• Login
    View Item 
    •   DSpace@RPI Home
    • Rensselaer Libraries
    • RPI Theses Online (Complete)
    • View Item
    •   DSpace@RPI Home
    • Rensselaer Libraries
    • RPI Theses Online (Complete)
    • View Item
    JavaScript is disabled for your browser. Some features of this site may not work without it.

    Investigations into multimodal chromatographic selectivity using in silico designed Fab fragment variants and column modeling

    Author
    Karkov, Hanne Sophie
    View/Open
    176004_Karkov_rpi_0185E_10629.pdf (12.72Mb)
    Other Contributors
    Cramer, Steven M.; Karande, Pankaj; Tessier, Peter M.; Ahmadian, Haleh; Sejergaard, Lars;
    Date Issued
    2015-05
    Subject
    Chemical and biological engineering
    Degree
    PhD;
    Terms of Use
    This electronic version is a licensed copy owned by Rensselaer Polytechnic Institute, Troy, NY. Copyright of original work retained by author.;
    Metadata
    Show full item record
    URI
    https://hdl.handle.net/20.500.13015/1478
    Abstract
    Downstream bioprocessing is continuously faced with challenges requiring the development of more efficient and robust bioseparation technologies. Multimodal chromatography has the potential to provide improved selectivity and efficiency over traditional single mode chromatography. Despite a growing interest in multimodal materials, the lack of fundamental understanding associated with the multiple interactions present in these materials is limiting their implementation in industrial bioprocessing. In this thesis, a multi-tool framework will be presented for improving the understanding of selectivity of complex biomolecules in multimodal chromatographic systems.; The use of in silico protein surface property analyses combined with molecular biology techniques, protein expression and chromatographic evaluations represents a previously undescribed and powerful approach for studying multimodal selectivity with complex biomolecules. Importantly, the studies open new avenues of employing in silico protein surface characterization techniques and column modeling to aid in process design for product variant separations using multimodal chromatography.; The Fab variants were employed to investigate the effects of changing the mobile phase conditions and multimodal ligand design. Using arginine as a mobile phase modifier, Fab variant elution was promoted at a lower ionic strength. In contrast, the presence of guanidine amplified the selective trends of the multimodal system and created a window of selectivity for the separation of Fab variants which could not be separated in the system without the modifier. A novel set of multimodal chromatographic ligands were employed to examine the effects of changing the ligand structure on Fab variant retention and selectivity. Interestingly, the multimodal chromatographic ligands exhibited different selectivity trends and were capable of differentiating between different Fab classes and related variants.; Unique libraries of Fab fragment variants were designed in silico to examine the relationship between protein surface properties and selectivity in multimodal chromatographic systems. Protein surface property characterization tools were employed to identify potential multimodal ligand binding regions on the Fab fragments and to evaluate the impact of mutations on surface charge and hydrophobicity. To broaden the analysis, a diverse set of Fab variants with various CDR loops and/or framework regions was included in the analyses. Forty different Fab variants were then generated by site-directed mutagenesis, transient expression in HEK293 cells and purification by affinity chromatography. Gradient column experiments were carried out with the resulting Fab variants in various chromatographic systems to enable detailed investigations into the relative importance of local protein surface properties on protein retention and selectivity. The retention data demonstrated that the multimodal chromatographic systems provided different selectivity than traditional single mode chromatography.; The utility of multimodal chromatography in large-scale bioprocessing was investigated through the development of a column simulation model. This modeling approach was successfully implemented to describe highly selective multimodal purification processes with model proteins and complex industrial protein mixtures in concert with mobile phase modifiers as the eluents.;
    Description
    May 2015; School of Engineering
    Department
    Dept. of Chemical and Biological Engineering;
    Publisher
    Rensselaer Polytechnic Institute, Troy, NY
    Relationships
    Rensselaer Theses and Dissertations Online Collection;
    Access
    Restricted to current Rensselaer faculty, staff and students. Access inquiries may be directed to the Rensselaer Libraries.;
    Collections
    • RPI Theses Online (Complete)

    Browse

    All of DSpace@RPICommunities & CollectionsBy Issue DateAuthorsTitlesSubjectsThis CollectionBy Issue DateAuthorsTitlesSubjects

    My Account

    Login

    DSpace software copyright © 2002-2022  DuraSpace
    Contact Us | Send Feedback
    DSpace Express is a service operated by 
    Atmire NV