The influence of bond chemistry on immobilized enzyme systems for ex vivo use

Abstract

The ideal derivatized support for the clinical use of an immobilized enzyme system should irreversibly bind active enzyme. We have investigated the behavior of heparinase and bilirubin oxidase immobilized via cyanogen bromide, tresyl chloride, epoxide, or carbodiimidazole activated natural and synthetic matrices. The protein bound to each activated support was 90% for cyanogen bromide (CNBr) activated agarose, 50-80% for tresyl chloride activated agarose, and 50% for oxirane activated acrylic (Eupergit C). The activity retention of immobilized heparinase was greatest (50%) with CNBr activated agarose while for the immobilization of bilirubin oxidase, the activity retention was greatest (25-30%) with tresyl chloride activated agarose and oxirane activated acrylic.The stability of the different covalent bonds was studied in vitro with radioiodinated enzymes. The leaching profiles showed the same trends for each support and chemistry. A plateau in protein leaching was reached after a few hours of incubation and the transient leaching period was well represented by a logarithmic function of time. The amount of enzyme released from the least stable support (CNBr activated agarose) in 24 h was injected intravenously in New Zealand white rabbits. Using an indirect enzyme-linked immunosorbant assay (ELISA), no immune response was detected. The transient leaching profile was shortened by washing the enzyme-support conjugate with 1M hydroxylamine, pH8.5 intermolecular cross-linking with glutaraldehyde also improves the enzyme-support stability. Tresyl chloride and oxirane activated supports produce bonds with improved stability without adversely affecting enzymatic activity.

Separation of hexahistidine fusion proteins with immobilized metal ion affinity chromatographic (IMAC) sorbents derived from MN+‐tacn and its derivatives

“…These conditions enable appropriate clean-in-place (CIP) procedures to be implemented under many operational conditions. In common with observations of other workers (Comfort et al, 1988;Sundberg and Porath, 1974;Thillaivinayagalingam et al, 2007) comparing the influence of different activation chemistries, the cumulative leakage of these low molecular weight ligands from the oxiraneactivated gels was uniformly low, for example, <<0.01% after 25 days at pH 7.0-11.0. Inclusion of 300 mM NaCl, 10% glycerol in the incubation buffer was intended to decrease the electrostatic component of the interactions between the proteins and the IMAC ligates and to stabilize the conformation of the recombinant protein.…”

An approach for the stable immobilization of proteins

“…The leaching in blood was observed after gel samples were initially washed with only 20 vol of buffer containing 0.5M NaC1. 3 The possibility that blood proteases could be responsible for some leaching cannot be ruled out.…”

Simulation of Bilirubin Detoxification in the Newborn Using an Extracorporeal Bilirubin Oxidase Reactor

“…(13) showed a single protein band indicating high enzyme purity. Immobilization of bilirubin oxidase onto tresyl-chloride activated agarose beads was performed as described by Comfort et al (12). Briefly, 1 mL of enzyme solution at a concentration of 3 mg/mL was incubated at 22°C for 4 h in the presence of I mL of active beads.…”

“…Lavin et al (7) used cyanogenbromide-activated agarose beads with the same amount of enzyme immobilized per volume of beads. However, the use of bilirubin oxidase immobilized onto tresyl-chloride-activated beads may be preferrable because tresyl-chloride chemistry has been shown to yield more stable protein-support bonds (12). CONCLUSION A simulation of bilirubin distribution in the body and subsequent removal by extracorporeal bilirubin oxidase treatment was developed to provide additional understanding of the detoxification and to predict the treatment efficacy in hyperbilirubinemic neonates.…”