The next generation of influenza vaccines will be cell-culture-based for seasonal influenza and for pandemics.
Leading manufacturers of vaccines and antiviral drugs are working hard to develop new and novel methods of preparing seasonal influenza vaccines, as well as pandemic vaccine candidates. Until recently, most efforts have been focused on improving currently licensed egg-based vaccines. Manufacturers have been racing to increase production capacities and automate portions of largely manual steps in egg-based vaccine technology to meet the demands of the next seasonal campaign and to generate prototypes of pandemic vaccines for clinical trials. At least 30 clinical trials of avian pandemic prototype vaccines are in progress, and manufacturers are working with international agencies, such as the World Health Organization, European Medicines Agency, and the National Institutes of Health, on the development, licensing, and production of pandemic vaccines on a global scale (IFPMA, 2006).
Currently, about 25 of these projects are based on classical egg-based technology, and six are based on cell-culture systems (Chiron has one cell-culture vaccine in the final stages of development and approval by regulatory agencies). Cell-culture technologies may offer distinct advantages over egg-based manufacturing methods.
- They eliminate the need for embryonated chicken eggs from managed, biosecure flocks.
- They combine and automate upstream and downstream processes.
- They reduce the potential for contamination by viable and nonviable particulates.
- They eliminate the four- to six-month lead times for the organization of egg supplies.
- They have faster, high-volume start-up times for production.
- They have higher initial purity.
- They could supplement seasonal vaccine supplies when multiple strain changes are necessary.
- They would substantially increase global stockpiles of pandemic influenza vaccines.
The cell-culture vaccine
process is suitable for
Cell-Culture-Based Vaccine Production
Cell-culture-based technology is robust and reliable and could become a practical alternative for the pharmaceutical industry in vaccine production. Once the virus is propagated and harvested, the downstream processing parameters for purification, filling, and packaging of the vaccine are similar to current pharmaceutical methodologies and egg-based methodologies. However, there are no lead times involved, because typical cell-culture processes use cell lines; once a cell line is infected with the seed virus in a fermenter, the process can begin. The critical step is the availability of the seed virus. The substrates or media for cell-line propagation are not susceptible to virulent virus strains as embryonated chicken eggs are.
The cell-culture vaccine process is suitable for large-scale manufacture, and the process parameters can be ramped up and run routinely and cost effectively. The typical cell-culture production process can be run in batch sizes of practical scale, sufficient to provide vaccine quantities for interpandemic periods and pandemics. However, to date, no vaccines have been licensed using this technology.
Chiron has already submitted “mock-up” dossiers to European Union (EU) regulatory authorities for review and approval of an avian influenza vaccine (currently in clinical trials) and a license application for a cell-culture-based vaccine. This paper is focused on the cell-culture vaccine manufacturing process used by Chiron, as well as the use of adjuvants to enhance immunogenicity and reduce dosage size.
Biochemical Challenges of the Haemagglutinin (HA) Protein
Besides the engineering issues associated with establishing a new vaccine manufacturing process, biochemical challenges unique to the influenza haemagglutinin (HA) protein must be met for manufacture by any process. The three-dimensional HA protein structure is both stable and functional. However, if during purification steps, the pH drops to less than 5.5, which is a common approach to improving purification recovery processes, the conformation of the HA protein changes. The resulting conformations provide the same micro-equivalence of protein, but the folding of the HA protein no longer provides antigenic properties for the vaccine. Because this change is irreversible, the entire lot must be discarded. Clearly, the biochemical properties of the HA molecule must be maintained.
begins with the cultivation of the virus in a fermenter equipped with numerous process parameters to control temperature, pH, dissolved oxygen, and other factors. Two methods of mass cultivation of cells are recognized in the industry today, microcarrier cultures
and free-cell suspension cultures
. Both systems begin cultivation of the cell line in a fermenter, which can be scaled up to thousands of liters.
In microcarrier systems
, the cells are first anchored to microbeads. In the presence of nutrient media, the cells grow and proliferate covering the beads uniformly. Microbeads provide a high surface-area-to-volume ratio, which can lead to high cell densities. Once a bead is covered, the cells are dislodged, dispersed, and allowed to reattach to achieve another round of cell growth on the surface of the bead.
