Mathematical Biology seminar

Keith Roper
Department of Chemical Engineering, University of Utah
"Adenovirus binding, Elution and Equilibrium Measured by Surface Plasmon Resonance"
October 20
3:05pm in LCB 215

Many diseases such as cancer, diabetes, hemophilia, cystic fibrosis, heart disease and musculoskeletal disorders have an underlying genetic basis. Using human genome sequence data, correlated DNA mutations can be identified that could be altered to correct or prevent gene-related disorders. One treatment approach is gene therapy: inserting correct copies of the altered gene into non-germline cells using nanometer-sized viral or synthetic liposome vectors as delivery vehicles. About 90% of gene vectors used clinically are virus derivatives, mostly retrovirus (35%), followed by adenovirus (27%). About six hundred gene therapy clinical trials are in progress to target cancer (60%), treat monogenic diseases like hemophilia or cystic fibrosis (10%) or combat infectious disease like HIV (6%). Manufacture of viral gene vectors begins with propagation in mammalian cells followed by recovery using consecutive size-selective and adsorptive purification steps. High-resolution chromatographic purification of adenovirus is vital, since CBER recommends <100 picograms of residual DNA per dose from mammalian-cell products and <100 virions per infectious unit. But chromatography of viruses is limited by size and complexity of viral vectors and availability of suitable media and protocols for virus adsorption. Existing resins have been optimized to purify small synthetic organic molecules (104 Da) or recombinant proteins (10^6 Da). Spherodex( resin, for example, has 100-nm pores that exclude particles >10^7 Da including adenovirus (1.65x10^8 Da). ResourceQ( resin has a detection limit for adenovirus that exceeds 1x10^8 particles. These limitations are cost-prohibitive and motivate development of more suitable media. Development of chromatographic media suited to viral gene vector adsorption requires understanding adsorption, desorption and equilibrium binding interactions. We measure adsorption and desorption rate and equilibrium binding constants of Adenovirus Type 5 on model gold and derivatized gold surfaces using surface plasmon resonance. Colloidal Ad5-surface interactions arise primarily from electrostatic potential and van der Waals forces captured by DLVO theory. Modulation occurs by (1) adsorbed polymers that cause attractive bridging at low doses and steric repulsion at higher doses; (2) attraction from free energy reductions that result from exclusion of water from approaching hydrophobic surfaces; (3) repulsion arising from dehydration of approaching hydrophilic surfaces; and (4) heterogeneity of surface charge and structure. We estimate the magnitude of these effects by adjusting solution components such as nonionic surfactants that accommodate apolar hydrophobic groups into the aqueous environment, and small single-charged ions like chloride or multivalent ions like Ca2+ or Mg2+ that promote hydrophobic adsorption by structuring water. Our measurements are useful to optimize adsorbate physicochemistry and binding conditions for Adenovirus Type 5 and allow screening of candidate surfaces.