Cell affinity separations using magnetically stabilized fluidized beds: Erythrocyte subpopulation fractionation utilizing a lectin-magnetite support

Abstract

A magnetically stabilized fluidized bed is used to separate erythrocyte subpopulations. Binding specificity was obtained by immobilizing the lectin Helix pomatia Agglutinin (HpA) or Griffonia simplicifolia I (GSI) onto a magnetite-containing support. Separation of type A and type O erythrocytes with the lectin HpA was particularly effective, leading to a 94% purity of retained type A erythrocytes. A 3.1 ± 0.6 log removal of type A erythrocytes was also accomplished leading to a 99.7% ± 0.4% purity and 95% ± 7% yield of type O erythrocytes in the collected effluent. Elution of the purified cells was accomplished using fluidization in the presence of a sugar competing for the lectin–erythrocyte binding site. A mathematical model based on the depth filtration model of Putnam and Burns (Chem Eng Sci 1997;52(1):93–105) was extended to include multicomponent cell adhesion. This filtration model is the first to take into account the finite binding capacity of the chromatographic support and is used to characterize the cell binding behavior and to determine optimal parameters and conditions that lead to high capacities and selectivities. Model parameter values and observations from in situ adsorption studies suggest that the non-spherical shape of the magnetite-based support allows for a more efficient utilization of the support surface area than the spherical shape. Using a 1.5-cm diameter laboratory column and realistic parameter values, the processing rates of the system are predicted to be at least an order of magnitude greater than the 10 8 /h cells that can typically be processed in packed bed cell affinity chromatography (CAC) systems. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 81: 650–665, 2003.