High-performance liquid chromatographic total particles quantification of retroviral vectors pseudotyped with vesicular stomatitis virus-G glycoprotein

Abstract

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 × 10 8 and 1.57 × 10 9 viral particles/mL, respectively. Linearity was between 3.0 × 10 9 and 1.0 × 10 11 viral particles/mL with a correlation coefficient of 0.9923 and a slope of 6 × 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 β-galactosidase gene.