recombinant virus can be reproducibly manufactured in a way that the final product is of 
sufficient therapeutic quality and free of replication competent virus or other adventitious 
agents. 
Virus production is performed in a BL2+ facility that is solely dedicated for purposes of 
recombinant adenoviruses. A schematic of the laboratory is provided in Figure 18. Within this 
laboratory there is a 4 ft laminar flow hood and double stack incubator that is dedicated to the 
production of Ad.CB-CFTR. The detailed standard operating procedures for producing virus are 
described below as is the strategy for assuring quality of the final product. 
IV.D.1. Strategy and Standard Operating Procedures 
The overall strategy for the production of Ad.CB-CFTR for therapy is summarized in Figure 18. 
A master cell bank (MCB) of 293 cells will be established that has been evaluated for 
performance, in terms of production of recombinant adenoviruses, and for the absence of other 
pathogenic contaminants. The MCB will be infected with our initial Ad.CB-CFTR virus to 
generate a lysate. Virus will be purified and cryopreserved in aliquots. The lysate and purified 
seed lot will be subjected to safety testing as described in section IV.D.2. 
The MCB will be plated and infected with the virus seed lot. Lysates will be harvested from the 
infected cells and evaluated for sterility and mycoplasma testing. Virus will be purified from 
the lysate, cryopreserved, and lots will be evaluated as described in section IV.D.2. 
Production of Ad.CB-CFTR 
Thirty 150mm-plates of about 90% confluent 293 cells were infected with Ad.CB-CFTR in 10 
ml of DM EM/1 % pen-strep with m.o.i. 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-CI, pH8.1. 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-CI, pH 8.1. Supernatants were 
pooled and loaded onto 20 ml of CsCI gradient made up of each volumes of 1 .45g/ml(density) and 
1.20 g/ml CsCI in 10 mM Tris-CI, pH 8.1. Following a two hr spin at 20,000 rpm at 4° C in a 
SW 28 rotor, the adenovirus band was removed by puncturing the tube with a needle, diluted 
with an equal volumes of 10 mM Tris-CI, pH 8.1, and loaded onto a new gradient (8ml). 
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- 
CI, pH8.1 , lOOmM NaCI, 0.1%BSA, 50% glycerol) or purified by gel filtration through a 
Sephadex G50 column for immediate use. Viral yield was determined by OD 260 
(Particles/ml=OD 260*dilutionx10 12 /ml). Generally, a total of 3x1 0 1 3 viral particles was 
achieved from 30 plates. 
IV.D.2. 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 
Recombinant DNA Research, Volume 16 
