We offer the following QC release tests:
Virus Identity Testing
The identity of a virus stock can be tested by:
- Restriction analysis of the virus genome extracted from purified virus particles or from infected cells
- PCR, by amplifying a specific region of the virus genome
- Sequencing the region of interest (e.g. expression cassette), or the entire virus genome.
Transgene Expression Testing
A reporter cell line is infected with various doses of the recombinant virus.Cells are harvested at various time points. Transgene expression is assessed by:
- mRNA detection by reverse transcription and end-point PCR
- mRNA detection and quantification by reverse transcription and real-time PCR
- protein detection by western blot
The purity of a virus stock is assessed by analyzing the A260/A280 and A320/A260 ratios (Vellekamp et al, 2001, Hum Gene Ther 12:1923-1936):
- The A260/A280 ratio reflects the relationship between nucleic acid and protein in a purified virus suspension. For CsCl-purified adenovirus, this ratio falls typically between 1.2 and 1.4. Values outside that range indicate contamination.
- The A320/A260 ratio is the scattering ratio. It reflects the amount of any aggregation in the purified virus. The typical range for the scattering ratio for purified virus is approximately 0.22-0.27 and can rise rapidly from 0.3 to 0.5-0.7 as virus aggregation is initiated.
The presence of replication-competent adenovirus (RCA) in a virus stock is assessed using a modified infectivity/PCR method, which combines the amplification of infectious RCA by cell culture with the sensitivity of detection of E1-specific sequences by PCR (Ishii-Watabe et al., 2003, Mol. Ther. 8,1009–1016). Parallel assays of the same vector doses with spikes of wild-type Ad5 are carried out to determine the sensitivity of our techniques.
Virus Particle/Infectious Unit Ratio
The VP/IU ratio is determined using the titers obtained by spectrophotometry (physical titer) and the TCID50 assay (infectious titer). Virus preparations with a ratio VP/IU <30 are acceptable.
Endotoxin levels in adenovirus stocks are measured using the Endosafe-PTS system (Charles River Laboratories). FDA-licensed cartridges with a detection limit as low as 0.01 EU/mL are used.
The presence of mycoplasma in the cell cultures and purified virus suspensions is assessed using a PCR-based method. The method requires as little as 5 fg of mycoplasma DNA corresponding to 5 mycoplasma per sample volume.
Pricing and Timing