Larger volumes of media are required to achieve the same results with free-cell suspension
because the cell line proliferates while growing freely suspended in the nutrient medium. However, the scaling up of the system is easier, and there is no limit to the volume.
is the process of mixing the product setting dose requirements, concentrations, and final volumes, and establishing secondary process parameters, such as materials, vials, syringes, caps, plugs, incubators, and so on. Filling
, often referred to as sterile filling, is part of formulation. Filling involves bringing together sterile vials or syringes with sterile filtered vaccine solution. The syringe or vial is filled in a controlled environment to ensure that the final product is sterile. Packaging
is also an important step for ensuring the integrity of the product for the duration of its shelf life.
Cell Line Selection
The cell line used to cultivate the virus must be able to propagate the virus in large quantities, must be rapid and efficient in expressing the desired virus, and must be suitable for a wide variety of flu strains. It is desirable that the cell line be able to grow in a chemically defined synthetic medium that does not contain animal-derived components. It should also be scalable for industrial processes. Equally important, if the cell line has not been previously approved by regulatory agencies, the requirements for licensing should be known and validated.
Serum-based media have some disadvantages. First, typical acceptance criteria for serum can vary as much as ?20 percent, which could contribute to batch-to-batch variations during fermentation. Second, contamination of serum with adventitious agents is always possible. Third, if the target protein is functionally, biochemically, or physically related to a serum protein, it can be difficult to separate the target protein from the serum protein during purification. Finally, a serum-based medium is not always available, especially for large-scale cell-culture use.
Synthetic, serum-free media have some important advantages. First they are much better defined than serum-based media. Second, the potential source of infectious agents has been removed. Third, there is much less lot-to-lot variability than for serum-based media. Fourth, the purification of the desired protein is easier, requires fewer steps, and costs less. Fifth, serum-free media contain readily available components that are usually non-animal derived and have relatively easy storage requirements. Finally, shortages are unlikely.
Removal of Residual DNA
A critical step in selecting a cell line for cell-culture vaccine production is the removal of residual DNA from the final product. Regulatory agencies provide guidance for specific data for continuous cell lines (as they do for new cell lines). Continuous cell lines must have a well documented “clean” history with no tolerance for adventitious agents or other contaminants. The removal and/or inactivation of DNA must be much more thorough than for therapeutics. Testing paradigms have been defined to assess potential risk and to ensure safe use by the public.
There are three stringent regulatory requirements for validating a new cell line for use as a substrate in cell-culture formulations. First, there must be documentation to support the complete removal of the cells from the final product and documentation to show that the cell line does not bring any transforming agent (oncogenic transformation) into the final product (Figure 1a - see PDF version
for figures). Second, documentation must show that no genetic material is left from the cell line in the final product that can cause tumors to be formed. All residual DNA must be removed or inactivated so it cannot give rise to tumors in animal models (Figure 1b - see PDF version
Finally, documentation must show the removal and/or inactivation of infectious and/or oncogenic agents from the final product, regardless of whether they originated in the media or the cell line (Figure 1c - see PDF version
). This requirement was developed in response to contamination of dura mater grafts (e.g., the outermost layer of the meninges surrounding the brain and spinal cord) in Creutzfeldt-Jakob disease (mad cow disease). These stringent requirements are intended to protect the public from changes in our immune systems caused by foreign DNA.
A complete characterization of the cell line is required to meet licensing requirements in any country, and selecting the most appropriate approach for a cell-culture vaccine process must be based on growth rates, yields, and regulatory obstacles. Current cell lines being used to express the influenza virus are: PerC.6?, a proprietary formulation by Crucell; EBx™, a stem cell line derived from chicken embryos by a member of the Sigma-Aldrich Group; VERO, a kidney cell from the African green monkey; and Madin-Darby canine kidney (MDCK) cells.
MDCK cells are known to produce large quantities of virus and require easy downstream purification. Although this cell line has not yet been approved by regulatory agencies, it would be a considerable biochemical-engineering accomplishment if an influenza vaccine candidate used cell-culture manufacturing that includes MDCK cells growing in suspension in a synthetic medium. VERO is currently licensed with regulatory agencies but does not express large quantities of virus.
Manufacturing and Formulation
Assuming that a cell line can propagate the virus and that regulatory agencies will approve it, the selection criterion then becomes whether the cell line can be industrialized. Can it be grown in a fermenter, and should a free-cell suspension culture or microcarrier culture be used? There are engineering challenges associated with both methods (Table 1).
Regardless of the cell-cultivation method, the cell line must be grown in a nutrient medium. A medium is a solution of either synthetic (serum-free) nutrient components or a complex substance of animal-derived protein or serum. There is less risk associated with synthetic media, provided they promote the growth of the cell line. The use of serum-free synthetic media has increased significantly, particularly when using serum presents a safety hazard and a potential source of unwanted contamination.
