Biomedical Engineering Reference
In-Depth Information
levels at which they are normally produced in the body. Large-scale purifi cation from sources
such as blood was non-viable. Furthermore, interferons exhibit species preference and, in some
cases, strict species specifi city. This rendered necessary the clinical use only of human-derived
interferons in human medicine.
Up until the 1970s interferon was sourced (in small quantities) directly from human leuko-
cytes obtained from transfused blood supplies. This 'interferon' preparation actually consisted
of a mixture of various IFN-
s, present in varying amounts, and was only in the regions of 1 per
cent pure. However, clinical studies undertaken with such modest quantities of impure interferon
preparations produced encouraging results.
The production of interferon in signifi cant quantities fi rst became possible in the late 1970s, by
means of mammalian cell culture. Various cancer cell lines were found to secrete interferons in
greater than normal quantities, and were amenable to large-scale cell culture due to their trans-
formed nature. Moreover, hybridoma technology facilitated development of sensitive interferon
immunoassays. The Namalwa cell line (a specifi c strain of human lymphoblastoid cells) became
the major industrial source of interferon. The cells were propagated in large animal cell ferment-
ers (up to 8000 l), and subsequent addition of an inducing virus (usually the Sendai virus) resulted
in production of signifi cant quantities of leukocyte interferon. Subsequent analysis showed this to
consist of at least eight distinct IFN-α subtypes.
Wellferon was the tradename given to one of the fi rst such approved products. Produced by
large-scale mammalian (lymphoblastoid) cell cultures, the crude preparation undergoes exten-
sive chromatographic purifi cation, including two immunoaffi nity steps. The fi nal product contains
nine IFN-α subtypes.
Recombinant DNA technology also facilitated the production of interferons in quantities large
enough to satisfy potential medical needs. The 1980s witnessed the cloning and expression of
most interferon genes in a variety of expression systems. The expression of specifi c genes obvi-
ously yielded a product containing a single interferon (sub)type.
Most interferons have now been produced in a variety of expression systems, including
E. coli
,
fungi, yeast and some mammalian cell lines, such as CHO cell lines and monkey kidney cell lines.
Most interferons currently in medical use are recombinant human (rh) products produced in
E.
coli. E. coli
's inability to carry out post-translational modifi cations is irrelevant in most instances,
as the majority of human IFN-
α
α
s, as well as IFN-
β
, are not normally glycosylated. Whereas IFN-
γ
is glycosylated, the
E. coli
-derived unglycosylated form displays a biological activity similiar to
the native human protein.
The production of interferon in recombinant microbial systems obviously means that any
fi nal product contaminants will be microbial in nature. A high degree of purifi cation is thus
required to minimize the presence of such non-human substances. Most interferon fi nal product
preparations are in the region of 99 per cent pure. Such purity levels are achieved by exten-
sive chromatographic purifi cation. While standard techniques such as gel fi ltration and ion ex-
change are extensively used, reported interferon purifi cation protocols have also entailed use of
various affi nity techniques using, for example, anti-interferon monoclonal antibodies, reactive
dyes or lectins (for glycosylated interferons). Hydroxyapatite, metal-affi nity and hydrophobic
interaction chromatography have also been employed in purifi cation protocols. Many produc-
tion columns are run in HPLC (or FPLC) format, yielding improved and faster resolution.
Immunoassays are used to detect and quantify the interferons during downstream processing,
although the product (in particular the fi nished product) is also usually subjected to a relevant
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