VLB. Production of Ad5-CB-CFTR 
Thirty 150 mm plates of about 90% confluent 293 cells were infected with 
Ad5-CB-CFTR in 10 ml of DMEM/1% pen-strep with MOI of 10 for two hr., then 
20 ml of DMEM/15% FBS/1% pen-strep was added. 36-40 hr post-infection, cells 
were harvested, pelleted, and resuspended in 10 mM Tris-Cl, pH 8.1. A viral lysate 
was generated by three cycles of freeze-thawing (ethanol-dry ice bath/37 0 C water 
bath). Cell debris was removed by centrifugation for 20 min at 3,000 rpm at 4 ° C, 
and washed once with 10 mM Tris-Cl, pH 8.1. Supernatants were pooled and loaded 
onto 20 ml of CsCl gradient made up of each volumes of 1.45 g/ml (density) and 
1.20 g/ml CsCl in 10 mM Tris-Cl, pH 8.1. Following a two hr spin at 20,000 rpm at 
4 0 C in a S W 28 rotor, the adenovirus band was removed by puncturing the tube 
with a needle, diluted with an equal volumes of 10 mM Tris-Cl, pH 8.1, and loaded 
onto a new gradient (8 ml). Following an overnight spin at 20,000 rpm at 4 ° C in a 
SW41 rotor, the viral band was removed and either stored at -20 °C by diluting 1:5 
into a glycerol/BSA solution (10 mM Tris-Cl, pH 8.1, 100 mM NaCl, 0.1% BSA, 
50% glycerol) or purified by gel filtration through a Sephadex G50 column for 
immediate use. Viral yield was determined by OD 250 (Particles/ml = OD 
260 x dilutionxl0^/ml). Generally, a total of 3 x lO^viral particles was achieved 
from 30 plates. 
VI.B.l. Adenovirus seed lot 
The adenovirus seed lot has been sent to a contract lab to be analyzed by 
numerous tests. The following summarizes those tests and their results. 
Test Result 
Sterility negative (final) 
Mycoplasma negative (preliminary) 
Parvovirus B-19 hybridization still on test 
Electron Microscopy still on test 
AAV Hybridization negative (preliminary) 
In vitro still on test 
VI.C. Quality Assurance and Quality Control 
A four-stage test program has been designed to assess the cell bank, product 
intermediate (cell lysate) and the purified product (virus). The 293 cells used to 
produce the adenovirus will be characterized prior to infection for possible 
microbial, adventitious viral, and select specific human viral contaminants. Testing 
of the adenovirus preparation used to infect the cells will include assays for 
microbial contaminants and adventitious virus. After expansion of the infected 
cells, the cell lysate will be evaluated for microbial contaminants. Product testing of 
the purified product for endotoxin, microbial contaminants, extraneous toxins and 
infectious adenovirus completes the test battery. Each step will be carefully tested 
for the presence of wild type adenovirus. The majority of tests will be performed by 
Microbiological Associates Inc. (MAI), an independent laboratory that has provided 
contract research and safety assessment for the pharmaceutical industry. A contract 
has been established with MAI for this project. 293 cells have been grown to 
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