The transition from vaccine research and development to manufacturing requires

a good manufacturing practice (GMP)−qualified cell line growing in a GMP-

compliant medium under GMP conditions. The cell line should be fully char-

acterized and free of adventitious agents (mycoplasms, prions, endogenous retro-

viruses) and should not be tumorigenic/cancerogenic. Furthermore, cell substrates

need to be accepted by the EMA (European Medicines Agency) or FDA (Food and

Drug Administration) [10,11]. Therefore, cell lines are often used that have already

been approved for other vaccines and have otherwise proven to be safe. This also

greatly enhances the possibility to be “first on the market” or to establish a vaccine

manufacturing in a country for self-supply. Together with the safety aspects dis-

cussed previously, this also explains the limited availability of new cell substrates

and obtaining a license covering manufacturing for such fully characterized new

cell lines is equally very expensive. Nevertheless, a license for a certain cell line

that covers the use for manufacturing is essential unless a company’s own cell line

has been developed. More details are found in Chapter 5.9.

5.4.2

CELL BANKING

One crucial step in the design of a vaccine manufacturing process is cell banking to

ensure the cell line availability. Preferably, cell banking is done reproducible and

under GMP conditions. Cryo tubes of cells are kept in liquid nitrogen for long-term

storage. Freezing and thawing of cells are very critical steps to guarantee high cell

survival and rapid growth. Therefore, appropriate protocols should be followed or

developed. First, a master cell bank (MCB) is created consisting of several cryo

tubes with cells of a low passage number. Based on the MCB, a working cell bank

(WCB) is prepared mostly with up to four more passages. The cells from the WCB

are then used for scale up with the final number of passages defined after com-

prehensive testing. Typically, cells are frozen in cryo tubes at 1.5 mL. By using

disposable cryobags to freeze the cells in a higher volume (and higher cell con-

centration) a high cell count for direct inoculation of bioreactors is possible and

with that the time to production scale can be reduced to allow for more efficient and

flexible manufacturing (see next chapter). Comprehensive characterization of the

cell banks is required for sterility (bacteria, viruses), absence of mycoplasm and

retroviruses, cell identity, and genetic stability [10,11]. Furthermore, cell banks

should be large enough to provide consistent access to cells for production over

many years. For establishing the cell banks, cell freezing is conducted during ex-

ponential growth phase of the respective cells. To avoid the formation of ice

crystals and osmotic stress, dimethyl sulfoxide (DMSO) (about 6−10% (v/v)) is

added as cryoprotectant to the cell suspension. Typically, adherent cells are frozen

at 1E06 cells/cryo tube and suspension cells at 1E07 cells/cryo tube. Freezing is

conducted either stepwise (−20°C, −80°C) or gradually (−1°C/min, −80°C) by

using a freezing container or dedicated equipment before aliquots are placed into

the gas or liquid phase of nitrogen. For cell thawing, a cryo tube is thawed until only

a small ice pellet is visible and the cell suspension is carefully transferred into a T-

flask or spin tube/shake flask. A medium exchange after thawing is highly re-

commended to outdilute any cell toxic DMSO. Before release of a MCB and WCB,

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Bioprocessing of Viral Vaccines