Recombinant blood products and therapeutic enzymes - Pharmaceutical Biotechnology: Concepts and Aapplications - page 350

surface is very slowly inactivated by α 1 -antiplasmin. The thrombolytic system has thus evolved

in a self-regulating fashion, which facilitates effi cient clot degradation with minimal potential
disruption to other elements of the haemostatic mechanism.

12.4.2 First-generation tissue plasminogen activator

Although tPA was fi rst studied in the late 1940s, its extensive characterization was hampered by
the low levels at which it is normally synthesized. Detailed studies were facilitated in the 1980s
after the discovery that the Bowes melanoma cell line produces and secretes large quantities of
this protein. This also facilitated its initial clinical appraisal. The tPA gene was cloned from the
melanoma cell line in 1983, and this facilitated subsequent large-scale production in CHO cell
lines by recombinant DNA technology. The tPA cDNA contains 2530 nucleotides and encodes a
mature protein of 527 amino acids. The glycosylation pattern was similar, though not identical, to
the native human molecule. A marketing licence for the product was fi rst issued in the USA to Ge-
nentech in 1987 (under the tradename Alteplase). The therapeutic indication was for the treatment
of acute myocardial infarction. The production process entails an initial (10 000 l) fermentation
step, during which the cultured CHO cells produce and secrete tPA into the fermentation medium.
After removal of the cells by sub-micrometre fi ltration and initial concentration, the product is
purifi ed by a combination of several chromatographic steps. The fi nal product has been shown to
be greater than 99 per cent pure by several analytical techniques, including HPLC, SDS-PAGE,
tryptic mapping and N-terminal sequencing.
Alteplase has proven effective in the early treatment of patients with acute myocardial infarction
(i.e. those treated within 12 h after the fi rst symptoms occur). Signifi cantly increased rates of patient
survival (as measured 1 day and 30 days after the initial event) are noted when tPA is administered
in favour of streptokinase, a standard therapy (see later). tPA has thus established itself as a fi rst-line
option in the management of acute myocardial infarction. A therapeutic dose of 90-100 mg (often
administered by infusion over 90 min) results in a steady-state alteplase concentration of 3-4 mg
l 1 during that period. However, the product is cleared rapidly by the liver, displaying a serum half-
life of approximately 3 min. As is the case for most thrombolytic agents, the most signifi cant risk
associated with tPA administration is the possible induction of severe haemorrhage.

12.4.3 Engineered tissue plasminogen activator

Modifi ed forms of tPA have also been generated in an effort to develop a product with an im-
proved therapeutic profi le (e.g. faster acting or exhibiting a prolonged plasma half-life). Reteplase
is the international non-proprietary name given to one such modifi ed human tPA produced in
recombinant E. coli cells and is sold under the tradenames Ecokinase, Retavase and Rapilysin
(Table 12.5). This product's development was based upon the generation of a synthetic nucleotide
sequence encoding a shortened (355 amino acid) tPA molecule. This analogue contained only the
tPA domains responsible for fi brin selectivity and catalytic activity. The nucleotide sequence was
integrated into an expression vector subsequently introduced into E. coli (strain K12) by treatment
with calcium chloride. The protein is expressed intracellularly, where it accumulates in the form