Cell culture based vaccine production

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.
Many clinical trials of 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.

The cell-culture vaccine process is suitable for large-scale manufacture.

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.

Production Steps
Bulk production 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.

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.

Formulation 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
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 Media
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.
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.

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. 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 a stem cell line derived from chicken embryos by a member of
the Sigma-Aldrich Group; VERO cell line, 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.

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.

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. All that
remains is to complete the licensing process. clinical trials.

Adjuvants
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.

Not all infectious viruses can grow in culture.