May 1, 2006 (Vol. 26, No. 9)

EB14 Suspension Cell Line Adapted to Serum-Free Media

Many human and animal vaccines currently being developed are produced in eggs or primary chicken embryonic fibroblasts (CEFs). Eggs and CEFs are used to manufacture both human and animal health vaccines. These processes have changed little in more than 60 years.

There are, however, known limitations to these platforms, including the time and effort involved in the manufacturing process, the number of fertilized eggs required, the potential for allergic reactions to eggs, and the inability to grow viruses that are lethal to eggs or chickens.

Another drawback to the current egg-based manufacturing methods for influenza vaccines requires the prediction of the strains most likely to be circulating a year in advance of the typical influenza season. Unlike an egg-based vaccine process, cell culture methods for generating vaccines allow a much quicker manufacturing response time.

However, there are concerns associated with cell culture platforms that require serum. Consequently, continuous cell lines and the use of serum-free, animal-component free media show promise for vaccine manufacturers.

SAFC Biosciences ( and Vivalis ( formed a scientific partnership to develop a cell culture-based vaccine platform. Vivalis has taken advantage of its expertise in avian biology and embryonic stem cells to develop fully characterized and documented cell lines that are permissive to a number of viruses. SAFC Biosciences has drawn on its knowledge and expertise in media development to generate an offering of media that support robust growth and viral productivity of these cell lines.

EB14 Cell Line

EB14 is a suspension cell line that was derived from avian embryonic stem (ES) cells using a process that specifically preserves some of the unique features of ES cells, such as a strong constitutive expression of telomerase. EB14 has been identified as a permissive host for a number of divergent viral strains, such as but not limited to pox and influenza viruses. It is a well-characterized and documented cell line that has been immortalized without using genotoxins, viral oncongenes, or genetic engineering.

Furthermore, EB14 cells are diploid, undifferentiated, nontumorigenic, and genetically stable. This cell line has been adapted to serum-free media and can be cultured using industrial processes, including stirred tank bioreactors. All of these factors make the EB14 cell platform a versatile and regulatory-compliant alternative to eggs, chicken embryonic fibroblasts, and mammalian cell culture platforms that are employed for vaccine production.

The initial phase to develop a robust growth formulation involved adapting the EB14 cell line to grow in the absence of serum and screening a number of the companys basal formulations to determine a base medium that would support cell growth. Cell line-specific consumption data was analyzed from spent media samples for components, such as amino acids, vitamins, and metabolites.

Fig.1: EB14 cells were seeded at 0.1, 0.2, or0.4e6/ml, in bioreactors. Viable cell density was monitored for five days following seeding.

Media Development for Growth

Based on the information obtained in the screening phase, further optimization efforts examined the effects of various components for negative or positive effects on cell growth and viability. Data generated was used to determine appropriate metabolite concentrations for optimal balancing of cell growth, metabolite consumption, and waste-product secretion.

Many components identified as having a positive effect on growth were titrated to determine the optimal condition. On the other hand, those identified as having a negative effect were either limited or removed. This work resulted in a robust medium that allowed a wide range of seeding densities in a variety of scales (i.e., Erlenmeyer 125-mL flasks up to 20-L stirred tank bioreactors).

As shown in Figure 1, EB14 cells were seeded at three different densities in 3-L bioreactors and analyzed for growth for five days. Not surprisingly, Bioreactor 1, seeded at 0.4e6 cells/mL, reached the highest cell density (6e6/mL) followed closely by Bioreactor 2, which was seeded at 0.2e6/mL (peak cell density 5.5e6/mL). Maximum cell density was reached in both reactors at three days post-seeding. Bioreactor 3, which was seeded at 0.1e6 cells/mL exhibited a delay in peak cell density, but by day five, over 3e6 cells/mL were obtained.

This flexibility in seeding density increases the attractiveness of the EB14 cell line as a platform for vaccine manufacturing processes. Potential users of this cell line are not locked into a specific seeding density around which they must adjust their protocol. Rather the seeding density may be adjusted to fit the preferred procedure.

Fig 2: EB14 cells wer seeded at 0.2e6/ml in growth medium.

Media Development for Viral Production

The base medium then was tested for viral production both at the flask and bioreactor level. Although the medium optimized in the experiments described above supported robust cell growth, we felt there was room for improvement in the production of infectious virus particles.

As factors that improve growth are not necessarily the same factors that will improve viral production, investigations into a two-part media system were initiated; one formulation would be used for cell growth and a different formulation would be used for viral propagation.

Additional optimization efforts concentrated on identifying key components that inhibit or increase viral production. Secondly, process issues were investigated, such as cell density at the time of infection, the timing and ratio of the production medium, and the addition of different feeds at the time of infection.

As many vaccines currently being produced in chicken eggs and CEFs are pox-based viruses, modified vaccinia ankara (MVA) was used as one of the model systems to investigate viral production. We found that the cell density at the time of infection did not appear to be as important in determining viral yields as the overall health of the cells (i.e., good growth of the cells following infection correlated with good viral titers).

Consistent with this, there was not a significant effect on the viral titers based on the time of the addition of the production media. Most importantly, as shown in Figure 2, the addition of feeds at the time of infection greatly increased the viral titers. The base formulations are the growth and production media in the absence of feeds, which result in a peak MVA viral titer of 7.2logTCID50/mL.

Feed 1 increased the viral titer by approximately one log, but more importantly, the addition of Feed 2 increased the viral titer by a little more than two logs, resulting in a peak viral titer greater than 9logTCID50/mL. This level of viral titer is more than competitive with the methods currently employed by vaccine manufactures. Furthermore, high viral titers can also be reached with other viruses, such as A and B strains of human influenza (Figure 3).

Fig.3: EB14 cells were seeded at 0.4e6/ml in growth medium.


The EB14 avian ES cell line represents a new cell-based platform for vaccine manufacturing. In combination with regulatory compliant, serum-free media, and feed formulations, the cell line is a powerful choice for vaccine manufacturers. The platform provides robust cell growth and production of high titers of infectious virus particles. The platform also provides the manufacturer with flexibility in terms of seeding density, density at the time of infection, and harvest of virus.

In the case of a pandemic, it will be critical to increase the vaccine production for what is likely to be a new strain of a virus. A system such as this could significantly decrease the amount of time it would take to manufacture a new vaccine strain.

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