2. 4. 4. 6 AvlCF2 Production Lots For Clinical Use: After evaluation and certification, Batches 
of CVL will be combined into Production Lots for final purification of AvlCF2 vector. Each 
Lot will be evaluated by the assays listed below. Production Lots of AvlCF2 vector "final fill" 
material will be accepted only if they pass all of the testing procedures listed below. 
General Safety; M.A. Inc, Protocol No. P1514.509: This assay is designed to detect 
extraneous toxic contaminants. Guinea pigs and mice are inoculated with test material and 
observed for overt signs of ill-health or unusual response. 
Sterility Test; M.A. Inc, Protocol No. 1514.510: See above (2. 4. 4. 3) for description. 
Limulus Amebocyte Lysate Assay; M.A. Inc, Protocol No. SPGT332070: The purpose 
of this study is to detect and quantitatively determine the gram negative bacterial endotoxin level 
of a test article or extract using the Limulus Amebocyte Lysate (LAL) gel-clot method for 
testing. 
Replication C< *ent Adenovirus: See above (2. 4. 4. 4) for description. 
Recombinant Auenoviral Vector-Derived Expression of Functional Human CFTR: See 
above (2. 4. 4. 4) for description. 
2. 4. 4. 7. Evaluation of AvlCF2 For Replication Competent Adenovirus: Purified 
preparations of AvlCF2 virus will be evaluated for the presence of Ela containing "wild type" 
virus using quantitative PCR (see appendix 1, Trapnell et.al., 1991a; Rosenfeld et.al., 1992; 
Crystal et.al., RAC proposal, 1992). In this assay, purified virions are heat denatured and 
assayed directly by PCR for the presence of Ela sequences to detect wild type or replicative 
virus. As a control, PCR amplification of the E2b region is performed in parallel. The current 
level of sensitivity of this detection system is 1 pfu of wild type virus per 10 8 pfu of AvlCF2. 
However, we are currently investigating ways to increase this sensitivity, but believe that the 
current levels of sensitivity are appropriate for this clinical study. 
Evaluation of the presence of infectious, wild type virus is possible using a plaque assay on 
indicator cells which do not complement the AE1 of Avl series vectors. However, these assays 
are difficult to perform, not yet validated for authenticity and are currently of unknown 
sensitivity and specificity. 
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