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The incidence of sepsis in the United States has risen in the past few years, and mortality from sepsis has risen over the past several decades. These increases have occurred despite the progress made in antibiotic therapy. The high mortality rate may not be related to the bacteria themselves but rather to the host's response. Of particular concern is the bacterial release of endotoxins, the development of endotoxic shock, and the resulting multiorgan failure, which has a high fatality rate and is the leading cause of death in patients admitted to intensive care units. Removing or neutralizing endotoxins are the two major therapeutic approaches to treating sepsis. To date, pharmacological methods have not been successful. Extracorporeal techniques are based on filtration, sorption, or both. Results with hemofiltration have not been definitive, in part because endotoxin levels have not been measured. Sorption devices, based on polymyxin B and anion exchange matrices, do remove endotoxins. Clinical studies in Japan on septic shock or multiorgan failure with polymyxin-B-bound fibers in direct hemoperfusion continue to suggest the utility of extracorporeal techniques for endotoxin removal. Continued effort is required and warranted to assess the clinical efficacy of these techniques.

A composite filter pad formed by embedding activated carbon powder in fibrous matrix was applied for removal of hormones from human serum. Enhanced adsorption was demonstrated through direct comparison between the stacked filter pads and the powered carbon packed in a column both having an equal weight of carbon. In addition, the composite structure offers the advantage of eliminating the carbon fines mixed in the filtrant, avoiding the flow channeling, and providing favorable rate of adsorption. The adsorption of both thyroid hormones T3 (triiodothyronine) and T4 (thyroxine), and steroid hormone, cortisol, by the carbon filter were studied. The effect of pH on hormone adsorption by activated carbon was verified as an important environmental factor, minimum adsorption occurs at the neutral pH. It was found that T4 carrying one more iodine atom than T3 is less effectively adsorped by carbon adsorption from the serum under competitive condition due most likely to their size differences. The filter pad containing 70% activated carbon as adsorbent was fabricated in disk form and stacked in a column for processing bulk volume of biological fluid. Such a device provides an effective means for removal of hormones from biological solutions.

Recombinant Protein A was immobilized on a cellulose and acrylic composite matrix through Schiff base formation. Various factors that could affect the binding of immunoglobulin by the Protein A molecules immobilized on the solid matrix were studied to achieve optimum affinity purification. The spacer arm length and ligand concentration of Protein A were verified as factors crucial to optimized IgG purification. Liquid-phase environmental conditions such as pH and salt concentration also play important roles in adsorption capacity by affecting the molecular interaction between IgG and the immobilized Protein A. The rate of interaction between Protein A and IgG is rather fast, with minimal differences observed at 10-fold increases in the cartridge loading rate. This paper describes a cellulose/acrylic composite matrix for immobilizing Protein A, at an optimized ligand concentration, installed on a spacer arm of adequate length, to purify immunoglobulins from animal plasma. The fast-flow property of the cartridge made from such a matrix and its simplicity in operation provide effective means for purifying immunoglobulins on a relatively large scale.

Endotoxin, a major pyrogen of concern to the biological industry, is a lipopolysaccharide containing a highly hydrophobic region, lipid A, in its structure. The effect of hydrophobic interaction on endotoxin adsorption from an aqueous solution was studied by covalently bonding aminoalkyl groups with varying hydrocarbon lengths to a cellulose and acrylic composite matrix. The amount of endotoxin adsorbed on the matrix increased with the increasing length of alkyl groups, demonstrating the contribution of hydrophobic interaction between endotoxin and the solid matrix. Both the hydrophobic and the charge interaction prove to be effective for endotoxin adsorption, and a synergistic effect from the dual chemical forces is achievable under specified conditions. The effect of solvent, pH and salts on endotoxin adsorption provides further evidence for the importance of hydrophobic force as a means of removing endotoxin from aqueous solutions.

A high level of heparin, infused into blood, often causes severe complications such as hemorrhage, especially when a drug is administered over a long period. The most common way of preventing a patient from bleeding after transfusion is by administering a heparin antagonist such as protamine. The complex molecules formed between heparin and protamine, if left in the bloodstream, may cause hypotension and other side effects. Protamine was immobilized as a bioligand on the affinity matrix formed by grafting an acrylic polymer on cellulose backbone. By flowing blood tangentially along the matrix surface immobilized with protamine, 70-90% heparin reduction was achieved from 1 L of blood containing 10 IU/ml of heparin studied in vitro. The acrylic gel surface avoids lysis of blood, the cellulose support sustains the flow of viscous blood at 50 ml/min, and the tangential flow design permits direct processing of blood without pressure buildup in the system. The example demonstrates the feasibility of applying such a device as a means of immunoadsorptive filter for the selective removal of disease-causing factors from blood.

