Adenovirus Amplification & Purification

Goal

A virus suspension: a purified stock (at least 10⁸ IU or PFU) or a crude virus lysate (the equivalent of a 6-cm dish of 293 cells infected with your virus).
Information about the nature of the virus to be amplified. A biosafety form will have to be filled.

Virus amplification of the virus in cell culture. We offer 4 scales:
- The small-scale amplification is based on the infection of ~ 10⁸cells and should yield about 10¹² virus particles (VP).
- The medium-scale amplification is based on the infection of ~ 10⁹ cells and should yield about 10¹³ VP.
- The large-scale amplification is based on the infection of ~ 10¹⁰ cells and should yield about 10¹⁴ VP.
- The pilot-scale amplification is based on the infection of ~ 10¹¹ cells in suspension in a bioreactor and should yield up to 10¹⁵ VP.

Virus purification on two-step and continuous CsCl gradients and/or anion-exchange column. CsCl gradients are used for small- and medium-scale preps. CsCl-purified virus can be further purified by anion-exchange chromatography in order to remove contaminating proteins, nucleic acids and traces of CsCl. This extra purification step should be considered for virus preps that will be used in vivo, especially in vaccination projects.
Virus dialysis or diafiltration against an isotonic buffer. The default buffer is GTS: 2.5% glycerol, 25 mM NaCl, 20 mM Tris-HCl, pH 8.0 (Hoganson, D., et al., Development of a Stable Adenoviral Vector Formulation. BioProcessing, 2002. 1(1): p. 43-48). We can also use the buffer of your choice.
Virus filtration (0.2 µm)
Virus aliquoting, labeling and quick-freeze. We usually make 100 µL aliquots, but we can change the volume according to your needs.
Determination of virus particle concentration by spectrophotometry (Abs260/280/320 nM in presence of SDS). This analysis will detect the presence of contaminating DNA or proteins in the preparation, as well as possible virus aggregation.

Virus concentration. In vivo virus injections often require stocks with high virus concentrations, because of the small volumes that must be injected, especially in small animals. We will concentrate your virus preparation using a gentle method that will not precipitate the virus.
Additional virus purification step by anion-exchange chromatography. This option should be considered if you will use the virus in toxicity or vaccination studies. The additional column purification step will remove contaminating proteins, nucleic acids, and CsCl.
Use of defined, gamma-irradiated fetal bovine serum and animal-free cell dissociation agent in cell culture. This should be considered if the virus will be used later in clinical trials and documentation of the reagents used in the process is necessary.

Purified, sterile adenovirus suspension. Typically 100 µL aliquots are provided in color-coded, labeled vials. Customers can request smaller or larger volumes, and we can personalize labels.

The amount of virus obtained depends significantly on the nature of the transgene.
The use of adenovirus as a genetic vector requires adequate containment equipment and practices. Biosafety Level 2 (BL2) is appropriate for most customers; however, for some, use of BL3 or higher level containment facility may be required. O.D.260 Inc. will not carry out projects that require BL3 (or above) containment.