Browsing by Subject "cell culture"

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    Investigating The Interactions Of Polycations With Nucleic Acid And The Mechanisms Of Delivery
    (2015-05) Sprouse, Dustin
    Polymers - large macromolecules composed from many smaller subunits - have ever-growing uses and potentials in our lives. More specifically, cationically charged polymers have been widely explored as non-viral vectors to deliver nucleic acid to cells in an effort to regulate gene and protein expression. The polymeric vehicle must not only bind and complex with DNA to protect and deliver it to the targeted site, but also efficiently dissociate from the DNA and be non-toxic to the cell or host organism. Herein lies the wide array of polymers that must be rationally designed and synthesized in order for the delivery vehicle to perform its specific function. At the turn of the century, two monumental achievements paved way for gene therapy. First, the Human Genome Project was completed. This milestone continues to unravel important information about the genetic basis of human health, disease, hereditary, and genetic dispositions. Second, RNA interference was discovered, an innate cellular pathway to control gene expression within cells. These discoveries afforded scientists the information necessary to move forward with controlling gene and protein expression profiles. More recently, CRISPR-cas technology was discovered, which allows scientists to permanently edit the genetic code by either regulating genes or adding, disrupting, deleting, or altering the specific base-pairs within the DNA sequence. Herein, we investigate several classes of polymers and macromolecules for the complexation and delivery of nucleic acid, including: amino acids, dendrimers, micelles, and linear homo- and block polymers. Initially, it was shown that polymer type, length, charge, dispersity, and composition greatly affect the efficacy of these therapeutic delivery vehicles. With this in consideration we set out to explore some of the fundamental properties of polymeric vectors. Diblocks, triblocks, and statistical copolymers were designed and synthesized with varying amounts of primary and tertiary amines. These were complexed with pDNA to from polyplexes and probed for their toxicity, stability, gene expression profiles, and mechanisms of membrane permeability. Amphiphilic polymers were also synthesized, which in aqueous environments spontaneously self assembled into core-shell structured micelles. These were probed for their ability to change size in different buffers and form different sized aggregates with DNA.
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    Systems Analysis In Mammalian Cell Biomanufacturing - Linking Energy Metabolism And Glycosylation
    (2018-05) Le, Tung
    Mammalian cells have been the major workhorse to produce therapeutic proteins owing to their capability to perform complex post-translation modifications that are essential to the pharmacological activities of these proteins. The performance of mammalian cell culture is greatly affected by cell metabolism, while the robustness of glycosylation patterns of the product proteins still needs improvement. A meta-analysis of cell culture bioprocess data revealed a correlation between lactate metabolism, productivity, and glycosylation patterns. In this study, we develop an integrated platform for generation and visualization of the O-glycosylation network. The platform was used to explore the heterogeneity of O-glycans produced in mammalian cells. In addition, we use a kinetic model of energy metabolism to explain the mechanism behind the increase in lactate production during the late stage of fed-batch cultures. Such a metabolic behavior of lactate can have negative impacts on the culture productivity and potentially, product quality because it occurs during the main production phase. Insights from this study can be helpful in contriving strategies for more robust control of cell metabolism and obtaining consistent glycan patterns of biologics.