Binding of glycosaminoglycans to cyano-activated agarose membranes: kinetic and diffusional effects on yield and homogeneity

Methods were developed for binding a glycosaminoglycan (GAG, a 50 kDa chondroitin sulfate) to thin agarose membranes using 1-cyano-4-(dimethylamino)pyridinium tetrafluoroborate (CDAP) as the activating agent. Process conditions were optimized to achieve high yields and spatially uniform concentrations of bound ligand. Yields were varied mainly by manipulating the duration and temperature of the aqueous washes prior to coupling, which affected the concentration of active sites available for subsequent GAG binding. The rate constants for degradation of the active cyanate esters in 0.1 M bicarbonate solutions were 0.24 ± 0.02 h−1 at 4 °C and 0.08 ± 0.03 h−1 at 0 °C. Steric limitations in the 3% agarose gels severely restricted binding, with only about 0.1% of active sites being accessible to GAG molecules. The GAG binding occurred primarily in the outer 50–70 μm of the membranes, so that coupling was homogeneous only for thin gels. A model of GAG diffusion and reaction in the coupling step was developed to explain the observed effects of parameters such as the GAG concentration in solution and the membrane thickness. An analysis of the key time scales in the synthesis provides design principles that should be useful also for other cyanylating agents, other ligands, and for beads as well as membranes.

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