hydrophilic and charged surface [33,34]. Typical microcarrier materials are dextran,

polyester and collagen and the surface is modified with DEAE (diethylethanola-

mine) or cations. Moreover, attachment factors are crucial to ensure the attachment,

which are either provided by the serum or addition of exogenously fibronectin under

serum-free conditions [33]. For optimal growth, homogenous cell attachment is

required, thus, the ratio of cells to microcarriers needs optimization. As second

phase, cell flattening occurs to enlarge the contact area for stable binding, a process

called cell spreading [32]. By using serum-free medium, the cells tend not to flatten,

resulting in easier cell detachment due to shear stress during agitation (Figure 5.2B).

To help cell attachment, intermediate stirring can be tried, in which a short stirring

for homogenization is followed by 30 minutes without stirring, repeated over about

2 hours in total. Moreover, using macroporous microcarriers, adherent cells are

attached within the carriers reducing the shear stress by agitation and sparging

[33,35]. Furthermore, volume can be reduced after cell seeding to increase the

chance of cell surface contact.

5.5

CULTIVATION MEDIA AND METABOLISM

5.5.1

MEDIA AND ADDITIVES

The choice of cell growth medium is essential for a successful cultivation of animal

cells and, consequently, the virus production. Media developed over many years

now allow growth of suspension cells in batch mode up to 2E07 cells/mL and in

perfusion mode up to 2E08 cells/mL. Traditionally, cell-culture−based viral vaccines

are produced in serum-containing media (e.g., fetal bovine serum, FBS). This may be

required even today as many adherent cells, and in particular primary cells, are the

only host cells that can be used to propagate some viruses at high yield. However,

serum-containing media pose numerous disadvantages for efficient viral vaccine

production by complicating the purification process and therefore increasing the costs.

Moreover, serum can be contaminated with adventitious agents and unwanted by-

products (i.e., antibodies against the virus to be produced). In addition, the quality of

serum is difficult to assess and batch-to-batch variation can cause significant incon-

sistencies in cell growth and virus yields. Thus, serum-free media were developed,

where only particular serum components, including hydrolysates, amino acids,

growth factors, hormones, lipids, adhesion factors, and other compounds are added at

a defined concentration (see Table 5.3). This trend is moving towards the use of

animal component-free (AFM) or even chemically defined media (CDM), where all

ingredients are defined and, thus, contribute to process robustness. To enable the

formulation of AFM variants of media, non-animal substitutes have been developed

for the various supplements derived from serum (see Table 5.5). Today, the preferred

media for production are chemically defined, free of animal components, and certified

for GMP production. The adaptation to serum-free variants of medium typically

follows two different protocols, the sequential adaptation (changing the ratio of

serum-containing and serum-free medium) or the adaptation by using conditioned

medium (also see “Adherent versus suspension cells”).

Upstream processing for viral vaccines

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