Purification and Characterization of Recombinant Produced Human APC

A novel method termed pseudo-affinity chromatography was developed for the purification of rHPC that took advantage of the conformational changes in HPC upon the addition or removal of calcium divalent ions [9,29]. This purification step proved to be economical, efficient, and highly selective, resulting in the recovery of only those molecules with full y-carboxylation of the light chain following activation [22]. The process for the activation of the recombinant zymogen protein C utilized immobilized bovine thrombin and was followed by repurification steps. APC from HEK293 cells purified by this method had complete y-carboxylation and displayed high functional activity comparable to or higher than that of plasma-derived HPC purified by amidolytic (protease) and anticoagulant assays. Plasma-derived HPC and the purified HEK293 cell-produced rHPCs both display apparent molecular masses from 62 to 66 kDa (as determined by SDS-PAGE). Like plasma-derived protein C, the HEK293 cell-derived rHPC was >90% two chain, and contained a similar pattern of the three heavy-chain a, p, y glycoforms, but with HEK293 cell-derived rHPC being more enriched equally in both a, p glycoforms, and plasma-derived

HPC having more of the a glycoform. This similarity to plasma-derived material was not seen in rHPC produced from other cell lines, for example, BHK-21 cells [30]. The respective N-terminal sequences of the heavy and light chains of rHPC were identical to those previously reported for plasma-derived HPC [31]. Thus, the HEK293 cell line, unlike others such as the C127 and CHO cell lines [16], was capable of proper proteolytic processing of the propeptide, even at high expression levels. There was no significant difference in the amount of P-OH-Asp in the rHPC and plasma-derived HPC. Thus, with respect to the posttranslational modifications of proteolytic processing and the two types of amino acid modification, the rHPC from the HEK293 cell line appears to be processed in a manner similar to plasma-derived material, but more completely in the case of y-carboxylation of the nine glutamate residues. y-Carboxylation of glutamates is dependent on an adequate supplement of vitamin K in the diet, avoidance of medications interfering with the vitamin K-dependent pathway, and the normal liver function of the large number of blood donors from which the plasma APC is derived. One advantage of deriving rHPC from a culture of HEK293 cells is that there is more consistency in the optimum requirement for y-carboxylation.

Unlike the other posttranslational modifications described above, the carbohydrates on rHPC differed from those on plasma-derived HPC. The structures of oligosaccharides on recombinant glycoproteins are determined to a large extent by the cell line used [32]. Oligosaccharides found on glycoproteins can influence parameters that are important to drug design, such as circulatory clearance, tissue targeting, immunogenicity, and efficacy [32]. In view of this, a detailed analysis of the oligosaccharide structures on rHPC from HEK293 cells was performed [9,33], and novel and rare Asn-linked oligosaccharides were identified (Figure 4.4). The novel trisaccharide epitope on rHPC was designated the PC293 determinant, and, along with the two rare disaccharide epitopes, GalNAcP(1 ^ 4)GlcNAcP(1 ^ 4) and NeuAca(2 ^ 6)GalNAcP(1 ^ 4), have been found in naturally occurring human glycoproteins [34-37]. Glycosyl composition analysis has shown that these GalNAc-containing rare oligosaccharides are not present in plasma-derived HPC. However, to date, clinical trial experience in healthy subjects and over 30,000 patients (both clinical trials and commercial use) suggests that these novel and rare oligosaccha-rides on HEK293-produced rhAPC probably do not pose any immunogenicity problem in humans. Studies in a primate arterial thrombotic model suggest that these rare Asn-linked oligosaccharides may slightly lessen the circulatory half-life and antithrombotic activity of rhAPC when compared with plasma-derived human APC for this model [38]. Because of the carbohydrate sequence homology of the PC293 determinant to the Lewis X determinant, Asn-linked oligosaccharides containing the PC293 determinants were

Manß (1^4)GlcNAcß(1^4)GlcNAc-Asn GalNAcß(1^4)GlcNAcß(1^2)Mana(1^3)/ Fuca(1 ^3)/

Manß(1^4)GlcNAcß(1^4)GlcNAc-Asn NeuAca(2^6)GalNAcß(1^ 4)GlcNAcß(1^2)Mana(l^3)/

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