AMEDIPLASE - Fusion Protein Technologies for Biopharmaceuticals: Applications and Challenges - page 487

FIGURE 33.1 Schematic composition of the most recent thrombolytics. In dark grey the tPA
portion, in light grey the sc-UPA portion. Dark diamond ¼ N glycosylation site, light diamond ¼ O-
glycosilation site.

of t-PA (to maintain fibrin affinity, while losing domains
responsible for fast plasma clearance) and the protease
domain of scu-PA, fused at the plasmin cleavage site
between Arg 275 of t-PA and Ile 159 of scu-PA (to form the
Arg 103 -Ile 104 bond in the chimera). The advantage of this
approach is that it eliminates from scu-PA two sites of
possible cleavage leading to fibrin-aspecific or inactive u-
PA forms (Glu 143 -Leu 144 and Arg 156 -Phe 157 ) [8].
This rationale design of the molecule should result in a
thrombolytic agent with relevant fibrinolytic potency (due to
the scuPa protease domain), remarkable safety (due to the
fibrin specificity linked to the kringle 2 of t-PA), prolonged
plasma half-life allowing single bolus administration (resist-
ance to metabolic degradation), and no immunogenicity
(humanized protein).

line CHO CGI-1, led to a modified expression vector and to
the new cell line, renamed CGI-2. The expression vector is a
pBR322-based plasmid containing a dihydrofolate reductase
selection marker in addition to the hybrid PA gene and
appropriate promoters, splicing signals, and so on required
for effective expression.
CGI-2 cell was then transferred to A. Menarini S.r.l.,
Florence, Italy, and Menarini Biotech S.r.l., Pomezia, Italy
for the production of amediplase (code MEN9036) for
further preclinical and clinical experimentations.

33.2.2 Drug Substance

Amediplase (Figure 33.2) is a hybrid PA consisting of the
kringle 2 domain of t-PA and the protease domain of u-PA.
Amediplase contains 356 amino acids, 18 of which are
cysteine residues forming 9 disulphide bonds. It has poten-
tially two glycosylation sites, one in each domain, whereas
the glycosylation site in the kringle domain is not or only
lightly occupied, the other site is fully glycosylated. Glyco-
sylation sites are occupied by N-linked complex oligo-
saccharide structures. No O-linked carbohydrates have
been detected on amediplase. Treatment of amediplase
with plasmin results in a single peptide bond cleavage
between residues Arg 103 and Ile 104 , leading to the formation
of two subunits, A and B, that are still connected through a
disulphide bridge. While amediplase is a zymogen, the
double chain form is the active form.
Production of amediplase drug substance is carried out at
Menarini Biotech (Pomezia, Italy), a company of the Menar-
ini Group. Currently, the production (in GMP compliance)
has been scaled-up to an industrial level in 1500-L fermen-
tors. A cycle consists of a semicontinuous fermentation in

33.2 SOURCE, PHYSICO-CHEMICAL
PROPERTIES AND FORMULATION

33.2.1 Production Cell Line

The product amediplase was initially derived from the tissue
culture supernatant of a permanent Chinese hamster ovary cell
line (CHO CGI-1), which was obtained by deliberate genetic
manipulation at Ciba Geigy Ltd, Basel, Switzerland. The
parent cell line DUKXBI (ATCC CRL 9096) was supplied to
Ciba Geigy by Professor L. Chasin [9]. Ciba Geigy modified
the parent cell line by recombinant DNA technology to obtain
the producer cell line CHO CGI-1, which produced and
secreted CGP 42935 in the culture supernatant.
Following studies performed at Ciba Geigy, aimed to
improving the stability and productivity of the original cell