Continuous purification of a clotting factor IX concentrate and continuous regeneration by preparative annular chromatography.

Preparative continuous annular chromatography, a method to separate proteins in a truly continuous manner, was investigated in an industrial environment. Plasma-derived clotting factor IX concentrate was used as model protein. Separation of vitronectin, a common impurity in commercial available factor IX concentrates, from factor IX was studied and compared to conventional packed bed chromatography in batch mode. As sorbent, Toyopearl DEAE 650M was used. Regeneration was performed simultaneously with the purification of factor IX in continuous mode. All required parameters applied for preparative annular chromatography such as feed flow-rate and elution flow-rate were first estimated from experiments on conventional batch columns. Then preparative annular chromatography and conventional packed beds were compared regarding enrichment, purity and productivity. Three different process scenarios, the optimal batch process,the preparative annular chromatography process and the batch process equivalent to the preparative annular chromatography process were investigated. The productivity of the optimal batch process was higher than that of the preparative annular chromatography and batch process equivalent to the preparative annular chromatography process. Therefore the throughput could not be increased by the use of the continuous chromatographic system.

Administration of human inter-alpha-inhibitors maintains hemodynamic stability and improves survival during sepsis.

OBJECTIVES: The major forms of human inter-alpha-inhibitor proteins circulating in the plasma are inter-alpha-inhibitor (IalphaI, containing one light peptide chain called bikunin and two heavy chains) and pre-alpha-inhibitor (PalphaI, containing one light and one heavy chain). Although it has been reported that a decrease in IalphaI/PalphaI is correlated with an increased mortality rate in septic patients, it remains unknown whether administration of IalphaI/PalphaI early after the onset of sepsis has any beneficial effects on the cardiovascular response and outcome of the septic animal. The aim of this study, therefore, was to determine whether IalphaI and PalphaI have any salutary effects on the depressed cardiovascular function, liver damage, and mortality rate after polymicrobial sepsis. DESIGN: Prospective, controlled, randomized animal study. SETTING: A university research laboratory. SUBJECTS: Male adult rats were subjected to polymicrobial sepsis by cecal ligation and puncture or sham operation followed by the administration of normal saline (i.e., resuscitation). MEASUREMENTS AND MAIN RESULTS: At 1 hr after cecal ligation and puncture, human IalphaI/PalphaI at a dose of 30 mg/kg body weight or vehicle (normal saline, 1 mL/rat) were infused intravenously over a period of 30 mins. At 20 hrs after cecal ligation and puncture (i.e., the late, hypodynamic stage of sepsis), cardiac output was measured by using a dye dilution technique, and blood samples were collected for assessing oxygen content. Oxygen delivery, consumption, and extraction ratio were determined. Plasma concentrations of liver enzymes alanine aminotransferase and aspartate aminotransferase as well as lactate and tumor necrosis factor-alpha also were measured. In additional animals, the necrotic cecum was excised at 20 hrs after cecal ligation and puncture with or without IalphaI/PalphaI treatment, and survival was monitored for 10 days thereafter. The results indicate that administration of human IalphaI/PalphaI early after the onset of sepsis maintained cardiac output and systemic oxygen delivery, whereas it increased oxygen consumption and extraction at 20 hrs after cecal ligation and puncture. The elevated concentrations of alanine aminotransferase, aspartate aminotransferase, tumor necrosis factor-alpha, and lactate were attenuated by IalphaI/PalphaI treatment. In addition, administration of human IalphaI/PalphaI improved the survival rate from 30% to 89% in septic animals at day 10 after cecal ligation and puncture and cecal excision. CONCLUSION: Human IalphaI/PalphaI appears to be a useful agent for maintaining hemodynamic stability and improving survival during the progression of polymicrobial sepsis.

Development and applications of surface plasmon resonance-based von Willebrand factor-collagen binding assay.

Von Willebrand factor (vWf) functions both as a carrier of factor VIII (fVIII) in plasma and as an adhesive protein providing the primary link between collagen of the extracellular matrix and platelets sequestered from blood flow. The functional activity of vWf correlates with the level of its binding to collagen, which is commonly measured in the enzyme-linked immunosorbent assay (ELISA). We developed an automated collagen-binding assay employing the surface plasmon resonance (SPR) phenomenon, which allows one to quantitatively measure the binding of purified vWf and vWf-containing therapeutic fVIII concentrates to collagen type III immobilized on a biosensor chip. The results of the SPR-based assay highly correlated (r = 0.987) with collagen-binding ELISA. The advantages of the SPR-based assay are its higher accuracy and reproducibility in comparison with ELISA. We applied the developed assay for monitoring structural changes in the vWf component of plasma-derived fVIII/vWf concentrates during a virus inactivation procedure performed by heat treatment. We determined the critical residual moisture content of 2% that can be present in lyophilized concentrates during heat-treatment procedures without causing deteriorative changes in vWf properties. Our data suggest that the SPR-based assay is a useful tool in the development of industrial virus-inactivation procedures, allowing one to preserve vWf activity and achieve the maximal therapeutic efficacy of fVIII/vWf concentrates.