Texas A&M University LibrariesTexas A&M University LibrariesTexas A&M University Libraries
    • Help
    • Login
    OAKTrust
    View Item 
    •   OAKTrust Home
    • Colleges and Schools
    • Office of Graduate and Professional Studies
    • Electronic Theses, Dissertations, and Records of Study (2002– )
    • View Item
    •   OAKTrust Home
    • Colleges and Schools
    • Office of Graduate and Professional Studies
    • Electronic Theses, Dissertations, and Records of Study (2002– )
    • View Item
    JavaScript is disabled for your browser. Some features of this site may not work without it.
    The full text of this item is not available at this time because the student has placed this item under an embargo for a period of time. The Libraries are not authorized to provide a copy of this work during the embargo period, even for Texas A&M users with NetID.

    EVALUATION OF MIXED-MODE CHROMATOGRAPHY RESINS FOR ISOLATION OF RECOMBINANT THERAPEUTIC PROTEINS

    Thumbnail
    View/Open
    RAVI-DISSERTATION-2019.pdf (5.314Mb)
    Date
    2019-07-12
    Author
    Ravi, Ayswarya
    Metadata
    Show full item record
    Abstract
    Biotherapeutics are lifesaving drugs, but their cost is prohibitively high for widespread use. Multiple chromatography operations required to meet stringent regulatory standards, contributes up to 70% of manufacturing cost. Mixed-mode resins are emerging chromatographic media having multimodal chemistries promising enhanced process selectivity. Their potential to reduce the number of chromatography operations employed, is key to improving affordability of biotherapeutics. This dissertation assessed the capabilities of mixed-mode resins, using two recombinant therapeutic proteins with challenging purification needs. Mixed-mode ceramic hydroxyapatite (CHT) resin, was tested on microalgae, Chlamydomonas reinhardtii lysate, containing phosphorylated bone regenerative protein, osteopontin, in low purity (0.1-0.2% w/w). Complete binding of osteopontin to CHT through electrostatic interactions and Ca^2^+ coordination required 1500 mM sodium phosphate followed by 100 mM NaOH elution buffers to recover 86% of osteopontin. CHT distinguished between phosphorylated and non-phosphorylated Escherichia coli osteopontin, however, also co-purified impurity proteins (Rubisco) with similar properties. Versatility of mixed-mode resins was tested with osteopontin lacking phosphorylation, expressed intracellularly in E. coli. High-throughput screening of mixedmode resins, established HEA HyperCel as the capture chromatography promising 14- fold increase in purity. Interestingly, ensuing protein elution with NaCl from HEA HyperCel, the interaction with Capto Q resin was orthogonal resulting in 95% purity and 44% overall recovery. For the potential treatment of cystic fibrosis, human thioredoxin, was expressed in E. coli in high titers. HEA HyperCel resin bound thioredoxin at moderate conductivity of 5 mS and provided highest elution purity. However, unlike osteopontin, elution with NaCl buffer had the caveat of 45% thioredoxin recovery. Molecular modelling revealed existence of electrostatic and van der Waals interactions, supplementing experimental results. Simultaneous targeting of all interactions with 0.1 M arginine in 1 M NaCl buffer resulted in 100% recovery of biologically active, monomeric thioredoxin. This study established that mixed-mode resins fare well in capturing therapeutic proteins from complex lysates, irrespective of the expression titer. Their flexibility in binding conditions eliminate the processing cost and time associated with conditioning operations. However, the protein-ligand interaction is driven by the properties of the target protein and process development with a thorough understanding of interaction mechanism is essential to realize their maximum potential.
    URI
    http://hdl.handle.net/1969.1/186497
    Subject
    Proteins
    chromatography
    mixed-mode chromatography resins
    osteopontin
    thioredoxin
    downstream process development
    Collections
    • Electronic Theses, Dissertations, and Records of Study (2002– )
    Citation
    Ravi, Ayswarya (2019). EVALUATION OF MIXED-MODE CHROMATOGRAPHY RESINS FOR ISOLATION OF RECOMBINANT THERAPEUTIC PROTEINS. Doctoral dissertation, Texas A&M University. Available electronically from http : / /hdl .handle .net /1969 .1 /186497.

    DSpace software copyright © 2002-2016  DuraSpace
    Contact Us | Send Feedback
    Theme by 
    Atmire NV
     

     

    Advanced Search

    Browse

    All of OAKTrustCommunities & CollectionsBy Issue DateAuthorsTitlesSubjectsDepartmentThis CollectionBy Issue DateAuthorsTitlesSubjectsDepartment

    My Account

    LoginRegister

    Statistics

    View Usage Statistics
    Help and Documentation

    DSpace software copyright © 2002-2016  DuraSpace
    Contact Us | Send Feedback
    Theme by 
    Atmire NV