virus production phase (2−4 days) (Figure 5.6). First, cells are seeded in the cul-

tivation vessel (<1E06 cells/mL) and optimal process parameters are set (tem-

perature, pH, pO2, rpm). In a batch process, cells grow up to a certain cell

concentration in the range of 1E06−1E07 cells/mL prior to infection. Taken into

account that virus production is inhibited by the limitation of substrates and by

accumulating by-products, a dilution or medium-exchange before time of infection

(TOI) is beneficial for some processes.

Typically, infection takes place at the end of the exponential growth phase (e.g.,

for lytic viruses such as IAV). However, for slow replicating lytic viruses (e.g.,

YFV, MVA), an infection in the middle of the exponential growth phase should be

considered, as cells continue to grow and the additional increasing cell con-

centrations might result in higher virus yields. For some viruses, like mink enteritis

virus (MEV), the infection is even carried out directly after cell seeding as MEV

replication only takes place in mitotic cells [77].

After infection, the viruses utilize the host cells to replicate their genome and

synthesize viral proteins. The assembly and release of progeny virions completes a

replication cycle. Infectious viruses released then infect uninfected cells until pre-

ferably the whole cell population is infected. Virus particles accumulate in the

vessel and highest virus titers are reached, depending on the replication time of the

TOI

Cell conc. (cells/mL)

Viability (%)

Total virus titer (units/mL)

Infect. virus titer (units/mL)

MOI

Cells infected (%)

0.0001

0.001

0.01

0.1

1

10

Time (d)

(a)

(b)

(c)

FIGURE 5.6 Typical time courses for a virus

production process. Typically, this process is a two

phase process with first cell growth (3−5 days) and

second virus replication phase (2−4 days) (here

indicated by the vertical dashed line at time of

infection (TOI)). (a): cell concentration and viabi-

lity, (b): total and infectious virus titer, (c): multi-

plicity of infection (MOI) and percentage of

infected cells. As a reference, cells are often

infected at 2E06 cells/mL with a MOI below

0.01. Due to the release of progeny virions, the

MOI in the vessel increases (>10) and the cyto-

pathic effect reduces cell concentration and viabi-

lity. Conditions should be selected to allow

productive infection of all cells to achieve the

maximum virus yield. Ratios between infectious

and non-infectious virus particles can be 1:10 and

lower than 1:10,000, depending on the virus strain.

Non-infectious viruses can be defective and inter-

fering (defective interfering particles, DIPs). These

DIPs need co-infections with standard virus parti-

cles for their replication; a high MOI scenario

within the vessel increases DIP production and

may reduce the infectious virus titer and the total

virus titer [ 76].

Upstream processing for viral vaccines

111