| Monoclonal antibodies and Fc-fusion proteins form the largest and most rapidly expanding category of biopharmaceuticals today with annual sales exceeding {dollar}8 billion and applications across a wide range of diseases. While Protein-A affinity chromatography has been universally applied as the primary purification step for process-scale production of these molecules, it suffers from disadvantages of high cost, limited throughput and the need for extensive methods development. This thesis seeks to improve fundamental understanding of Protein-A chromatography and to address these issues. To circumvent the traditional trade-off between cost and throughput during Protein-A chromatography, a novel dual flow-rate loading strategy has been developed to enable simultaneous improvements in both capacity and throughput. In the first work of its kind, an explanation has been obtained for elution pH differences among antibodies during Protein-A chromatography. These were found to be the result antibodies interacting with the Protein-A ligand through their variable regions in preference to conventional interactions through their Fc-regions. Eliminating variable-region interactions by use of an engineered Protein-A ligand resulted in a similar elution pH for all molecules, thus enabling the use of generic process conditions for Protein-A purification. In addition, for the first time, differences in binding capacity observed among molecules have been explained through steric hindrance to binding exerted by the non-binding domain of the molecules. This work also provides the first critical exploration of selectivity and binding mechanism on commercially available "alternatives" to Protein-A chromatography (HCIC and mimetic systems). A novel formalism to explain both preparative and analytical behavior in these systems has been developed.; This thesis provides an improved understanding of molecular level interactions with Protein-A chromatographic supports. The strategies developed here enable the rapid development of optimal process-scale separations, and also bear significant implications for the economics of antibody production. The development of generic operating conditions for this mode of chromatography are expected to result in significant savings in time and resources during industrial process development and to remove bottlenecks for the rapid introduction of these drug candidates into the clinic. This work has been the result of a joint collaboration between Amgen, the world's largest biotechnology company, and Rensselaer. |
