Design of anion exchange cellulose hydrogel for large proteins
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In our previous studies, uncross-linked large diameter cellulose beads were optimized for solids content, bead size, pressure-flow limits, molecular accessibility and performance as an immunosorbent. Here, anion exchange (DEAE) cellulose beads were derivatized by two different procedures (defined as A and B) and the changes in bead morphology were correlated with transport and sorption kinetics. The kinetic characteristics clearly defined a minimum of two different types of protein binding site architecture. DEAE cellulose beads exhibited molecular exclusion of BSA near the edge of the bead in contrast to greater permeability seen in underivatized beads. Thus, accessible BSA binding sites are present only on the surface of the derivatized beads. DEAE cellulose beads derivatized by procedure B gave higher density of DEAE ligand as compared to beads derivatized by procedure A, as well as higher static and dynamic capacity for BSA. Even though DEAE cellulose beads (DP 2070, 450 μm diameter derivatized by procedure B) have lower small ion capacity than DEAE cross-linked agarose beads, as well as 1/4 the surface area, they exhibit equivalent binding capacity for BSA per volume of support. Thus, DEAE cellulose beads possess more sites per surface area as well as have lower ligand density per BSA site. Furthermore, BSA adsorption sites on DEAE cellulose beads derivatized by procedure B exhibit slow binding kinetics as compared to those derivatized by procedure A and also compared to DEAE crosslinked agarose beads. Thus, the rate limiting step for the adsorption of BSA on DEAE cellulose beads was not diffusion as suggested by the large diameter of the bead. Feasibility studies were performed for process scale applications to fixed and expanded bed anion exchange purification. The large diameter DEAE cellulose beads of this study maybe useful for process scale anion exchange as evident from purification of immunoglobulins from hybridoma cell culture in fixed bed. The balance of large diameter and density of these DEAE cellulose beads enable stable expanded bed purification of proteins such as recombinant human protein C from transgenic porcine whey.
- Doctoral Dissertations