In this study, we describe a new approach for the characterization of process-related impurities along with an in silico tool to generate orthogonal, integrated downstream purification processes for biological products. A one-time characterization of process-related impurities from product expression in Pichia pastoris was first carried out using linear salt and pH gradients on a library of multimodal, salt-tolerant, and hydrophobic charge induction chromatographic resins. The Reversed-phase ultra-performance liquid chromatography (UPLC) analysis of the fractions from these gradients was then used to generate large data sets of impurity profiles. A retention database of the biological product was also generated using the same linear salt and pH gradients on these resins, without fraction collection. The resulting two data sets were then analyzed using an in silico tool, which incorporated integrated manufacturing constraints to generate and rank potential three-step purification sequences based on their predicted purification performance as well as whole-process "orthogonality" for impurity removal. Highly ranked sequences were further examined to identify templates for process development. The efficacy of this approach was successfully demonstrated for the rapid development of robust integrated processes for human growth hormone and granulocyte-colony stimulating factor.

Integrated designs of chromatographic processes for purification of biopharmaceuticals provides potential gains in operational efficiency and reductions of costs and material requirements. We describe a combined method using screening and in silico algorithms for ranking chromatographic steps to rapidly design orthogonally selective integrated processes for purifying protein therapeutics from both process-and product-related impurities. IFN-α2b produced in Pichia pastoris containing a significant product variant challenge was used as a case study. The product and product-related variants were screened on a set of 14 multimodal, ion exchange, and hydrophobic charge induction chromatography resins under various pH and salt linear gradient conditions. Data generated from reversed-phase chromatography of the fractions collected were used to generate a retention database for IFN-α2b and its variants. These data, in combination with a previously constructed process-related impurity database for P. pastoris, were input into an in silico process development tool that generated and ranked all possible integrated chromatographic sequences for their ability to remove both process and product-related impurities. Top-ranking outputs guided the experimental refinement of two successful three step purification processes, one comprising all bind-elute steps and the other having two bind-elute steps and a flowthrough operation. This approach suggests a new platform-like approach for rapidly designing purification processes for a range of proteins where separations of both process-and product-related impurities are needed. K E Y W O R D Shigh throughput process development, integrated downstream processing, mixed mode chromatography, process development tool, protein chromatography, straight-through processing

A diverse array of experimental techniques was used to compare protein sorption and transport on agarose-based resins with and without dextran extenders. Van 't Hoff plots indicated similar trends for lysozyme on the resins, with enthalpically dominated sorption. Salt breakthrough curves demonstrated significant exclusion of salt from the dextran, consistent with Donnan equilibrium predictions. Polarization-sensitive confocal microscopy and fluorescence recovery after photobleaching (FRAP) exhibited little effect of dextran modification on the respective rotational and translation mobilities of adsorbed protein at equilibrium. Finally, confocal microscopy imaging of intraparticle elution behavior showed that slower elution can occur for dextran-modified resins.

BACKGROUND: In this study, we have demonstrated the design, screening and selection of peptide ligands for the affinity capture of human growth hormone (hGH) from yeast cell cultures. RESULTS:Ligand design was carried out using multiple approaches based on primary sequence and structures of natural binding partners of hGH. Screening of potential affinity peptides was conducted using high throughput microarray platforms followed by assessment of in-solution binding to hGH using fluorescence polarization. Peptide leads were subsequently immobilized on chromatographic resins and the binding and desorption behavior was examined using batch adsorption studies. A lead candidate was examined in further details in column chromatography studies which indicated that while high purity was attained, further refinement was necessary for improved product recovery. Histidine scanning was employed to successfully improve the recovery of hGH from cell culture fluid while still maintaining high purity. Finally, proof-of-concept was demonstrated in the column format using complex feed stock where a product purity of 95% was attained at 80% yield. CONCLUSION: The approaches presented here can be translated to other biologics of interest for the rapid development of affinity based purification processes.

A fully-mechanistic understanding of protein transport and sorption in chromatographic materials has remained elusive despite the application of modern continuum and molecular observation techniques. While measuring overall uptake rates in proteins in chromatographic media is relatively straightforward, quantifying mechanistic contributions is much more challenging. Further, at equilibrium in fully-loaded particles, measuring rates of kinetic exchange and diffusion can be very challenging. As models of multicomponent separations rely on accurate depictions of protein displacement and elution, a straightforward method is desired to measure the mobility of bound protein in chromatographic media. We have adapted fluorescence recovery after photobleaching (FRAP) methods to study transport and exchange of protein at equilibrium in a single particle. Further, we have developed a mathematical model to capture diffusion and desorption rates governing fluorescence recovery and investigate how these rates vary as a function of protein size, binding strength and media type. An emphasis is placed on explaining differences between polymer-modified and traditional media, which in the former case is characterized by rapid uptake, slow displacement and large elution pools, differences that have been postulated to result from steric and kinetic limitations. In conclusion good qualitative agreement is achieved predicting flow confocal displacement profiles in polymer-modified materials, based solely on estimates of kinetic and diffusion parameters from FRAP observations.