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A Mechanistic Dissection of Polyethylenimine Mediated Transfection of Chinese Hamster Ovary Cells

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A Mechanistic Dissection of Polyethylenimine Mediated Transfection of Chinese Hamster Ovary Cells A thesis submitted to The University of Sheffield Department of Chemical and Biological Engineering For the degree of Doctor of Philosophy A Mechanistic Dissection of Polyethylenimine Mediated Transfection of Chinese Hamster Ovary Cells Olivia Louise Mozley 2013 Declaration I, Olivia Louise Mozley, declare that I am the sole author of this thesis and that the research presented within is a result of my own efforts and achievements. Where this is not the case, this has been clearly stated. I confirm that this work has not been submitted for any other degrees. ii ACKNOWLEDGEMENTS Thank you to my supervisor, Professor David James, without whom this thesis would not exist. Thank you for your guidance and inspiration throughout the project. Throughout the course of the project, laboratory colleagues/ friends have made the experience an enjoyable one (you know who you are). Special thanks to Dr Ben Thompson for his expert and invaluable guidance on my project. In addition, thanks to Dr Rhian Grainger, Dr Claire Bennett, Dr Christa Walther and Dr Esther Karunakaran for all helping with laboratory techniques. Last but not least, thank you to Neil Johnson, for everything. FUNDING Thanks to the Sheffield A*STAR Research Attachment Programme. iii LIST OF ABBREVIATIONS ACA anti-clumping agent AF auto-fluorescence bPEI branched PEI BSA bovine serum albumin CPV culture percentage viability CDR complimentarity determining region CHO Chinese Hamster Ovary CS CellScrub CTB cholera toxin B DHFR dehydrofolate reductase DMEM Dulbecco’s modified Eagle Medium DMSO dimethyl sulfoxide DNA deoxyribonucleic acid EST exostosin FBS fetal bovine serum FC ferric (III) citrate FGF fibroblast growth factor FITC fluorescein isothiocyanate GAG glycosaminoglycan Gal galactose GALT galactosyltransferase GalNac n-acetylgalactosamine GFP green fluorescent protein GlcA glucuronic acid GLCAT glucuronyltransferase GlcNAc n-acetylglucosamine GPI glycosylphosphatidylinositol GS glutamine synthetase HACA human anti chimeric antibody HAMA human anti-mouse antibody HS heparan sulphate HSPG heparan sulphate proteoglycan HSnST 2-O-sulphotransferase IdoA iduronic acid lPEI linear PEI LDC limiting dilution cloning mAb monoclonal antibody MβCD methyl beta cyclodextrin MSX methionine sulphoximine MTX methotrexate iv NDST n-sulphotransferase OFAT one factor at a time PBS phosphate buffered saline PCR polymerase chain reaction PEG polyethylene glycol PEI polyethylenimine PFA paraformaldehyde PLL polylysine rDNA recombinant DNA RNS reactive nitrogen species ROS reactive oxygen species RPMI Roswell Park Memorial Institute (culture medium) PEG poly(ethylene glycol) rProtein recombinant protein RSM response surface model SEAP secreated alkaline phosphatase SEC size exclusion chromatography SFM serum free medium siRNA small interfering ribonucleic acid SPION superparamagnetic iron oxide nanoparticles TGE transient gene expression VCD viable cell density XYLT xylosyltransferase Lipids use for transfection DOPE dioleoylphosphatidylethanolamine DOTMA -N,N,N-trimethyl-2,3-bis(z-octadec-9-enyloxy)-1- propanaminium chloride DOTAP 1,2-dioleoyl-3-trimethylammonium-propane (chloride salt) DOSPA -N,N-dimethyl-N-[2-(sperminecarboxamido)ethyl]-2,3- bis(dioleyloxy)-1-propaniminium pentahydrochloride DDAB Dimethyldioctadecylammonium (Bromide Salt) DMRIE -N-(2-hydroxyethyl)-N,N-dimethyl-2,3- bis(tetradecyloxy)-1-propanaminium bromide v ABSTRACT Biopharmaceutical production through transient gene expression (TGE) is used within industry for the rapid supply of product for early stage testing. A key requirement of the process is the large scale transfection of mammalian cells, for which the cationic polymer, polyethylenimine (PEI), is widely used. In this thesis, the mechanism of PEI mediated transfection of CHO-S cells is explored at the cell surface, a fundamental barrier to successful transgene delivery. By approaching the question from first principles, exploring the kinetics of transfection at the cell surface, bio-physical and bio-molecular interactions governing polyplex binding to the cell surface, three key findings were made. Firstly, polyplex uptake was biphasic. Initial, rapid endocytosis of polyplex and heparan sulphate proteoglycans (HSPG) was followed by a slower phase of polyplex uptake, on depletion of cell surface HSPGs. Enzymatic depletion of cell surface HSPGs was found to reduce TGE by 25%, whereas sequestration of cholesterol using methyl-β-cyclodextrin abrogated TGE. Taken together, the data indicate that HSPGs mediate maximal TGE (via an early, rapid phase of endocytosis) but that the predominant mechanism of polyplex uptake is through the clustering