Phase 3 study of rhAPC in severe sepsis — 11 countries 164 hospitals


Phase 3 study of rhAPC in severe sepsis — 11 countries 164 hospitals


End of phase 3 testing of rhAPC in sepsis

1/2CC1 11/2CC1 8/2CC2

rhAPC FDA BLA approval submission to FDA & EMEA

Approvals: EU, etc.

FIGURE 4.3 Summary of the development history of drotrecogin alpha (activated) (see text for details).

modification of the Gla domain. Point mutagenesis studies have shown that elimination of P-hydroxylation or incorrect processing of the propeptide essentially eliminated all the anticoagulant activity [10].

In the mid-1980s, we found that an engineered, adenovirus-transformed HEK293 cell line was capable of producing fully active rHPC [8,9,13,14]. There were substantial efforts both in academic and industrial laboratories to express and secrete active vitamin K-dependent proteins. However, with the exception of the HEK293 cell line, all cell lines thus far engineered secrete only partially active material at reasonable expression levels [8,13,15-20]. The human 293 parent cell line, into which the protein C expression vectors were introduced, is a permanent line of primary HEK. The cell line was isolated in 1973 by in vitro transfection of HEK cells with sheared human adenovirus type 5 (Ad5) DNA, as described by Graham et al. [21]. We found that the adenovirus transformation of a cell was critical for the secretion of highly functional, correctly modified protein in part because of the induction of the carboxylase enzyme [22]. In fact, it was possible to engineer improved y-carboxylation in cells via viral transformation methods, although other important posttranslational processes such as correct proteolytic cleavage were not affected by transformation [22]. The inability to express correctly processed rHPC in commonly used cell lines such as Chinese hamster ovary (CHO), baby hamster kidney (BHK)-21, etc., required the development of new expression vectors and methods for the efficient secretion of rHPC in the HEK293 cells [23-28]. The vectors were designed with elements that could be transactivated by the endogenous E1A protein of adenovirus, which is constitutively expressed by the parent line. This eliminated the need for the commonly used method of gene amplification to drive highlevel expression. Although the HEK293 cell line primarily secreted fully processed protein, the next challenge was to overcome a rate-limiting step in secretion, slow glycosyl processing in the endoplasmic reticulum [7]. To overcome this hurdle, a variant 293 line was developed by high-volume clonal selection, followed by reintroduction of a second strong E1A-dependent expression vector [11,26]. The resulting cell line showed no evidence of genetic instability or alterations, as determined by Southern-blot hybridization, RT-PCR analysis, and sequencing of the protein C mRNA by the RT-PCR in the original cell bank through postproduction cells. Extensive biosafety testing has failed to identify any evidence for the presence of adventitious agents in the recombinant derivative of this cell line.

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