title: Development of stable liquid formulations for adenovirus-based vaccines.
abstract: We have evaluated the stability profiles of adenovirus type-5 (Ad5)-based vaccine formulations to identify liquid formulations that are stable during long-term storage at 4 degrees C. By identifying the major physiochemical inactivation pathway(s) during storage, formulations of Ad5 were designed with specific pharmaceutical excipients leading to greatly enhanced stability. For example, results indicate that Ad5 is stabilized by non-ionic surfactants and cryoprotectants as well as excipients known to inhibit free-radical oxidation. A non-ionic surfactant is necessary to prevent adsorption of adenovirus to glass surfaces during storage, and a cryoprotectant is needed to prevent freeze-thaw-induced virus inactivation. In a base formulation (A105) containing sucrose as the cryoprotectant and polysorbate-80 as the non-ionic surfactant, metal-ion catalyzed free-radical oxidation is an important mechanism of Ad5 inactivation. The free-radical oxidation inhibitors ethanol and histidine, combined with the metal-ion chelator ethylenediaminetetraacetic acid (EDTA), were determined to be effective stabilizers of Ad5. Arrhenius plots of stability data are consistent with a first-order inactivation mechanism with apparent activation energies for virus inactivation of 26.5 +/- 0.9 and 28.7 +/- 0.6 kcal/mol in the absence and presence of free-radical oxidation inhibitors, respectively. Optimization of formulation pH, as well as the EDTA and ethanol concentrations, allowed for the identification of formulations that further enhanced long-term storage stability. For example, Ad5 in an optimized liquid formulation (A195) lost <0.1 logs of infectivity after 24 months of storage at 4 degrees C. The immunogenicity of a recombinant Ad5-based human immunodeficiency virus (HIV) vaccine candidate expressing HIV-1 gag (MRKAd5gag) formulated in A195, was shown to be equivalent to the same vaccine formulated in A105. Therefore, the use of EDTA, ethanol, and histidine did not significantly alter the immunogenicity of the vaccine in mice. The identification of 4 degrees C stable liquid formulations should significantly enhance the utility of Ad5 as a vector for vaccines and gene therapy.
authors.affiliation: Department of Vaccine Pharmaceutical Research, Merck Research Laboratories, WP78-302, Sumneytown Pike, West Point, PA 19486, USA. robert.evans@merck.com
authors.forename: Robert K
authors.initials: RK
authors.lastname: Evans
authors.forename: Denise K
authors.initials: DK
authors.lastname: Nawrocki
authors.forename: Lynne A
authors.initials: LA
authors.lastname: Isopi
authors.forename: Donna M
authors.initials: DM
authors.lastname: Williams
authors.forename: Danilo R
authors.initials: DR
authors.lastname: Casimiro
authors.forename: Stephen
authors.initials: S
authors.lastname: Chin
authors.forename: Minchun
authors.initials: M
authors.lastname: Chen
authors.forename: De-Min
authors.initials: DM
authors.lastname: Zhu
authors.forename: John W
authors.initials: JW
authors.lastname: Shiver
authors.forename: David B
authors.initials: DB
authors.lastname: Volkin
mesh_terms: D000260:Adenoviruses, Human / Q000235:genetics / Q000276:immunology*; D000818:Animals; D002460:Cell Line; D004355:Drug Stability; D004356:Drug Storage; D004492:Edetic Acid; D000431:Ethanol; D005456:Fluorescent Dyes; D006639:Histidine; D006801:Humans; D006863:Hydrogen-Ion Concentration; D051379:Mice; D008807:Mice, Inbred BALB C; D009930:Organic Chemicals; D010084:Oxidation-Reduction; D019999:Pharmaceutical Solutions / Q000737:chemistry; D016133:Polymerase Chain Reaction; D013501:Surface-Active Agents; D013696:Temperature; D013997:Time Factors; D014614:Vaccines, Synthetic / Q000737:chemistry / Q000276:immunology; D014765:Viral Vaccines / Q000737:chemistry* / Q000276:immunology
chemical_list: D005456:Fluorescent Dyes; D009930:Organic Chemicals; D019999:Pharmaceutical Solutions; C099476:PicoGreen; D013501:Surface-Active Agents; D014614:Vaccines, Synthetic; D014765:Viral Vaccines; D000431:Ethanol; D006639:Histidine; D004492:Edetic Acid