Title page for ETD etd-06062008-170317
|Type of Document
||Kaster, Jeffrey Allen
||Optimization of large beaded cellulose as a chromatographic support
|Velander, William H.
|Conger, William L.
|Davis, Richey M.
|Drohan, William N.
|Glasser, Wolfgang G.
|Date of Defense
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.
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