An improved anion-exchange HPLC method for the detection and purification of adenoviral particles.
We have developed an anion-exchange high-performance liquid chromatography (HPLC) method using Q Sepharose XL (Amersham Pharmacia Biotech) as adsorbent to analyze samples containing adenovirus. This method has several major advantages over the HPLC method previously described for quantitating particles, namely (1) a >10-fold improvement in the detection limit of adenovirus in crude preparations; (2) absence of interferences originating from nucleic acids and proteins which usually contaminate crude samples; (3) unprecedented sharpness and symmetry of adenovirus peak, rendering the identification of the viral peak unambiguous, even in extremely crude and dilute preparations; and (4) no enzymatic treatment required even for crude samples. This assay was used to quantitate particles in samples taken at the transfection and amplification stages of production of various recombinant adenovirus, and in cultures of wild-type adenovirus of different serotypes. A modification of this analytical method was also developed for the purification of infectious adenovirus particles, including fiber-modified and third-generation recombinant viruses, giving highly purified preparations from low-titer crude lysates with an excellent overall recovery (50-74%).
Blanche
F
F
Process Development, Gene Therapy, Centre de Recherche de Vitry/Alfortville, Vitry/Seine Cedex, France.
Cameron
B
B
Barbot
A
A
Ferrero
L
L
Guillemin
T
T
Guyot
S
S
Somarriba
S
S
Bisch
D
D
D000256:Adenoviridae / Q000145:classification / Q000302:isolation & purification*; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D005822:Genetic Vectors; D006801:Humans; D012703:Serotyping; D014162:Transfection