Immunotherapy for Infectious Diseases

Production of Igs and MAbs Targeting Infectious Diseases 89
vation systems is the art of determining the optimal compromise between engineering
and reactor performance in order to avoid chemical or physical stress on the cells and
to allow mass transfer to and from the cells. Such a compromise is easily achieved for
small bioreactor units. However, if production units for manufacturing several hundred
kilogram quantities per year are necessary, most of the currently used small-scale production
devices are no longer useful. Only a few bioreactor configurations are applicable
to large-scale, mass cell propagation and biologic manufacture. If the suspension
type of cell culture is used for production, both the stirred tank reactor, the air lift reactor,
and the packed bed reactor (133) can be used for large scale. If the adherent type
of cell culture is necessary, the fluidized bed reactor is a good choice (134).
It should be noted at this point that the standard cell lines such as hybridomas and
NSO are preferentially grown in suspension, whereas CHO and BHK cells can be grown
and propagated in both versions, adherent and in suspension. If the stirred tank reactor is
used for animal cells, axial flow impellers with large blades are preferable, as they lead
to good mixing with low mechanical shear forces. For aeration, direct sparging of air can
be applied. Both reactor types (the airlift and the stirred tank reactors) have been used
for up to 10,000-L working volume in animal cell suspension culture. Batch-, batch-fed,
and continuous culture methods can be applied. Although the airlift reactor performance
is optimal, with a constant filling volume slight modifications of the inner draft tube also
allow its use with variable filling for batch-fed culture (135). In batch culture the average
cell densities are in the range of 1–4 � 10 6 cells/mL, whereas batch-fed culture
allows a slight increase in cell density and maintenance in a productive state for longer
time. Thus, increased yields of antibody in the culture supernatant are achieved. The
batch-fed culture is defined by the increase of osmolarity due to the feed of substrates
and by the accumulation of metabolites such as lactate and ammonia (136,137).
On the basis of ultrafiltration principles, devices have been developed that give the reactor
a kind of kidney function to remove low-molecular-weight metabolites and ammonia,
while the large biomolecules are retained. Thus cell viability and density are improved and
the yield of product is increased. Other possibilities to increase productivity are found with
devices that allow continous perfusion with fresh media and cell retention in the reactor.
Various unit operations such as ultrasonic devices (138), special filters (139), cartrifuges,
or backlooping of cells into the reactor can increase cell retention. Such high-density continuous
perfused systems can accumulate cell densities beyond 10 8 cells/mL (140).
Depending on the expression rate of the production cells and the cultivation methods
applied, antibody titers above 1 g/L crude culture harvest can be accumulated.
Downstream Processing and Purification
Antibodies are applied therapeutically in high doses and at high concentrations. The
process steps downstream from the bioreactor must therefore establish a product of the
highest possible purity. Furthermore, the single process steps must allow safe sanitization
procedures since downstream processing usually cannot be performed under sterile
conditions. In addition to the purification of the antibody from impurities contained
in the matrix of the culture supernatant, the downstream process steps have to be
designed and validated to remove and inactivate potential viral contaminations.
A typical downstream processing procedure usually starts with removal of the cells
and cell debris from the crude culture supernatant. Cell sedimentation combined with
filtration or centrifugation are generally applied. The following process steps usually