Recombinant DNA Advisory Committee - 12/3-4/92 
Discussion 
Dr. Parkman said that the long-term culture of production lines, which was proposed by 
Drs. D. Miller and Geiduschek, should be the most appropriate and simple assay for the 
detection of RCR. How long would the production line have to be fed and passaged? 
What would the additional cost be to an investigator if long-term culture were required? 
Dr. D. Miller stated that the production line would have to be passaged every 3 days; 
therefore, a 4-week culture would require 10 passages. 
Dr. D. Miller explained that there are probably as many as 100 different murine viruses 
that could be assayed. The reconstruction assays have to be designed to yield optimal 
information. Does the FDA want investigators to assay for all 100 possible murine 
viruses? These viruses all have different tropisms. What about the number and types of 
helper viruses? He proposed the following scenario: what if 1 weakly-replicating virus is 
present but it takes 3 months to obtain a positive readout. Does the FDA want 
investigators to aim at this target as well? The testing process is not as simple as it 
appears. Tests can be performed for the detection of amphotropic viruses, i.e., the 
culture could be spiked with amphotropic virus and cultured for 3 weeks. RCR should 
be readily detectable after this period of time. 
Dr. Parkman noted that an amphotropic virus was the agent that produced the monkey 
lymphomas. Therefore, amphotropic viruses represent definable risks as opposed to the 
theoretical risks imposed by other viruses. Testing should focus on those viruses that 
have the potential to become RCR. There is more confidence in assaying the entire 
production lot for potentially hazardous agents, amphotropic viruses, than to perform 
multiple tests on an aliquot of the production lot as proposed by the FDA. 
Dr. Post said that the degree of confidence imposed by testing an aliquot of the 
production lot depends primarily on the volume of that aliquot. Dr. Kahn responded 
that the FDA is proposing that the amount of the production lot that should be tested 
should be approximately 5% of the total lot. This testing volume for a 5 liter lot would 
be approximately 100 ml. Based on the FDA's calculations, the co-cultivation assay is 
approximately 10 times more sensitive than testing the supernatant directly; therefore, 
0.5% of the total pool sample is recommended. 
Dr. D. Miller asked if the FDA has tested the co-cultivation technique, i.e., have 
reconstruction experiments been performed in which Mus dunni cells have been co- 
cultivated with packaging cell lines. Dr. Kahn said that the actual reconstruction 
experiments have not been performed by the FDA; however, co-cultivation has generally 
been proven to be a more sensitive assay than other methods in retrovirology. Similar 
data has been derived from experiments with the human immunodeficiency virus (HIV). 
Dr. D. Miller said that HIV is a very unique virus and should not be compared to this 
Recombinant DNA Research, Volume 16 
[595] 
