Factors that Influence Stability of Recombinant Adenoviral Preparations for Human Gene Therapy

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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 CaCl2combination showing a 3-unit drop in pH. 4-[2-Hydroxyethyl]-l-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°C with lyophilized sorbitol'gelatin and sucrose preparations. Both formulations provided adequate stability for the adenovirus, with 2.6 and 5.6° 1011Ifu/ml detected 150 days after drying, respectively.

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