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Journal of Virology, June 2004, p. 6200-6208, Vol. 78, No. 12
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.12.6200-6208.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Common Structure of Rare Replication-Deficient E1-Positive Particles in Adenoviral Vector Batches

Pete Murakami,¹ Menzo Havenga,² Farah Fawaz,¹ Ronald Vogels,² Giuseppe Marzio,² Erno Pungor,¹ Jim Files,¹ Linh Do,¹ Jaap Goudsmit,^2,3 and Michael McCaman^1*

Process Development Department, Berlex Biosciences, Richmond, California,¹ Crucell Holland BV, 2301CA Leiden,² Center for Poverty-Related Communicable Diseases, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands³

Received 7 November 2003/ Accepted 10 February 2004

The use of the PER.C6 adenovirus packaging cell line in combination with a designated vector plasmid system, whereby the cell line and vector with E1 deleted have no sequence overlap, eliminates the generation of replication-competent adenovirus during vector production. However, we have found cytopathic effect (CPE)-inducing particles in 2 out of more than 40 large-scale manufacturing lots produced in PER.C6 cells. The CPE inducer was detected at a frequency of 1 event in 7.5 x 10¹² vector particles. Despite amplification, it was not readily purified, indicating that the agent itself is replication deficient and requires the parental recombinant adenovirus serotype 5 (rAd5) vector for replication and packaging. Therefore, we designated the agent as a helper-dependent E1-positive region containing viral particle (HDEP). Here, we report the molecular structure of the HDEP genome, revealing an Ad comprised of E1 sequences derived from PER.C6 cells flanked by inverted terminal repeat, packaging signal, and transgene sequences. These sequences form a palindromic structure devoid of E2, E3, E4, and late genes. Since only 5 bp were shared between E1 sequences in the PER.C6 genome and viral vector sequences, the data strongly suggested that insertion of genomic DNA into an adenoviral genome had occurred essentially via nonhomologous recombination. HDEPs have been found in unrelated virus batches and appear to share a common structure that may explain their mechanism of generation. This finding allowed development of an HDEP assay to screen batches of rAd5 produced on the PER.C6 cell line and resulted in detection of seven HDEP agents from four different transgene-virus vector constructs in separate batches of Ad.

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* Corresponding author. Mailing address: Berlex Biosciences, 2600 Hilltop Dr., Richmond, CA 94804. Phone: (510) 669-4576. Fax: (510) 669-4920. E-mail: mike_mccaman{at}berlex.com.

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Journal of Virology, June 2004, p. 6200-6208, Vol. 78, No. 12
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.12.6200-6208.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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