Strategies for the Development of Integrated Purification Processes for Non-Platform Biologic Therapeutics

The design of chromatographic purification processes for non-platform biologic therapeutics is a complex problem due to the multitude of species involved and the difficulty of predicting their interactions with the separations media. This design problem is made even more challenging if the target therapeutic is to be manufactured in an integrated fashion, in which hold tanks, buffer conditioning, and other steps which allow the design of each unit operation on an individual basis are removed. This thesis explored several strategies to develop purification processes for non-platform biologics which were to be manufactured in a small-scale, integrated system with no conditioning steps or hold tanks between chromatographic unit operations. In the first approach, a multiscale platform for the discovery of peptide affinity chromatography ligands was developed and applied for the purification of human growth hormone (hGH) from yeast cultures. A high-throughput batch screening approach using RP-UPLC was created to rapidly sample portions of the competitive adsorption isotherms for the biologic, allowing the selection of peptide leads based on their abilities to both bind the product from representative mixtures and subsequently desorb it from the chromatographic resin. A peptide ligand, SMWRTYH, was selected as the final candidate for the purification of hGH and in column-scale experiments was shown to purify hGH from yeast (Pichia pastoris) cultures with a product recovery of 80% and purity of 95% after a single step. The second process development approach made use of a novel method for characterizing the host-related impurities in Pichia cultures. This characterization data was analyzed by an in silico tool which synthesized tentative chromatographic purification processes and ranked them based on predicted impurity clearance. The utility of the approach was demonstrated by employing it to develop integrated, 3-step purification processes for two therapeutic proteins using only commercially available separations media. This impurity characterization based approach could have applications in the development of highly constrained purification processes for a wide range of non-platform biologics. Finally, a more straightforward approach was used to develop an integrated purification process for an Interferon mutant. This approach combined the lessons learned from the impurity characterization experiments with the results from a gradient elution screen on the product and its key variants to guide process development. These results demonstrate that integrated purification processes can also be designed for molecules with significant (>30%) product variant separation challenges and that a characterization of the host-related impurities can be used to guide decisions in the development process, even without the use of the in-silico process synthesis tool. The successful implementation of the strategies presented in this thesis provides evidence that a characterization of both the product and the impurity interactions with the separations media can enable the development of highly constrained, integrated purification processes for non-platform biologics. Moreover, this body of work offers perspective on the relative merits and drawbacks of the various strategies that can be employed to design these processes