Biomedical Engineering Reference
In-Depth Information
Although earlier factor VIII preparations were relatively crude (i.e. contained lower levels of
various other plasma proteins), many of the modern preparations are chromatographically puri-
fi ed to a high degree. The use of immunoaffi nity chromatography has become widespread in this
regard since 1988 (Figure 12.7). The extreme bioselectivity of this method can yield a single-step
purifi cation factor of several thousand-fold.
Although fractionation can reduce very signifi cantly the likelihood of pathogen transmission, it
cannot entirely eliminate this possibility. Blood-derived factor VIII products, including those pre-
pared by immunoaffi nity chromatography, generally undergo further processing steps designed
to remove/inactivate any virus present. The raw material is often heated for up to 10 h at 60
C or
treated with solvent or dilute detergent prior to chromatography. Recombinant factor VIII is also
often treated with dilute detergent in an effort to inactivate any viral particles potentially present.
Production of recombinant factor VIII (Table 12.2) has ended dependence on blood as the only
source of this product, and eliminated the possibility of transmitting blood-borne diseases specifi -
cally derived from infected blood. In the past, over 60 per cent of haemophiliacs were likely to be
accidentally infected via contaminated products at some stage of their life.
Several companies have expressed the cDNA coding for human factor VIII:C in a variety of eu-
karyotic production systems (human VIII:C contains 25 potential glycosylation sites). CHO cells
and BHK cell lines have been most commonly used, in addition to other cell lines, such as various
mouse carcinoma cell lines. The recombinant factor VIII product generally contains only VIII:C
(i.e. is devoid of vWF). However, both clinical and preclinical studies have shown that administra-
tion of this product to patients suffering from haemophilia A is equally as effective as administer-
ing blood-derived factor VIII complex. The recombinant VIII:C product appears to bind plasma
Factor VIII complex
Spacer arm
Epitope against
which antibody
was raised
Anti-factor VIII
antibody
Bead support
Additional blood factors
or other plasma proteins
which fail to bind to the
antibody
Figure 12.7
Purifi cation of factor VIII complex using immunoaffi nity chromatography. The immobilized
anti-factor VIII antibody is of mouse origin. Antibodies raised against specifi c epitopes on both the VIII:C
and vWF components have both been successfully used. Industrial-scale columns would often exhibit a bed
volume of several litres. Note that the different elements in this diagram are not drawn to the correct scale
relative to each other
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