Biomedical Engineering Reference
In-Depth Information
Genzyme Corporation was granted marketing authorization in 1991 for a glucocerebrosidase
preparation to be used for the treatment of Gaucher's disease. This Genzyme product (tradename
Ceredase) was extracted from placentas (afterbirths) obtained from maternity hospital wards.
The enzyme displays a molecular mass of 65 kDa and four of its fi ve potential glycosylation sites
are glycosylated. It had been estimated that a 1-year's supply of enzyme for an average patient
required extraction of 27 000 placentas, which rendered treatment extremely expensive. Genzyme
then gained regulatory approval for a recombinant version of glucocerebrosidase produced in
CHO cells. This product (tradename Cerezyme) has been on the market since 1994, and the total
world market for glucocerebrosidase is estimated to be in the region of US$200 million.
Cerezyme is produced in a CHO cell line harbouring the cDNA coding for human β-glucocer-
ebrosidase. The purifi ed product is presented as a freeze-dried powder, which also contains man-
nitol, sodium citrate, citric acid and polysorbate 80 as excipients. It exhibits a shelf life of 2 years
when stored at 2-8
C.
An integral part of the downstream processing process entails the modifi cation of cerezyme's oli-
gosaccharide components. The native enzyme's sugar side-chains are complex and, for the most part, are
capped with a terminal sialic acid or galactose residue. Animal studies indicate that in excess of 95 per
cent of injected glucocerebrosidase is removed from the circulation by the liver via binding to hepatocyte
surface lectins. As such, the intact enzyme is not available for uptake by the affected cell type, i.e. the
tissue macrophages. These macrophages display high levels of surface mannose receptors. Treatment
of native glucocerebrosidase with exoglycosidases, by removing terminal sugar residues, can expose
mannose residues present in their sugar side-chains, resulting in their binding to and uptake by the mac-
rophages. In this way, the 'mannose-engineered' enzyme is selectively targeted to the affected cells.
12.5.4
α
-Galactosidase, urate oxidase and laronidase
Recombinant α-galactosidase, urate oxidase and laronidase represent additional biopharmaceuti-
cals recently approved for general medical use.
-Galactosidase is approved for long-term enzyme
replacement therapy in patients with Fabry disease. Like Gaucher's disease, Fabry disease is a
genetic disease of lipid metabolism. Sufferers display little or no liposomal
α
-galactosidase-A
activity. This results in the progressive accumulation of glycosphingolipids in several body cell
types. Resultant clinical manifestations are complex, affecting the nervous system, vascular en-
dothelial cells and major organs. Although the condition is rare (500-1000 patients within the
EU), untreated sufferers usually die in their 40s or 50s.
Two recombinant α-galactosidases are now on the market (Fabrazyme, produced by Genzyme
and Replagal, produced by TKT Europe). Fabrazyme is produced in an engineered CHO cell
line, and downstream processing entails a combination of fi ve chromatographic purifi cation steps
followed by concentration and diafi ltration. Excipients added include mannitol and sodium phos-
phate buffering agents, and the fi nal product is freeze-dried after fi lling into glass vials. Replagal
is produced in a continuous human cell line and is also purifi ed by a combination of fi ve chroma-
tographic purifi cation steps, although it is marketed as a liquid solution.
Human
α
-galactosidase is a 100 kDa homodimeric glycoprotein. Each 398 amino acid mono-
mer displays a molecular mass of 45.3 kDa (excluding the glycocomponent) and is glycosylated
at three positions (asparagines 108, 161 and 184). After administration (usually every second
week by a 40 min infusion), the enzyme is taken up by various body cell types and directed to
the lysosomes. This cellular uptake and delivery process appear to be mediated by mannose-
α
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