Batches of CVL will be evaluated (see 2.4.4. 5 Cell Harvest) and combined for purification 
as "Lots" of AvlCF2 virus. Cell debris will be removed from the CVL by low speed 
centrifugation (7000 rpm, 5 min, 4°C). Cleared CVL is then layered onto a cesium chloride step 
gradient (1.25 g/ml and 1.4 g/ml in 10 mM Tris-HCl pH 7.4, 1 mM MgC12) and centrifuged 
(20,000 rpm, 2 hr, 4°C, SW 28 rotor). The viral band is collected away from other cellular 
components and re-purified by isopycnic centrifugation. To accomplish this, the virus from the 
first gradient is layered onto cesium chloride (1.33 g/ml in 10 mM Tris-HCl pH 7.4, 1 mM 
MgCl 2 ) and centrifuged (20,000 rpm, 18 hr, 4°C). Intact AvlCF2 virions are collected away 
from empty or partly filled capsids by side puncture and dialyzed to remove the cesium chloride 
(10 mM Tris-HCl, pH 7.4, 1 mM MgCl 2 , 10% (v/v) glycerol; 4 x 1 L, 1 hr each). Virus is 
then aliquoted, labeled and stored at -70°C. 
2.4.2 Production of the 293 Cell Master Cell Bank: 293 cells were obtained at passage 31 
from the American Type Culture Collection. Cells were thawed and seeded into T-175 flasks 
(Falcon) in IMEM supplemented with 10% fetal bovine serum (parvovirus and mycoplasma 
screened), 1% glutamine and cultured at 37°C in a humidified 5% C0 2 atmosphere. Medium 
was changed every 3 days and the cells were split using trypsin/versine (parvovirus and 
mycoplasma screened) immediately prior to confluency. Sufficient cells were available after 3 
passages and were frozen using standard techniques. 
2.4.3 Production of the AvlCF2 Master Virus Bank: A Master Virus Bank (MVB) of 
AvlCF2 vector will be prepared as a seed stock for production of Lots AvlCF2 vector (Figure 
12). To accomplish this, AvlCF2 DNA will first be "sterilized" by phenol chloroform 
extraction, and then collected by ethanol precipitation and re-solubilized in sterile solution. The 
DNA will then be transfected into an aliquot of 293 cells from the 293 cell (MCB). Infectious 
virus will be isolated and amplified to high titer by repeated infection and purification cycles 
using 293 cells from the MCB. This will allow generation of a large amount of viral seed stock 
(the MVB) which is free of adventitious pathogenic agents. 
2.4.4 Quality Assessment and Quality Control: The evaluation and quality control measures 
used in the production of AvlCF2 virus will occur at each stage of the process. Many of the 
quality assessment procedures will be performed, independently, by Microbiologic Associates, 
Inc. or Quality Biotech, Inc. under contract and all procedure will be carried out in compliance 
with FDA/GLP guidelines. First, the MCB will be characterized and evaluated extensively for 
the presence of adventitious pathogenic agents (see 2.4.4. 3 Master Cell Bank). AvlCF2 virus 
is thrice plaque-purified to ensure a pure clone of AvlCF2 virus completely free of wild type 
or parental (Ad-df327) virus. Second, the MVB generated from the MCB and "sterile" (phenol- 
chloroform extraction and ethanol precipitation) AvlCF2 DNA will then be characterized 
structurally, functionally, and for transfer and expression of functional CFTR to cells defective 
in CFTR function and evaluated extensively for the presence of adventitious pathogenic and 
other agents (see 2. 4. 4. 4 Master Virus Bank). The MVB will be extensively evaluated for the 
presence of wild type virus or parental (Ad-d]327 adenovirus (see 2. 4. 4. 4 subsection replication 
competent adenovirus). Third, each batch of AvlCF2 containing CVL (cell harvest) will be 
evaluated for sterility prior to purification into Lots of AvlCF2 vector. Fourth, product testing 
on the final fill material (purified AvlCF2 virus) will consist of functional evaluation, and the 
presence of adventitious pathogenic and other agents as well as for the presence of wild type 
virus (see 2. 4. 4. 6 AvlCF2 Production Lots for Clinical Use). 
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