hand efficiency, stability, ratio of infectious to non-infectious particles, and glyco-

sylation. Furthermore, all these product quality parameters need to remain throughout

purification and formulation as well as with storage, transport and final application as

only few side effects will be tolerated by the vaccinated person. To be able to consider

TABLE 5.7

Points to consider for process optimization either for adherent cells or for

suspension cell lines

Evaluation/

optimization

Adherent cells

Suspension cells

Cell growth, small-

scale

T-flasks, roller bottles, spinner flasks

with microcarriers in SCM or SFM up

to 2E06 cells/mL (good cell attachment

rate and cell flattening); split ratio, split

timing, inoculation concentration,

maximum number of passages

Shake flasks in SCM, SFM or CDM at

different rpm and pH up to 1E07 cells/

mL (single cells/aggregates, cell size);

split ratio, split timing, inoculation

concentration, maximum number of

passages

Cell growth,

bioreactor

Growth on microcarriers or

macrocarriers up to at least 2E06

cells/mL; inoculation concentration,

trypsinization

Inoculation concentration, rpm, pH up

to 1E07 cells/mL

Cell counting

Complete cell detachment

No aggregates

Cell banking

Check best medium, cell conc. and

successful thawing

Check best medium, cell conc. and

successful thawing, check freezing at

high cell concentration

Process

intensification

Check packed bed or HFBR

Check semi-perfusion in shake flasks

and perfusion devices up to 2-5E07

cells/mL

Cell characterization Check for sterility, mycoplasmas

(PCR), etc. depending on

requirements (FDA, EP, WHO)

Check for sterility, mycoplasmas

(PCR), etc. depending on

requirements (FDA, EP, WHO)

Seed virus

Adapt virus strain to host cells, 3−5

passages should be fine

Check for sterility, mycoplasmas

(PCR), identity, etc. depending on

requirements (EP, FDA, WHO)

Adapt virus strain to host cells, 3−5

passages should be fine

Check for sterility, mycoplasmas

(PCR), identity, etc. depending on

requirements (EP, FDA, WHO)

Virus production

Check virus replication at 2E06 cells/mL

Check TOI and MOI

Check yield at 37°C and reduced

temperature (32-34°C)

Check virus replication at 2E06 cells/mL

Check virus replication at >2E06 cells/

mL for high cell density effect

Check TOI and MOI

Check yield at 37°C and reduced

temperature (34°C)

SCM: serum containing medium; SFM: serum free medium; CDM: chemically defined medium; EP:

European Pharmacopeia; FDA: Food and Drug Administration; WHO: World Health Organization;

TOI: time of infection; MOI: multiplicity of infection.

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Bioprocessing of Viral Vaccines