title: Factors that influence stability of recombinant adenoviral preparations for human gene therapy.
abstract: This report identifies formulation and processing factors that influence stability of viral preparations such as selection of appropriate buffer systems, cryoprotectants, and cooling rates. Adenovirus type 5 containing the lacZ marker gene was suspended in combinations of trehalose, sorbitol, sucrose, mannitol, glycine, CaCl2, and gelatin. X-gal stains of 293 cells were used to determine the lac-forming units (lfu)/ml of each preparation before and after treatments. Phosphate-buffered solutions (except those containing sucrose or trehalose) demonstrated a drop of 3 pH units upon freezing regardless of cryoprotectant used. Tris-buffered solutions demonstrated a variation in pH which was dependent upon chosen cryoprotectant, with 1 M trehalose exhibiting no change and a 5% mannitol/10 mM CaCl2 combination showing a 3-unit drop in pH. 4-[2-Hydroxyethyl]-1-piperazine ethanesulfonic acid (HEPES)-buffered solutions showed little change in initial pH when frozen regardless of cryoprotectant chosen. In solution, adenovirus was not affected by incubation for 24 hr in buffers ranging from pH 4 to 8. However, when the solutions were frozen, the number of remaining infectious virions was dependent upon the final pH of the suspending medium. Cryoprotectant solutions that significantly maintained viral stability during a single freeze--thaw cycle were 0.5 M sucrose, 0.5 M trehalose, and 10% sorbitol/0.4% gelatin. Long-term stability studies were performed at 4 degrees C with lyophilized sorbital/gelatin and sucrose preparations. Both formulations provided adequate stability for the adenovirus, with 2.6 and 5.6 x 10(11) lfu/ml detected 150 days after drying, respectively.
authors.affiliation: College of Pharmacy, University of Michigan, Ann Arbor 48109-1065, USA. macroyle@mail.med.upenn.edu
authors.forename: M A
authors.initials: MA
authors.lastname: Croyle
authors.forename: B J
authors.initials: BJ
authors.lastname: Roessler
authors.forename: B L
authors.initials: BL
authors.lastname: Davidson
authors.forename: J M
authors.initials: JM
authors.lastname: Hilfinger
authors.forename: G L
authors.initials: GL
authors.lastname: Amidon
mesh_terms: D000256:Adenoviridae / Q000235:genetics*; D002461:Cell Line, Transformed; D005612:Freeze Drying; D005615:Freezing; D015316:Genetic Therapy; D006801:Humans; D006863:Hydrogen-Ion Concentration; D011995:Recombination, Genetic