Howard Bernstein

[46] Immobilized heparin lyase system for blood deheparinization

Publisher Summary This chapter illustrates immobilized heparin lyase system for blood deheparinization. The properties of immobilized heparinase are discussed. The activity retained based on activity bound is between 50–90%. The beads are stored in sterile phosphate-buffered saline at 4° until they are used. Heparinase immobilized to agarose via cyanogen bromide has an enhanced thermal stability. An immobilized heparinase filter could be used in a variety of clinical situations. For example, it might be used at the end of a clinical procedure to eliminate heparin without the toxic effects of heparin-neutralizing substances, such as protamine. Alternatively it might be used continuously in such situations, such as kidney dialysis, continuous arteriovenous hemofiltration, or extracorporeal membrane oxygenators, to prevent high levels of heparin from ever entering the patient. Blood filters as large as 2 liters are used at the effluent of extracorporeal devices to remove microemboli. Heparinase might be bonded to the biomaterials of these filters.

Removal of the anticoagulant activities of the low molecular weight heparin fractions and fragments with flavobacterial heparinase

Recently, the development of low molecular weight heparin fractions and fragments (LMHF) as potential antithrombotic agents has gained increased attention. However, the lack of antagonists to neutralize the anticoagulant effects of these drugs may seriously exclude them from possible uses in extracorporeal therapy. This is mainly because of the concern that the high dosage of the drugs employed in extracorporeal therapy could lead to serious bleeding risks. Our earlier work has demonstrated that immobilized heparinase can remove polydisperse heparin both in vitro and in vivo. To examine whether such a system may be used as a novel approach to neutralize the anticoagulant effects of LMHF, different LMHF were tested using heparinase. In vitro data showed that both the APTT and anti-FXa activities of the LMHF including Kabi 2165, PK 10169, Cy 216 and CY 222 were nearly completely eliminated by heparinase in less than 20 min. This study suggests that an immobilized heparinase system may be an useful element for the acceptance of the LMHF for their use in extracorporeal therapy.

An investigation of heparinase immobilization

A systematic investigation of the parameters that affect the efficiency of immobilizing heparinase onto cyanogen bromide activated crosslinked 8% agarose beads was conducted. Two experimental measures, the “fraction bound” and the “fraction retained,” were used to monitor the coupling efficiency. The fraction bound is the portion of the total initial enzyme that is bound to the agarose gel. The fraction retained is the fraction of bound enzyme that is active. The product of the two measures indicates the coupling efficiency. The activity of the immobilized heparinase was measured under conditions free of both internal and external mass transfer limitations, and thus, the fraction retained represents the true immobilized enzyme activity.Increasing the degree of activation of the beads results in an increase in the fraction bound, the fraction retained, and consequently, the coupling efficiency. As the ratio of enzyme solution to gel volume increases from 1.5 to 2.2, the fraction bound remains constant but the fraction retained decreases (heparinase concentration; 0.15 mg/mL and degree of activation; 9.5 μmol of cyanate esters/g of gel). At volume ratios greater than 2.2, both the fraction bound and the fraction retained decline continuously. Changing the heparinase concentration in the coupling solution changes the coupling efficiency in a manner similar to that of the volume ratio change.When heparin is added during the coupling process, the fraction bound declines as the heparin concentration increases, whereas the fraction retained increases up to a heparin concentration of 12 mg/mL and decreases thereafter. When arginine, lysine, and glycine are used to block the unreacted cyanate ester groups after the coupling process, the immobilized heparinase shows different pH optima of 6.5, 6.9, and 7.2, respectively. Based upon these findings, a protocol to optimize heparinase immobilization is developed.

Large Scale Preparation and Characterization of Mucopolysaccharase Contamination Free Heparinase

By a combination of hydroxylapatite chromatography and negative adsorption on QAE-Sephadex at pH 8.3, heparinase (E.C.4.2.2.7) can be successfully isolated from all the other mucopolysaccharase contaminants present inFlavobacterium heparinum. Hydroxylapatite isolates heparinase primarily from chondroitinases, hyaluronidase, and most glycuronidases. QAE-Sephadex chromatography at pH 8.3 further separates heparinase from heparitinases, sulfatases, and the remaining glycuronidases. The heparinase preparation thus obtained contains no statistically significant levels of other contaminating mucopolysaccharases except for heparitinases that are present at an apparent maximum level of 3.4%. Owing to the presence of a crossreaction of heparinase on heparitin sulfate at conditions employed for the assay of heparitinase, the heparitinase level of 3.4% could be misleading because of the action of heparinase on heparitin sulfate. Characterization of this heparinase preparation shows that the enzyme has an optimum salt concentration of 0.08M NaCl, an optimum pH of 6.5, an activation energy of 5 kcal/mol, and a Km of 7.95 x 10-6 M. These parameters are almost identical to those displayed by a homogeneous heparinase preparation. The method described here is suitable for scale-up purposes using batch Chromatographic procedures.

Kinetics of immobilized heparinase in human blood

Immobilized enzyme reactors can form the basis of useful blood detoxification systems. One such reactor was developed for heparin neutralization by immobilized heparinase. In this article, reactor kinetics were studied under clinically relevant conditions. Heparin neutralization was assessedin vitro in whole human blood using (a) a well-mixed batch reactor, and (b) an oscillating, continuous-flow reactor. The kinetics of heparin neutralization in human blood were first order over the entire range of heparin and enzyme concentrations and particle fractions tested. The kinetic rate was not sensitive to physiological variations in the concentration of antithrombin, a heparin binding protein in blood. Enzyme activity did not decrease significantly over the 2 hour test period. Kinetic control of the system with minimal intraparticle diffusional limitations was suggested by the Thiele moduli (0.11–0.67) and effectiveness factors (0.98±0.01). The ratio kcat/Km obtained in batch studies was 0.0028±0.0008 cm3/μg-min. A continuous-flow oscillating reactor within a closed recirculation loop performed as a single well mixed batch reactor; there was a short mixing time of recirculating blood when compared to reaction time. A model based on this mixing pattern and the kinetics obtained in independent batch studies accurately predicted heparin neutralization profiles observed in the continuous-flow system.

Heparinase Immobilization Characterization and Optimization

Lheparinase de flavobacterium heparinum est fixee sur differents supports de reticulation et de porosites differentes (sepharose, cellulose, sephadex, polyacrylamide, acrylate). Lagent de couplage est selectionne pour que le nombre de groupements actives soit suffisant a la fixation de lenzyme. Optimisation de la technique pour utiliser le biocatalyseur dans une circulation extracorporelle