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Updated: Jul 17, 2026
An Efficient Method for Adenovirus Production
Published on: June 10, 2021
A replication-competent adenovirus assay for E1-deleted Ad35 vectors produced in PER.C6 cells
G Marzio1, E Kerkvliet, J A Bogaards
1Crucell Holland BV, P.O. Box 2048, 2301CA Leiden, The Netherlands. g.marzio@crucell.com
Abstract:
The presence of replication-competent adenovirus (RCA) is a safety concern for biologics based on recombinant adenoviruses and RCA testing is therefore mandatory for release of clinical material. RCA, which arises from homologous recombination between Ad5 vectors and HEK-293 cells, can be eliminated by the use of PER.C6 cells in combination with a matched vector. However, little is known on RCA formation with vectors based on adenovirus serotypes other than Ad5 and reliable RCA assays to test them are generally lacking. Here we report on the development and qualification of a sensitive RCA assay for Ad35, a promising alternative to Ad5 vectors. The assay is able to detect 1 RCA in 3x10(10) vector particles with 95% confidence, thus meeting current FDA requirements, and can discriminate between RCA and other rare CPE-causing entities, including helper dependent E1 positive particles (HDEP). Using this assay, the first batches of Ad35 vectors produced in PER.C6 cells were analysed and found to be free of RCA and HDEP. Based on the statistical model used, we anticipate that our approach to RCA assay development can be broadly applicable to other adenoviral vectors.
Insights
Replication-competent adenovirus (RCA) poses a safety risk in adenovirus-based biologics. A new assay for Ad35 vectors detects RCA with high sensitivity, ensuring product safety and meeting regulatory standards.
Area of Science:
- Biotechnology
- Virology
- Molecular Biology
Background:
- Replication-competent adenovirus (RCA) is a critical safety concern for recombinant adenovirus-based biologics, necessitating mandatory testing for clinical material release.
- While RCA formation is understood for Ad5 vectors using HEK-293 cells, data on other serotypes, like Ad35, and reliable detection assays are limited.
Purpose of the Study:
- To develop and qualify a sensitive assay for detecting RCA in Ad35 vectors.
- To ensure the safety of Ad35-based biologics by establishing robust RCA testing methods.
Main Methods:
- Development and qualification of a sensitive assay for Ad35 replication-competent adenovirus (RCA).
- Assay validation to detect 1 RCA in 3x10^10 vector particles with 95% confidence, meeting FDA requirements.
- Discrimination of RCA from other cytopathic effects, including helper-dependent E1 positive particles (HDEP).
Main Results:
- The developed Ad35 RCA assay demonstrated high sensitivity, meeting stringent FDA requirements.
- The assay successfully distinguished RCA from other rare cytopathic entities like HDEP.
- Initial batches of Ad35 vectors produced in PER.C6 cells were analyzed and found to be free of RCA and HDEP.
Conclusions:
- A sensitive and reliable RCA assay for Ad35 vectors has been successfully developed and qualified.
- This assay ensures the safety of Ad35-based biologics by detecting potential RCA contamination.
- The methodology is anticipated to be applicable for RCA assay development for other adenoviral vectors.
