Assay method for detection of Replication-Competent Adenovirus

Replication-defective adenovirus vectors are deficient in early viral gene E1 and are generally grown in cell lines such as HEK-293, which contain and express an integrated copy of E1 for the purpose of complementing the defective adenoviral genome. Although the host cell E1 gene is required for complementation, its presence can also be problematic because recombination between vector and host cell genomes may lead to the reacquisition of E1 by the vector and the formation of replication-competent adenoviruses (RCAs).

This problem has long been recognized, and approaches, such as the minimization of the sequence homology between the adenovirus genes inserted into the host chromosome DNA and viral sequences remaining in the 5' end of the vector genome, have been taken to reduce recombination and RCA formation. However, in the clinical setting, it is important to ensure that the vector dose given to patients contains no more than one RCA infectious particle per patient dose. Emergence of the RCA in the El -deleted replication-defective rAd vector products is a concern for both regulatory agencies and manufacturers.

QVirus^TM Platform has developed several methods to determine RCA levels in purified adenovirus batches, including direct PCR analysis for the presence of El sequences and analysis of cytopathic effect (CPE) caused by RCA and infectivity-linked PCR for infectious RCA.

Assay method for detection of Replication-Competent Adenovirus

• CPE-based assay

The CPE-based assay involves inoculation of the test articles onto a cell line that supports replication of RCA but not of the defective vector, culturing the cells for a specified period of time, creating a cell lysate and then using that lysate to inoculate a new cell culture. The lysate passage is repeated to allow sufficient amplification of any RCA to meet the detection limits set out during assay validation. RCA is determined by the observance of the cytopathic effect.

• RCA assay by PCR

The DNA purified from viral particles is used as a template for a PCR procedure in which E1 gene specific primers are used to detect the presence of the E1 gene. The sensitivity of this method is limited by the number of template DNA that can be added. Greater sensitivity (10-fold) can be achieved through doing 10 replicate PCR reactions. The PCR procedure to detect the presence of RCA is faster and more sensitive and is recommended before amplification of a viral stock.

Based on years of experience and in-depth investigation, QVirus^TM Platform can provide a quick and complete system to detect replication-competent adenovirus in compliance with FDA, ICH, and CPMP guidelines in your viral prep.