Biomedical Engineering Reference
In-Depth Information
Mice injected intraperitoneally
with 5 x 10 - 1 x 10
hybridoma cells
Harvesting of ascites 14-30
days post innoculation
6
7
Centrifugation, followed by
filtration (0.2m) of antibody-
containing fluid
Storage at -80°C if processing
is not undertaken immediately
Pepsin digestion; generates
F(ab) fragment and F fragment
2
Antibody purification (two
ion-exchange and one affinity
step)
c
Reduction of F (ab) fragment
using cysteine as reducing agent
(i.e. generation of 2 x Fab
fragments)
2
Final Fab purification by gel
filtration
Addition of excipients
(sucrose), filtration and
aseptic filling
Lyophylization of
finished product
Figure 13.5 Outline of the production strategy of CEA-SCAN. The antibody-producing hybridoma cell line
was originally obtained by standard methods of hybridoma generation. Spleen-derived murine B-lymphocytes
were fused with murine myeloma calls. The resulting stable hybridomas were screened for the production of
anti-CEA monoclonals. The clone chosen produces an IgG anti-CEA antibody. Note that the fi nished product
outlined above is not radiolabelled. The freeze-dried antibody preparation (which has a shelf life of 2 years at
2-8 C) is reconstituted immediately prior to its medical use. The reconstituting solution contains 99m Tc, and
is formulated to facilitate direct conjugation of the radiolabel to the antibody fragment
diphtheria toxin. After binding to the cell surface, the antibody-toxin conjugate is often internal-
ized via endocytosis. It is presumed that, rather than being destroyed, the toxin is subsequently
made available inside the cell, such that it can induce its toxic effects. One such antibody-based
product now approved for general medical use is Mylotarg (Table 13.2). The product consists of an
engineered antibody (a 'humanized' antibody, as described later), conjugated to a cytotoxic anti-
tumour antibiotic, calicheamicin (Figure 13.6). The antibody binds specifi cally to a cell surface
antigen, CD33. This is a sialic-acid-dependent adhesion protein found on the surface of leukaemic
cells in more than 80 per cent of patients suffering from acute myeloid leukaemia. The product
production process entails initial culture of the antibody-producing mammalian cell line with sub-
sequent purifi cation of the antibody by a series of chromatographic steps. Downstream processing
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