this already in upstream processing and process development is not always easy.

Therefore, process optimization focuses on maximum yield. Nevertheless, as early as

possible produced material should be evaluated in its efficacy. The selection of the

optimum process conditions (e.g., temperature, pH, pO2, rpm) is key (see Table 5.8).

The pH typically ranges between 7.0−7.6, as a more acidic pH often results in virus

inactivation or degradation. Optimal temperature for cell growth and virus production

is mostly found at the physiological temperature of 37°C. However, a lower tem-

perature (32−34°C) in the virus production phase is sometimes beneficial. Here, cell

growth and virus propagation is inhibited, hence, maximum cell concentrations

and virus titers peak delayed. Nevertheless, lower temperature might boost the

maximum virus yield and prevent temperature-induced inactivation. Moreover, cold-

adapted virus strains (attenuated virus strains for live vaccine manufacturing) also

require a cultivation temperature lower than body temperature for efficient propa-

gation in cell culture.

To optimize infection conditions, different TOIs and MOIs should be tested.

Usually, virus infection takes place at low MOI in the range of 1E-02 to 1E-05, at

which high virus yields are obtained and the risk of DIP propagation by unwanted

co-infections with standard virus is low (see below). However, this optimum must

be identified for every cell/virus combination and production process. At low MOI,

it takes longer before maximum titers are reached. This poses the risk of early

media depletion, which could have a negative impact on further virus propagation.

Thus, special care for sufficient nutrient supply has to be taken. In contrast, in-

fection at high MOI (>1E-01) is not cost-efficient, as a high volume of seed virus is

required. Moreover, early virus-induced cell death might occur due to the high virus

input, further limiting yields. Moreover, infection at very high MOI leads for almost

all viruses to an accumulation of defective particles, that might even interfere with

virus replication (defective interfering particles: DIPs). Overall, defective particles

and, in particular, DIPs can significantly reduce yields or lead to non-optimal seed

virus. Due to an internal deletion in their genome, DIPs cannot replicate on their

own. They need a co-infection with standard virus (STV), where they rely on the

complementing genome of the STV for their replication. But DIPs replicate faster

and interfere with STV replication and, thus, may limit the overall virus yield.

(However, DIPs might work as adjuvant in the final vaccine product by stimulating

the immune response after vaccination.) One approach to reduce the level of DIPs

in the virus seed is a serial passaging at low MOI [79].

In general, different attributes are determined by the host cell line, virus, and the

process (see Table 5.9). Cell growth and virus production is heavily dependent on

nutrients. During the cell growth phase, the main substrates glucose and glutamine

are consumed for cell growth. The metabolization of these substrates leads to the

accumulation of the metabolic by-products lactate, ammonia, and glutamate

(Figure 5.3). At TOI a medium exchange is favored (fully or at least 50%) to

improve substrate supply and dilute by-products released into the supernatant

(ammonia is toxic at higher concentrations, lactate lowers the extracellular pH).

Overall, cell growth and virus production continues until substrates are depleted,

by-products reach inhibiting levels or cell death occurs by virus-induced apoptosis

and lysis. During cultivation, a metabolic shift may occur, after which lactate is no

Upstream processing for viral vaccines

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