Large‐Scale Production and Properties of Immunoaffinity‐Purified Human Activated Protein C Concentrate

Abstract

Activated protein C (APC) is a highly specific serine proteinase which functions as an important naturally occurring antithrombotic enzyme. APC also has anti-inflammatory properties. We have developed a large-scale process for the production of APC for therapeutic use starting with cryoprecipitate-poor human plasma. This report describes the process, its performance at the pilot plant scale, and the characteristics of immunoaffinity-purified human APC concentrate referred to as APC (human). The process consists of three chromatographic steps, an enzymatic conversion step, and incorporates a solvent/detergent treatment step for the inactivation of lipid-enveloped viruses. Solvent/detergent was shown to rapidly inactivate spiked HIV-1, as well as three marker viruses to nondetectable levels under process conditions. The immunoaffinity-purified protein C (PC) intermediate was enriched 13,600-fold over plasma and had a specific activity of 231 U/mg. The overall yield of the process following enzymatic conversion of the PC intermediate to APC and its processing by anion exchange chromatography was 36%. APC (human) was shown to be highly purified, functional and stable.

A Sensitive and Facile Assay for the Measurement of Activated Protein C Activity Levels in Vivo

“…Protetns. Protein C was purified from human cryoprecipitate-poor plasma by anion exchange followed by immunoaffinity chromatography (14,22). APC was produced by incubating protein C with purified human cr-thrombin and purification of the resulting APC by an additional anion exchange step.…”

“…Thus, the available surface area is probably not limited to the surface of the beads, but better represented by a shell of some finite (but unknown) thickness. 22,1993…”

Removal of poliovirus type 1 from a protein mixture using an immunoaffinity chromatography column

“…Large-scale purifications of enzymes to homogeneity have been performed (Robinson et al, 1972;Nicholas et al, 1972;Holroyde et al, 1976). In addition, large-scale immunoaffinity chromatography is used to purify HBsAg from a 335 L Saccharomyces cerevisae culture in the preparation of the recombinant HBV vaccine (McAleer et al, 1984), insulin (Sofer & Nyström, 1989), FVIII and FIX (Weinstein, 1989;Lawrence, 1993;Tharakan et al, 1990), interferon (Knight and Fahey, 1981;Mikulski et al, 1980;Dembinski and Sulkowski, 1986;Staehlin et al, 1981) and protein C (Orthner et al, 1995). The system developed in this study could be developed on a manufacturing scale, however further analysis of suitable matrix types and scale-up considerations would have to be incorporated into the analysis.…”

Large‐scale production and properties of human plasma‐derived activated Factor VII concentrate

“…For preparation of mAb‐immobilized gel, Sepharose Fast Flow (Amersham Pharmacia Biotech) was activated with cyanogen bromide (Nakarai Tesque, Kyoto, Japan) and the mAb was coupled to the activated gel at a mean density of 5 mg of mAb/ml of gel [15]. Anti‐FVII mAb was quantified by sandwich ELISA for murine IgG, using a modification of a previously described procedure [16]. The limit of detection for anti‐FVII mAb was 0·25 ng/ml.…”