Development of a highly efficient purification process for recombinant adenoviral vectors for oral gene delivery.
Recently, replication-deficient adenoviruses have received increasing attention as vector for gene delivery and as potential vaccine carriers. With the increased use of the vector in vivo and in clinical trails, the demand for a safe, rapid, and cost effective purification process has been heightened. In this report, a simple and efficient method for the purification of large quantities of live adenoviral vectors was developed. The process involved the replacement of cesium chloride (CsCl) gradients with sucrose gradients. Ultracentrifugation times were reduced and the desalting step eliminated, decreasing total preparation time by 15 hr. A 20-80% linear sucrose gradient provided optimal recovery of infectious viral particles and positioning of the viral band in the gradient. Purification with this gradient system produced a preparation containing 1.39 x 10(14) lac-forming units (lfu)/ml. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed that the process also removed all associated cellular proteins from the preparation. Studies have shown that direct lyophilization of the vector in sucrose after purification produces a product containing 1.4 x 10(12) lfu/ml. Minimal degradation was seen in the lyophilized preparation. A viral concentration of 6 x 10(11) lfu/ml was detected in the product after 150 days in storage at -20 degrees C. This approach will not only simplify the preparation of adenoviral vectors for in vivo studies and clinical trials, but will facilitate production of stable adenoviral formulations for oral gene delivery.
Croyle
M A
MA
College of Pharmacy, University of Michigan, Ann Arbor 48109-1065, USA. macroyle@mail.med.upenn.edu
Anderson
D J
DJ
Roessler
B J
BJ
Amidon
G L
GL
D000256:Adenoviridae / Q000235:genetics / Q000302:isolation & purification*; D000284:Administration, Oral; D002499:Centrifugation, Density Gradient; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D011995:Recombination, Genetic; D013395:Sucrose / Q000008:administration & dosage
D013395:Sucrose