Optimization of large beaded cellulose as a chromatographic support
Kaster, Jeffrey Allen
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The design of existing beaded adsorbent materials for column-mode protein purification has emphasized the impact of diffusional transport phenomena upon adsorbent capacity. A design model is presented that optimizes molecular accessibility of proteins relative to the mechanical stability of the material by manipulation of size and solids content for uncross-linked cellulose beads. Cellulose beads of various sizes ranging from about 250 to 1000 pm diameter and having different solids contents were evaluated. Cellulose beads (1.2 mm diameter) gave pressure drops of less than 1 psi per cm of bed at superficial fluid velocities of 100 cm/min in a 1 5 cm bed. Solids content of greater than about 9% cellulose greatly reduced the permeability of large proteins such as thyroglobulin and p-Amylase into the beaded matrix at bead contacting times of 5 and 50 seconds. The amount of permeation in 3% cellulose beads by thyroglobulin at bead contacting times of 5 seconds was about tenfold larger than predicted by diffusion models using the diffusivity of the protein in water. The utility of a low solids content, large bead cellulose support was shown with immobilized IgG (Mr 155 kDa) capturing recombinant human Protein C (M, 62 kDa). The amount of immobilized antibody was varied and immunosorptive capacity of 1 mm cellulose beads was found to be equivalent to that of 0.1 mm cross~linked agarose beads. The immobilization of antibodies to these supports was studied by photomicroscopy of cross-sectioned beads containing immobilized fluorescent labeled antibodies. While 75% of the antibody was immobilized within 0.07 mm of the cellulose bead surface at an antibody density of 1 mg antibody per ml of beads, an appreciable amounts of antibody immobilized deeper into the bead may have been utilized in order to yield capacities equivalent to the smaller agarose beads. The beaded cellulose supports derivatized to form either immunoaffinity or anion exchange matrices exhibited very low non-specific binding. Thus, the particle size, solids content, and extent of derivatization of cellulose matrices can be engineered so as to create matrices that provide high flow rates with low pressure drops while also having desirable adsorptive capacity for proteins.
- Doctoral Dissertations