Purification of non-mAb biologics : development of integrated purification processes

Abstract

The first strategy employed for purification process development consisted of identifying affinity peptides for use as chromatographic ligands to selectively capture desired product directly from cell culture fluid. In this strategy, epitope mapping of known target binding partners was used to generate an initial library of peptides. Further, phage display screening with negative selection was used to identify additional peptides to supplement the library of initial peptide candidates. Using immobilized peptide microarrays, the library of candidates was screened to evaluate the affinity and selectivity of the peptides towards granulocyte colony-stimulating factor. Lead peptide candidates identified by this screening method were further evaluated in fluorescence polarization assays to determine their ability to be utilized as affinity ligands.

The second strategy employed for process development consisted of a process screening approach that characterized the chromatographic retention profile for the contaminants from Pichia pastoris cell culture fluid as well as product related materials. Linear gradient screens, 96-well plate batch screens, and high throughput UPLC analytics were used together to obtain retention behavior profiles for the process related impurities and the products. Utilizing a database of these retention behavior profiles, integrated purification process sequences were generated using an in house software and further developed at lab scale for validation. Through this screening method, integrated purification processes for granulocyte colony-stimulating factor, interferon alpha-2b, and various single-domain camelid antibodies were generated that proved capable of recovering product at high yields while reducing impurities such as host cell proteins and DNA to target levels.

Process and product related impurities are critical to remove and characterize during the purification of therapeutic biologics. For mAb based therapeutics, the development of purification processes has the benefit of being able to follow a standard platform template due to the nature of the conserved Fc region on the biologic. However, for non-mAb recombinant protein therapeutics it’s rare that such standard platforms for purification exist, making the development process for purification of these molecules from crude cell culture fluid complex. This work seeks to establish a standard method for the development of purification processes for a variety of non-mAb proteins. The application of this work is primarily in the development of chromatographic capture and polishing processes for biologics produced in Pichia pastoris, specifically granulocyte colony-stimulating factor, interferon alpha-2b, and various single-domain camelid antibodies. Two different strategies for development have been investigated and will be presented.