Improved high-performance liquid chromatographic method in the analysis of adenovirus particles.
We developed a HPLC method on a novel continuous bed matrix (UNO Q, Bio-Rad) for the direct quantification of adenoviral type 5 (Ad5) particles produced in 293S Human Embryonic Kidney cells and compared this with an existing HPLC method on a conventional ion-exchange resin (Resource Q, Pharmacia). The 293S cell extract contained large amounts of DNA. This contaminated the viral peak on the Resource Q column and only after Benzonase treatment was it possible to quantify the viral particles in the cell extract. In contrast, the virus peak on the UNO Q column was resolved from the DNA which eliminates the need for pretreatment of the sample with Benzonase. Cross-analysis of the Ad5 fraction from the UNO Q column using a size-exclusion HPLC column revealed no additional contaminating peaks. We conclude that the purity of the Ad5 virus peak on the continuous bed matrix UNO Q column was superior to the purity of the virus on the conventional Resource Q column, which is essential for reliable quantification.
Klyushnichenko
V
V
Biotechnology Research Institute, NRC, Montreal, Quebec, Canada. vklyushnichenko@altus.com
Bernier
A
A
Kamen
A
A
Harmsen
E
E
D000256:Adenoviridae / Q000737:chemistry / Q000302:isolation & purification*; D002457:Cell Extracts / Q000737:chemistry; D002460:Cell Line; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D002852:Chromatography, Ion Exchange; D004279:DNA, Viral / Q000302:isolation & purification; D004720:Endonucleases; D006801:Humans; D014771:Virion / Q000737:chemistry / Q000302:isolation & purification*
D002457:Cell Extracts; D004279:DNA, Viral; D004720:Endonucleases