High-performance liquid chromatographic total particles quantification of retroviral vectors pseudotyped with vesicular stomatitis virus-G glycoprotein.
A novel and rapid method for the total particles quantification of murine leukemia virus derived retroviral vectors pseudotyped with vesicular stomatitis virus-G glycoprotein was developed using high performance liquid chromatography. Virus particles were detected by absorbance at 260 nm and quantified using a calibration curve generated from highly purified and concentrated viral stock characterized by negative stain electron microscopy. The method requires Benzonase digestion and concentration of the supernatant prior to analysis. The virus eluted in 12.55 min at a flow rate of 1 mL/min in 20 mM Tris-Cl, pH 7.4 + 1.1 M NaCl. The limits of detection and quantification of this assay were 4.71 x 10(8) and 1.57 x 10(9) viral particles/mL, respectively. Linearity was between 3.0 x 10(9) and 1.0 x 10(11) viral particles/mL with a correlation coefficient of 0.9923 and a slope of 6 x 10(-6). The assay precision was <5% and <10% for intra- and inter-day analysis, respectively. This assay was used for the total particles quantification of a 7-day, large-scale perfusion culture production of a retroviral vector grown in 293 cells expressing the beta-galactosidase gene.
Transfiguracion
Julia
J
Animal Cell Technology Group, Bioprocess Sector, Biotechnology Research Institute, National Research Council Canada, 6100 Royalmount Avenue, Montreal, Que. Canada H4P 2R2.
Coelho
Helene
H
Kamen
Amine
A
D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005822:Genetic Vectors; D008562:Membrane Glycoproteins / Q000235:genetics*; D015203:Reproducibility of Results; D012190:Retroviridae / Q000235:genetics*; D012680:Sensitivity and Specificity; D014759:Viral Envelope Proteins / Q000235:genetics*
C028391:G protein, vesicular stomatitis virus; D008562:Membrane Glycoproteins; D014759:Viral Envelope Proteins