Recombinant blood products and therapeutic enzymes - Pharmaceutical Biotechnology: Concepts and Aapplications - page 351

of an inclusion body. Owing to the prokaryotic production system, the product is non-glycosylated.
The fi nal sterile freeze-dried product exhibits a 2-year shelf life when stored at temperatures be-
low 25

C. An overview of the production process is presented in Figure 12.12.
The lack of glycosylation, as well as the absence of the EGF and K 1 domains (Table 12.6),
confers an extended serum half-life upon the engineered molecule. Reteplase-based products
display a serum half-life of up to 20 min, facilitating its administration as a single bolus injec-
tion as opposed to continuous infusion. Absence of the molecule's F 1 domain also reduces the
product's fi brin-binding affi nity. It is theorized that this may further enhance clot degradation,
as it facilitates more extensive diffusion of the thrombolytic agent into the interior of the clot.
Tenecteplase (also marketed under the tradename Metalyse) is yet an additional engineered
tPA now on the market. Produced in a CHO cell line, this glycosylated variant differs in
sequence to native tPA by six amino acids (Thr 103 converted to Asn; Asn 117 converted to
Gln and the Lys-His-Arg-Arg sequence at position 296-299 converted to Ala-Ala-Ala-Ala).
Collectively, these modifi cations result in a prolonged plasma half-life (to between 15 and 19
min), as well as an increased resistance to PAI-1 (plasminogen activator inhibitor 1, a natural
tPA inhibitor).

Cellular disruption
and recovery of
inclusion bodies

Fermentation
(1000 litre vessel)

Cellular harvesting

Affinity chromatography
(Erythrina trypsin
inhibitor - sepharose)

Solubilization of
inclusion bodies under
reducing conditions

Renaturation and
acidification

Removal of low
molecular weight
species (diafiltration)

Ion-exchange
chromatography
(x 2)

Concentration
(Ultrafiltration)

Excipient addition
(arginine, phosphoric
acid, polysorbate 20)

Aseptic filling
(20 ml glass vials)

Sterile filtration

Freeze-drying
and sealing

Figure 12.12 Production of Ecokinase, a modifi ed tPA molecule that gained regulatory approval in Europe in
1996. The production cell line is recombinant E. coli K12, which harbours a nucleotide sequence coding for the
shortened tPA molecule. The product accumulates intracellularly in the form of inclusion bodies