Immunoaffinity interactions can play a vital role in the purification of therapeutic proteins. In most cases immunoaffinity chromatography is used as the final clean up step to yield a highly pure product. The customized avidity of the parent antibody for a given antigen or hapten can make immunoaffinity chromatography an indispensable tool in the field of bioseparations. However, the use of immunoaffinity chromatography at a large scale is frequently precluded by the high capital costs of antibody columns. This is in part due to the low functional activity of immobilized antibodies and the high cost of pathogen free antibody. The objective of this research was to detail the impact of orientation, multipoint attachment and high local density upon the functional activity of immobilized antibodies. Immobilization methods were designed to affect changes in both orientation and local density. Both metal and pH dependent murine monoclonal antibodies against human Protein C were used as model systems. The character of the antigen and antibody were modified to study the impact of orientation, local density and multipoint attachment. For example, modified antigens of various sizes having a single binding epitope were synthesized from polymer-peptide adducts. The lysyl residues of recombinant hPC were chemically modified to reduce chemical reactivity with the matrix while maintaining avidity for the Mab. The synthetic and recombinant antigens were used to mask the antigen binding regions (Fab) of a monoclonal antibody (Mab) prior to covalent immobilization on a porous membrane and beaded supports. In addition, lysyl residues of Mabs against hPC were chemically modified to reduce multipoint covalent attachment. A 2-step method consisting of permeation and immobilization of antibodies was developed to manipulate local Mab density. Conventional single step immobilization by simultaneous reaction and diffusion produced highly local shell-like density. Immunosorbents made with the 2-Step method at greater than 3 mg Mab / ml gave 2-3 fold higher antigen binding efficiencies and a more uniform distribution of immobilized antibody. Immunosorbents made from Mabs which were immobilized at low local density having masks consisting of large synthetic or recombinant antigens gave the highest antigen binding efficiency and the most accessible Fab domains to pepsin digest. Mabs at high or low densities containing modified or unmodified lysyl residues gave no difference in antigen binding efficiencies or accessible Fab domains to pepsin digests. In summary, for Mabs immobilized at low densities "orientation" of the antibody on the support was identified as having the most negative impact on immunosorbent performance with multipoint attachment being less important. At high antibody loadings the functional activity of antibodies is also impacted by high local density superimposed with orientation. / Ph. D.