longer produced but consumed. Furthermore, both cell growth and virus replication

depend strongly on availability and performance of certain commercially available

media. Therefore, screening experiments are necessary to identify a medium that

supports fast growth to high cell concentrations (first phase) and maximum virus

yields. Furthermore, supplementation (e.g., concentration of glucose and glutamine

or other additives) should be optimized.

Osmolality also impacts cell growth and virus production. For adenovirus pro-

duction using HEK293 cells in suspension culture, hyperosmotic conditions during

the cell growth phase were reported to lead to a reduced specific growth rate and a

lower maximum cell concentration, but stimulated subsequent virus production

(higher CSVY) [80]. A similar finding was already reported for antibody production

[65,81,82]. Thus, osmolality should be screened during media development to

determine the right balance between cell growth and virus production to ultimately

optimize yields.

The harvest time point not only affects maximum virus yields, but also virus

quality. As already mentioned, virus yields peak at a maximum in the range of 1−5

days post infection depending on the virus strain, host cell system and MOI. The

optimal harvest time was reported for different viruses as: IAV (24 h post infection

(hpi)) [16], YFV (48−96 hpi) [19], and MVA (72 hpi) [83]. However, infectious

virus titer and total virus titer are typically not reaching their maximum at the same

time (Figure 5.6). For IAV, the total virus titer typically further increases, while the

infectious titer is stationary at its maximum or already decreases due to virus de-

gradation [84]. This is due to the circumstance that at late time points, the pro-

portion of non-infectious viruses, including DIPs, increases significantly. Moreover,

depending on the harvest time point the level of contaminating DNA and protein

might change. Typically, the total protein and host cell DNA level is most pro-

nounced at later production time points due to cell lysis and the associated release of

cell contents. This poses a major problem for DSP to achieve values lower than the

TABLE 5.9

Parameters for virus replication determined either by cell line, virus, or

process conditions

Determined by cell line

Determined by virus

Determined by process

Membrane composition of virus

Range of permissive cell lines

Volumetric virus

productivity (VVP)

Glycosylation of viral proteins

Generation of DIPs

pH, temperature

Ratio infectious/non-infectious

virions

Virus stability, virus

degradation rate

Virus stability, virus

degradation rate

Replication time

Replication time

Cell-specific virus yield (CSVY)

Cell debris, cell lysis, apoptosis

Amount of cell debris

VVP: Volumetric virus productivity, virions produced per total volume medium and total process time

(e.g., virions/L/d).

Upstream processing for viral vaccines

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