Recombinant DNA Advisory Committee - 12/3-4/92 
a production lot (5 liters per lot) are added to cells. Any RCR that is present will be 
amplified. Standard assays will yield positive or negative RCR results. The problem is 
that the level of detection is 1 RCR particle per 100 ml, not in the 5 liter production lot. 
What are the chances that there are zero RCR in the 100 ml sample, but there are still 
viral particles in the remaining 4,900 ml? Statistically, there is the probability that 
between 5 and 20 undetected RCR particles could be present in the untested portion of 
the production lot. Knowing that approximately 1 x 10 7 RCR are required to produce 
pathology, do 5-10 undetectable particles represent a risk? 
Dr. Anderson explained that Dr. Geiduschek's concern about inaccurate calculations 
regarding the monkey data was due to poor wording in the report. The number of viral 
particles produced in the monkeys that resulted in a clonal event (at the time of the 
clonal event, not at the end) was approximately 10 12 particles. In other words, an 
inoculating dose of 2 x 10 7 PFU is sufficient to give a 30% chance of getting 10 12 PFU 
and a clonal event. An additional safety margin exists in that patients can be tested for 
viremia. If a viremia occurs, a patient could be treated with antibodies against the 
murine retrovirus; therefore, the patient would presumably never reach the 10 12 particle 
level. 
Dr. Anderson stated that it is important for the RAC to remember that sooner or later a 
patient will probably develop a leukemia from a retroviral insertion. It is bound to 
happen. Will it take 10 12 patients? The number of patients is unknown. The point is 
that the present RCR assays are adequate and have to be maintained conscientiously. It 
is certainly rational to discuss the inclusion of better assays as long as they are 
reasonable. 
Dr. Anderson said that Dr. Geiduschek has suggested the requirement for an assay that 
would detect breakout. Breakout can be detected by adding a defined number of virus- 
producing cells to a defined number of production cells. These cells are then co- 
incubated for a period of time, e.g., several weeks. If no breakout is detected in the 
aliquot that has been co-cultured, then the frozen production lot is assumed to be safe. 
This assay is reasonable and straightforward and should be performed for all retrovirus 
vector production lots. 
Discussion 
Dr. D. Miller said that culturing the production run cells and splitting them at the 
appropriate rate to preserve any helper virus is an enormous undertaking. Dr. Anderson 
stated that the entire production run would not be tested, only a portion of the cells. Dr. 
D. Miller expressed concern that a representative sampling might miss a breakout. Dr. 
Anderson agreed that there is a statistical risk; however, these are severely immune 
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Recombinant DNA Research, Volume 16 
