Recombinant DNA Advisory Committee - 12/3-4/92 
Dr. Post asked for a description of the generation of the clinical grade vector 
preparations. Dr. Crystal explained that his laboratory has had several years experience 
making adenovirus vector lots that have highly reproducible titers and that are free of 
contaminants. Dr. Post asked about the degree of experience with the detection of 
helper virus. Dr. Crystal stated that helper virus assays have been performed routinely 
for a long period of time. Dr. Post asked if an Ela positive recombinant virus has ever 
been detected. Dr. Crystal said that his laboratory has detected positive Ela 
recombinant virus on several occasions, and that these lots were rejected. Dr. Post asked 
how often these positive recombinants are detected. Dr. Crystal said that the critical 
question is where is the recombinant virus coming from? In most cases, the Ela positive 
virus comes from the 293 cells. The recombinant virus probably results from pieces of 
DNA, not the entire virus. All vector preparations that have been used for the in vitro 
and in vivo experiments have been Ela negative. Dr. Post asked if recombinants have 
ever been detected, not Ela sequences. Dr. Crystal said that recombinants have never 
been detected. 
Dr. D. Miller inquired whether Ela positive samples were tested in bioassays to 
determine the presence of replication-competent helper virus. Dr. Crystal said that these 
bioassays have not been incorporated as part of the routine production scheme due to 
concerns about HeLa cells, etc. However, bioassays have been performed at random 
and replication-competent viruses have never been detected. 
Dr. Geiduschek said that he is aware that there are practical limits to biological assays 
for the detection of replication-competent virus posed by the ability of Ela minus virus 
to replicate at high MOIs. However, it is possible to change the lower contamination 
limit by performing serial plaque purifications. Therefore, it should be possible to 
specify precisely that specific lots of virus will be at a contamination level of X or lower, 
where X is defined and consistent between the various lots. Dr. Crystal said that he 
would perform these assays if required to do so by the RAC. However, Dr. Crystal 
explained that the assays that are outlined in the protocol are the experiments that are 
most appropriate. It is preferable to perform only the assays listed. 
Dr. Ginsberg agreed with Dr. Geiduschek's recommendation that serial plaque 
purifications be performed on a non-permissive cell line and suggested that the 
investigators include this experiment in the vector testing procedures. Dr. Crystal agreed 
that his laboratory would perform the suggested assays. Dr. Ginsberg suggested that the 
seed virus used for the production lots should be assayed in addition to a sample 
obtained after 6 cycles of production. 
Dr. D. Miller suggested that a stipulation be included that there be less that 1 
adenovirus helper particle per 20 ml of vector preparation. Since the investigators 
cannot obtain enough airway epithelial cells to test 10 11 particles, how will the biological 
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