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Abstract: Resins containing immobilized Staphylococcal Protein A (PA) are widely used in the commercial purification of recombinant human monoclonal antibody (rhuMAb IgG) biotherapeutics. Therefore, a sensitive assay for leached PA is needed to ensure that PA is not present at unacceptable levels as an impurity in the final product. PA impurities are measured by an ELISA using chicken anti-PA antibodies. However, PA in the presence of IgG product forms a PA/IgG complex that interferes in the assay. In this report a multi-product PA ELISA is described, wherein the PA/IgG complex is dissociated by heating in the presence of detergents and chelators prior to the ELISA. The dissociation facilitates the accessibility of the anti-PA antibodies to bind to PA in the immunoassay. Heat is provided by a novel microwave technology which allows brief heating time and high sample throughput using a microtiter plate for sample heating. Thus, broadly applicable dissociation conditions, suitable for all 21 rhMab IgGs tested to date were identified. This approach streamlines the measurement of leached PA, allows higher sample testing throughput, facilitates application across multiple products, and facilitates assay automation. Data comparing in-process samples tested with both the former product-specific ELISA and this new multi-product assay are shown. [Copyright &y& Elsevier]

Objective: Transplanted hearts subjected to prolonged ischemia develop ischemia-reperfusion injury and graft coronary artery disease. To determine the effect of δ-protein kinase C and ϵ-protein kinase C on ischemia-reperfusion injury and the resulting graft coronary artery disease induced by prolonged ischemia, we used a δ-protein kinase C-selective inhibitor peptide and an ϵ-protein kinase C-selective activator peptide after 30 or 120 minutes of ischemia. Methods: Hearts of piebald viral glaxo (PVG) rats were heterotopically transplanted into allogeneic August Copenhagen Irish (ACI) rats. After cardioplegic arrest of the donor heart, ϵ-protein kinase C activator was injected antegrade into the coronary arteries. Hearts were procured and bathed in ϵ-protein kinase C activator, and before reperfusion, δ-protein kinase C inhibitor was injected into the recipient inferior vena cava. Controls were treated with saline. To analyze ischemia-reperfusion injury, grafts were procured at 4 hours after transplantation and analyzed for superoxide generation; myeloperoxidase activity; tumor necrosis factor α, interleukin 1β, and monocyte/macrophage chemoattractant protein 1 production; and cardiomyocyte apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling and caspase 2, 3, 8, and 9 activity. To analyze graft coronary artery disease, another set of animals underwent equal ischemic times and treatment strategies and then after 90 days were analyzed for graft coronary artery disease indexes. Results: All measures of ischemia-reperfusion injury and graft coronary artery disease after 120 minutes of ischemia in the saline-treated group were significantly increased relative to those observed after 30 minutes of ischemia. It is important to note that all ischemia-reperfusion injury parameters and graft coronary artery disease indexes decreased significantly in the protein kinase C regulator-treated group in comparison to saline-treated controls; additionally, these values were equivalent to those in saline-treated controls with 30 minutes of ischemia. Conclusions: Combined treatment with ϵ-protein kinase C activator and δ-protein kinase C inhibitor reduces ischemia-reperfusion injury and decreases the resulting graft coronary artery disease induced by prolonged ischemia. [Copyright &y& Elsevier]

Background: Prolonged cold ischemia is thought to exacerbate ischemia–reperfusion injury and graft coronary artery disease (GCAD). We investigated the effect of varying lengths of cold ischemia on inflammation and apoptosis during ischemia–reperfusion injury and correlated this with the degree of GCAD in rat cardiac allografts. Methods: PVG rat (RT1c) hearts subjected to 30, 60, 90, 120, or 150 minutes of cold ischemia were heterotopically transplanted into ACI rats (RT1a). Grafts were procured after 4 hours of reperfusion and analyzed for superoxide generation, myeloperoxidase activity, tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and monocyte chemoattractant protein-1/chemokine (C-C motif) ligand 2 (MCP-1/CCL2) production, cardiomyocyte apoptosis, and caspase-2, -3, -8, -9 activities. Additional transplanted animals received cyclosporine A (7.5 mg/kg/day) for 10 days as chronic rejection models. Indices of GCAD were determined at 90 days. Results: A direct linear correlation was found between cold ischemic time, ischemia–reperfusion injury, and GCAD. Superoxide generation, myeloperoxidase activity, TNF-α, IL-1β, MCP-1/CCL2 production, cardiomyocyte apoptosis, and caspase-2, -3, -8, and -9 activities increased with ischemic time, peaking at 120 minutes and plateauing at 150 minutes. GCAD, assessed by the percentage of luminal narrowing, the intima/media ratio, and the percentage of diseased vessels, worsened with increased ischemic time, peaking at 120 minutes and plateauing at 150 minutes. All tested variables in both the acute and chronic phases were significantly increased with 120-minute ischemia compared with 30-minute ischemia. Conclusions: These data indicate that the degree of cardiomyocyte apoptosis and inflammatory response in cardiac allografts during ischemia–reperfusion injury depends on the duration of cold ischemia. More important, that prolonged cold ischemia correlates with increased GCAD. [Copyright &y& Elsevier]