TABLE 5.8

Parameters for optimization of cell culture-based virus production processes

Parameter

Examples: Stirred Tank, Batch

Comment

Choice of cell line,

engineering cell line

Adherent (continuous, primary)/

suspension; e.g., improve

metabolism or reduce apoptosis

Adherent Vero cells often allow to be

fast on the market; e.g., AGE1.pIX

cells designed for better MVA

replication

pH value

Optimal 7.0−7.6

Below pH 7: risk of virus inactivation

pO2

Above 40%

Hypoxia might be beneficial for virus

replication of some viruses [ 78]

rpm

100−250

Impacts cell growth

Temperature shift

Reduce to 32−34°C for virus

infection phase

Can increase virus stability and yield

TOI

Often at 2E06 cells/mL to avoid any

cell density effect

Cell concentration, cell cycle phase?

MOI

Between 1E-02 and 1E-05

As low as possible

Seed virus (DIPs,

titer, engineering?)

Patient isolate?, egg or cell culture-

derived, chimeric viruses

(dengue/YFV)

Generate at low MOI, adaptation

needed? aim for high titers, low DIP

contamination level, engineering

could have risk of creating a more

dangerous virus?

Virus adaptation

3−5 passages in host cell

For IAV, switch from eggs to cell

culture, adaptation to a new cell line

Infection mode

Washing step with PBS to remove

serum at TOI, medium exchange,

low volume infection, change to

medium supporting virus

propagation (e.g., by cell

aggregation)

Complete medium exchange might

be beneficial for virus replication

Time of

harvest (TOH)

At max. virus titer (infectious or

total virus particle concentration

depending on vaccine type)

Amount of cell debris/DNA and

protein contamination levels versus

virus titer; cell disruption (e.g.,

freeze-thaw cycles) needed for

virion release?

Multiple harvests

For non-lytic viruses and unstable

viruses

Can reduce DIP production, limits

virus degradation

Reduce inhibitors,

medium selection

Lactate, ammonia, trypsin inhibitors,

released interferons

Addition of so called “virus booster”

(e.g., lipid cocktails) or virus

medium to induce cell aggregation

(e.g., MVA)

Additives

Trypsin, “virus booster,” cholesterol,

nucleosides, Pluronic F68

Needs to be optimized for medium,

vessel and possible shear, multiple

additions? stability, pH?

MOI: Multiplicity of infection; T: temperature; TOI: time of infection; DIPs: defective interfering

particles.

116

Bioprocessing of Viral Vaccines