SummaryActivated protein C (APC) is a serine protease which plays an important role as a naturally occurring antithrombotic enzyme. APC, which is formed by thrombin-catalyzed limited proteolysis of the zymogen protein C, functions as an anticoagulant by proteolytic inactivation of the coagulation cofactors VIIIa and Va. APC is inhibited by several members of the serpin family as well a by α2-macroglobulin. APC is being developed as a therapeutic for the prevention and treatment of thrombosis. We have developed an assay to quantify circulating levels of enzymatically active APC during its administration to patients, in healthy individuals, and in various disease states. This assay utilizes an EDTA-dependent anti-protein C monoclonal antibody (Mab) 7D7B10 to capture both APC and protein C from plasma, prepared from blood collected in an anticoagulant supplemented with the reversible inhibitor p-aminobenzamidine. Mab 7D7B10-derivatized agarose beads are added to the wells of a 96-well filtration plate, equilibrated with Tris-buffered saline, and incubated for 10 min with 200 μl of plasma. After washing, APC and protein C are eluted from the immunosorbent beads with a calcium-containing buffer into the wells of a 96-well microtiter plate containing antithrombin III (ATIII) and heparin. The amidolytic activity of APC is then measured on a kinetic plate reader following the addition of L-pyroglutamyl-L-prolyl-L-arginine-p-nitroanilide (S-2366) substrate.The rate of substrate hydrolysis was proportional to APC concentration over a 200-fold concentration range (5.0 to 1,000 ng/ml) when measured continuously over a 15 to 30 min time period. The coefficient of variation was 5.9% at 35 ng/ml and 8.8% at 350 ng/ml APC. The sensitivity of the assay could be increased by measuring the amount of color produced after longer incubation times in the endpoint mode. The measured APC activity levels were little affected by varying protein C or prothrombin over the extremes of 0 to 150% of normal plasma concentrations. By constructing the standard curve in protein C-deficient plasma, the concentration of APC activity in normal pooled plasma was determined to be 2.8 ng/ml (45 pM), which represents 0.08% of the protein C concentration. The assay was approximately 50-fold more sensitive than the identical assay, but using Mab-coated microtiter wells rather than immunosorbent beads as the capture step.

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.

Activated protein C (APC) is a highly specific serine proteinase which functions as an important naturally occurring antithrombotic enzyme. APC also has antiinflammatory 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 immunoaffmity-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 immunoaffmity-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.

Heat stability of lyophilized C1 inactivator (C1-INA) concentrates of intermediate and high purity has been investigated under several heat treatment protocols that include heating for 96 and 192 h at 68 degrees C and for 10 h at 80, 90 and 100 degrees C. Both types of concentrate showed high stability in functional activity, with not more than 5% loss in any of the time-temperature combinations evaluated. However, the C1-INA antigen from both concentrates showed small but progressive changes in crossed immunoelectrophoretic pattern, in proportion to the intensity of heat treatment. High-pressure size-exclusion chromatography revealed only minimal signs of aggregation in the high-purity concentrate, but a significant and progressive aggregation of nonspecific protein contaminants present in the intermediate-purity concentrate, making the high-purity concentrate preferable for heat treatment.

Heat stability of lyophilized Cl inactivator (Cl-INA) concentrates of intermediate and high purity has been investigated under several heat treatment protocols that include heating for 96 and 192 h at 68 °C and for 10 h at 80, 90 and 100°C. Both types of concentrate showed high stability in functional activity, with not more than 5% loss in any of the time-temperature combinations evaluated. However, the C1-INA antigen from both concentrates showed small but progressive changes in crossed immunoelectrophoretic pattern, in proportion to the intensity of heat treatment. High-pressure size-exclusion chromatography revealed only minimal signs of aggregation in the high-purity concentrate, but a significant and progressive aggregation of nonspecific protein contaminants present in the intermediate-purity concentrate, making the high-purity concentrate preferable for heat treatment