During the formulation and manufacturing stages of the cell-culture process, the decision of whether to make a whole-virus vaccine, a split vaccine, or a subunit vaccine must be finalized, qualified, and validated. Today, most egg-based vaccines are split or subunit vaccines. A vaccine for pandemic influenza is most likely to be a whole-virus vaccine.
Preparation of a cell line for propagation begins with the thawing of the cell line “seed” lot (e.g., PerC.6?, EBx™, VERO, or MDCK). (In contrast, it can take up to six months to organize the egg supply for initial inoculation.) “First-pass” cell line propagation begins with the small-scale pre-culture propagation of seed cells after thawing. The cells are then introduced to the fermenter vessel with the selected nutrient medium. When the cell line reaches a predetermined cell density, the virus is introduced and begins to propagate in the cell line; after approximately three days the virus is harvested. After treatment of the infected cell line, the virus is released into the supernatant, and the cellular debris is centrifuged away. This occurs in a clean, closed environment, whereas harvesting of an egg-based virus is largely a manual process that requires extracting infected cells, breaking down cell walls, and then collecting the virus.
After inactivation, the whole virus can be purified, split, and ultrapurified as a “subunit.” Initial chromatography with ultrafiltration is often followed by treatment with beta-propiolactone, which deactivates the virus; final splitting of the virus is followed by ultracentrifugation. This ultrapurification technology is basically similar to the egg-based vaccine ultrapurification process, and the resulting purified subunit vaccine is identical in composition to egg-based vaccine.
At this point, the development phase of an influenza cell-culture vaccine is complete (Figure 2 - see PDF version
). All that remains is to complete the licensing process. Phase III clinical trials in Europe have shown equivalent safety and immunogenicity of cell-culture influenza vaccine strains and established egg-based vaccines. The protective line is well above the minimum limit for both formulations. Phase I and II studies were completed in the United States in 2005.
Adjuvants are substances added to vaccines to improve antibody production and the immune response of the recipient or to decrease the amount of antigen (dose size) required in the vaccine. The latter is the most effective way to increase global vaccine manufacturing capacity. So far, the only two adjuvants that have met regulatory standards for safety are aluminum compounds (which have been used safely for many years) and M59 (licensed in most European countries). More than 25 million doses with M59 have been administered since commercial operations began.
Chiron selected MF59 adjuvant because in mouse model experiments, older mice challenged with an influenza virus after they had been vaccinated with the HA protein plus MF59 had a better immune response than younger mice vaccinated with the HA protein without the adjuvant. In addition, clinical data on the use of MF59 adjuvant with H9N2 in a new trial in 2004 confirms the potency of MF59 against avian influenza (Figure 3 - see PDF version
). The most recent work on M59 in influenza vaccines shows that persistent antibodies against different influenza virus strains are boosted by a third immunization. Thus, MF59 adjuvant also offers protection from antigenic drift in influenza strains. This cross protection is only afforded when the adjuvant is present (Figure 4 - see PDF version
The efficacy of cell-culture-based influenza vaccine production has been demonstrated to have many advantages over egg-based vaccine production and should be licensed by regulatory agencies in the near future. Adjuvants have been shown to be not only effective, but also to provide a method of increasing global vaccine manufacturing capacities through antigen sparing.
I believe we could initiate immunization campaigns without waiting for the latest virus variant to be identified and before a pandemic occurs. Cell-culture vaccines can be manufactured over a longer period of time, with the assurance that they can be administered months before a pandemic occurs and remain effective in creating an effective immune response.
IFPMA (International Federation of Pharmaceutical Manufacturers and Associations). 2006. IFPMA Clinical Trials Portal. Available online at: http://clinicaltrials-dev.ifpma.org/
TABLE 1 Biomedical Engineering Challenges for Cell Cultivation Methods Microcarriers Free Cell Suspension
|Microcarriers||Free Cell Suspension|
|Cells are anchorage dependent and grow on solid or macroporous microcarriers. Cell expansion often occurs in roller-flasks.||Cells are suspended and grow freely.|
Culture is homogenous and sampling is easy. Process is simple, scalable, robust. No additional factors or proteins are necessary for adhesion or spreading. Costs are lower.
|Major Challenges||Major Challenges|
- Shear stress and mixing adaptation of stirrer speed for attachment.
- Lengthen cell-culture lead time.
- Continuous perfusion of fresh culture media.
- Microcarrier preparation (swelling and autoclaving).
- Trypsin for cell-passaging.