioFlo 320 allows precise control of the cell culture environment throughout the culture period to enable reliable cell expansion. Rapid growth of Expi293F in suspension culture was observed at levels like Chinese hamster ovary (CHO) cells, the accepted industry standard.

The results from the study demonstrate that the Expi293F cell line has great potential in both vaccine and gene therapy method development when used in conjunction with Eppendorf’s advanced stirred-tank bioreactors.

5. Easy Perfusion for Anchorage-dependent Cell Culture using an Eppendorf Vessel equipped with Microcarrier Spin Filter

The demand for vaccine products for viral diseases has necessitated the development of more sophisticated production techniques based on bioreactor cell culture systems, such as perfusion or continuous cell culture. Perfusion cell culture utilizes a cell retention device and continuous media exchange to achieve and maintain high cell densities and viabilities over extended culture periods compared to conventional batch or fed-batch processes. The cell retention device keeps cells inside the bioreactor in perfusion mode, allowing fresh media to be added to replenish nutrients and remove toxic byproducts.

In this application note, the suitability of a microcarrier spin filter as cell retention device was tested with Vero cells adhered to Cytodex® 3 microcarrier beads. The microcarrier spin filter—which is coupled to Eppendorf’s low-shear Cell-Lift Impeller shaft in the bioreactor—is a cylinder-shaped cage covered with a large 75µm screen designed to prevent microcarriers from being collected with the waste media during perfusion. The process was controlled with a BioFlo® 320 system with cell density, viability and metabolite analysis conducted throughout the culture period.

At a modest microcarrier loading density of 10 g/L, Vero cell density of 8.0 × 10⁶ cells/mL was reached in 9 days, which is sufficient to innoculate a 40 L CelliGen® 510 packed-bed bioreactor designed for vaccine production, assuming > 50% cell recovery. The data demonstrates that the BioFlo 320 vessel equipped with spin filter for microcarrier is an excellent platform for high density Vero cell culture with great potential for attachment cell-based vaccine production scale-up.

6. High-Density Vero Cell Perfusion Culture in BioBLU® 5p Single-Use Vessels

In stirred-tank bioreactors, anchorage-dependent Vero cells are typically grown on microcarriers to achieve high cell densities, which translates to high virus yield. Fibra-Cel® disks are a promising alternative attachment matrix with a high surface-to-volume ratio capable of providing a three-dimensional low-shear environment that protects cells and helps to achieve high cell densities. Eppendorf’s BioBLU 5p Single-Use Vessels are pre-packed with Fibra-Cel disks. Its performance for Vero cell culture in perfusion mode is evaluated in the final application note of the eBook.

After 21 days of culture, a very high Vero cell density of approximately 4.31 x10⁷ cells/mL was achieved in the 3.5 L vessel. Since viral particles are much smaller than cells, they can be directly harvested from packed-bed cell culture vessels without removal of cells.

To download a copy of the eBook in full, please visit: Solutions for Vaccine Development