Bacterial endotoxin, a major quality of parenteral pharmaceuticals, is negatively charged lipopolysaccharide (LPS). A depth filter chemically modified to carry positively charged functional groups is applied to remove endotoxin from aqueous solutions and buffers through charge interaction. The environmental conditions, such as pH and electrolytes, known to affect the efficiency of endotoxin removal by the filter were studied. The charge character of the solid filter was titrated potentiometrically. Other factors such as pyrogen concentration and flow rate were also studied. The effectiveness of depyrogenating the aqueous solution by endotoxin removal through charge interaction with the depth type filter cartridge is fully demonstrated. An optimized cartridge performance can be achieved by following the conditions illustrated in this report. A multi-cell cartridge capable of processing bulk volume of fluid at or over 5 GPM makes it an effective depyrogenating device for the pharmaceutical industry.

The effects of anion-exchange polymeric matrices on endotoxin removal from albumin and gamma-globulin solutions are evaluated. The positively charged cellulose acrylic media carrying DEAE or QAE functional groups remove significant amounts of endotoxin from tap water, but are less effective in protein solutions. With properly controlled pH levels and salt concentrations, the endotoxin level in a protein solution can be reduced; however, low endotoxin concentrations, less than 100 pg/ml, are more difficult to remove. The endotoxin removal capacity depends on the number of functional groups existing in the matrix, expressed as the number of milliequivalents (meq), and on the pH operable range, which is directly related to the pK alpha value of the matrix. The effects of pH and salt on endotoxin removal from albumin and gamma-globulin solutions by an anion-exchange polymeric matrix were evaluated statically in test tubes. In addition, a dynamic flow was performed under statically defined conditions on a 250-ml DEAE cartridge for the removal of endotoxin from albumin at a flow rate of 40 ml/min. A greater than 75% reduction in the endotoxin can be achieved, with protein loss occurring only in the early stage of removal. Such processes are useful for the reduction of endotoxin from biological solutions produced by natural sources or recombinant DNA technology.

To design a bioreactor for removing the potential cancer nutrient L-tryptophan from blood, the L-tryptophan degrading enzyme tryptophan side chain oxidase (TSO) was chemically bound to glutaraldehyde activated gamma amino silane silica and to Zetaffinity microcolumns consisting of a glutaraldehyde activated polyacrylic-cellulose copolymer. Five experiments were carried out in sheep and six experiments in rabbits using a closed circuit plasmapheresis bioreactor system. L-tryptophan in sheep was degraded by the silica bioreactor in a single pass to undetectable levels as measured by high performance liquid chromatography (HPLC). Zetaffinity bioreactors degraded L-tryptophan in rabbits to more than 95% in a single pass. Whole blood L-tryptophan levels changed little throughout the experiment indicating a vast extravascular tryptophan pool. Enzyme leakage from the bioreactor was less than 10(-5) IU TSO per ml plasma. The procedures were tolerated well by the animals without any change in vital signs.

Crude urokinase from human urine processed through foam flotation and ammonium sulfate precipitation containing 720 National Health Institute Committee on Thrombolytic Agents U/mg activity was purified by an SP cation exchanger followed by a zinc-chelated affinity chromatographic cartridge. The cartridges were of a radial-flow type formed by using acrylic and cellulose composite matrices. The high rigidity of the matrix structure permits fast flow of protein solutions (liters per minute) and thus allows processing of a large volume of crude urokinase under low operating pressures. A greater than six-fold increase in specific enzyme activity of urokinase was achieved by adsorbing and eluting 1 l of a 3 mg/ml crude urokinase solution on an SP cartridge. The eluent was further purified by passing through a zinc-chelated affinity cartridge to achieve greater than a eighteen-fold increase in urokinase specific activity. This report demonstrates the combined use of a cation exchanger with zinc-chelated chromatographic cartridges in purifying urokinase on a relatively large scale. The relationship between the amount of zinc chelated in the matrix to its effect on urokinase purification is also discussed.