of lipid rafts, occurring at depleted cell surface HSPG levels. Secondly, the role of both electrostatic and hydrophobic interactions in polyplex binding to the cell surface was investigated. These experiments revealed that at statistically optimized conditions for TGE (with respect to PEI:DNA ratio) the net charge of the polyplex in chemically defined medium was approximately neutral. Under these conditions polyplexes bound to the cell surface, predominantly, via a hydrophobic interaction, independent of cell surface HSPGs. Accordingly polyplex binding to the cell surface was disrupted by both non-ionic surfactant and depletion of plasma membrane cholesterol by methyl-β-cyclodextrin. An increase in polyplex zeta potential at elevated polyplex PEI:DNA ratio increased polyplex binding to the cell surface, but was accompanied by increased cytotoxicity with elevated PEI internalization. A decrease in polyplex zeta potential using ferric (III) citrate resulted in decreased polyplex binding to the cell surface. Both alterations in polyplex charge reduced TGE. Taken together, vi these data indicate that hydrophobic binding of polyplexes to cell surface lipid rafts (bearing passenger HSPGs) is the primary molecular interaction that promotes subsequent lipid raft clustering and polyplex micro/macropinocytosis to facilitate maximal TGE. Lastly, in order to engineer increased binding and endocytosis of recombinant DNA, alkylated PEIs varying in alkyl chain length and degree of substitution were chemically synthesized in order to increase polyplex hydrophobicity. Compared to unmodified PEI in TGE processes, optimized by Design of Experiments Response Surface Modelling, propyl-PEI was found to mediate more efficient TGE at similar reporter gene titre via a reduction in plasmid DNA load. Propyl-PEI formed polyplexes were found to mediate enhanced polyplex uptake relative to polyplexes formed of unmodified PEI. vii CONTENTS CHAPTER 1: Biopharmaceuticals and their production Chapter Overview................................................................................ 1 1.1 Biopharmaceuticals........................................................................ 1 1.1.1 Recombinant Protein Technology.................................... 2 1.1.2 Antibody Therapeutics..................................................... 2 1.2 Production of Biopharmaceuticals................................................. 5 1.2.1 Expression Systems......................................................... 5 1.2.2 Engineering Strategies to Improve Production................. 6 1.2.3 Stable Cell Line Generation............................................. 8 1.2.4 Stable Transfectant Pool Technology.............................. 10 1.2.5 Transient Gene Expression.............................................. 11 1.2.5.1 Cell Line and Vector Engineering...................... 12 1.2.5.2 Yield Enhancing Additives................................. 15 1.2.5.3 Optimization of Culture Modality........................ 15 CHATER 2: Transfection Chapter Overview................................................................................ 17 2.1 Methods of Transfection................................................................ 17 2.1.1 Viral.................................................................................. 17 2.1.2 Physical............................................................................ 18 2.1.3 Chemical.......................................................................... 18 2.1.3.1 Co-precipitation.................................................. 18 2.1.3.2 Cationic lipids..................................................... 18 2.1.3.3 Cationic polymers.............................................. 20 2.1.3.3.1 Polyethylenimine............................................. 20 2.2 Engineering PEI mediated transfection to improve TGE process.. 21 2.3 PEI:DNA Polyplexes: Cyto-Delivery and Cyto-Trafficking............. 22 (1) PEI:DNA polyplex formation................................................ 23 (5) Endosomal Release............................................................. 23 (6) Nuclear Trafficking............................................................... 25 (7) Nuclear Uptake.................................................................... 25 (8) Transcription of plasmid DNA.............................................. 26 2.4 Thesis overview............................................................................. 27 CHAPTER 3: Materials and Methods 28 3.1 Mammalian cell culture.................................................................. 28 3.2 Cell fixing....................................................................................... 29 3.3 Plasmid DNA preparation.............................................................. 29 3.4 Fluorescent labelling of plasmid DNA...........................................
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