Biomedical Engineering Reference
In-Depth Information
principles and practice of animal cell culture have been overviewed in Chapter 5. To date, bioreactor
size of 100 l or less have been used, which are suffi cient to satisfy clinical trial demand. The pack-
ing cells are then seeded with the replication-defi cient virus, allowing vector propagation (see also
Figure 14.5). After a fi xed time the viral-infected cells are collected (harvested) by microfi ltration
or centrifugation and the cells are then homogenized in order to release the viral vector. Tradition-
ally, adenoviral vectors (and indeed many other animal cell viruses) were purifi ed from such a crude
mixture by caesium chloride density-gradient centrifugation. While appropriate to laboratory-scale
operations, this method is unsuitable for large-scale viral recovery due to scale-up issues and cost.
Alternative purifi cation methods based upon column chromatography are thus employed on an
industrial scale. Major 'contaminants' present in this crude viral vector preparation include some
intact animal cells, cellular debris, and intracellular molecules, most notably animal cell protein
and nucleic acid. Intact cells/cellular debris is removed by fi ltration. The release of large amounts
of cellular DNA increases the solution viscosity and complicates downstream processing. The
purifi cation protocol, therefore, usually entails the physical degradation of DNA by addition of a
nuclease enzyme. A solvent/detergent treatment step is then undertaken as a safety step in order to
inactivate any enveloped contaminant viruses that might also be present. High-resolution purifi ca-
tion is usually achieved by a combination of ion-exchange and gel-fi ltration chromatography, with
product concentrations steps being undertaken by ultrafi ltration if necessary. The fi nal product is
then fi lter sterilized and fi lled into glass vials. The purifi ed vectors generally may be stored either
refrigerated or frozen, and they display useful shelf lives of 2 years or more.
14.3.4 Non-viral vectors
Although viral-mediated gene delivery systems currently predominate, a substantial number of
current clinical trials use non-viral-based methods of gene delivery. General advantages quoted
with respect to non-viral delivery systems include:
•
their low/non-immunogenicity;
•
non-occurrence of integration of the therapeutic gene into the host chromosome (this eliminates
the potential to disrupt essential host genes or to activate host oncogenes).
The initial approach adopted entailed administration of 'naked' plasmid DNA housing the gene
of interest. This avenue of research was fi rst opened in 1990, when it was shown that naked plas-
mid DNA was expressed in mice muscle cells subsequent to its i.m. injection. The plasmid DNA
concerned housed the β-galactosidase gene as a reporter. Subsequent expression of β-galactosi-
dase activity could persist for anything from a few months to the remainder of the animal's life.
The transfection rate recorded was low (1-2 per cent of muscle fi bres assimilated the DNA), and
the DNA was not integrated into the host cell's chromosomes.
Up until this point, it was assumed that naked DNA injected into animals would not be sponta-
neously taken up and expressed in host cells. This fi nding vindicated the cautious approach taken
by the FDA and other regulatory authorities with regard to the presence of free DNA in biophar-
maceutical products (Chapter 7).
Scientists have also since demonstrated that DNA (coated on microscopic gold beads) pro-
pelled into the epidermis of test animals with a 'gene gun', is expressed in the animal's skin cells.
Furthermore, the introduction in this fashion of DNA coding for human infl uenza viral antigens
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