M.J. Welsh and A.E. Smith, RAC Application 
components rather than contaminating proteins. The identity of the major protein bands is 
presently being established by N-terminal sequence analysis. 
12. Contaminating Materials 
The material to be administered to the patients will be 2 x 10 6 PFU, 2 x 10 7 PFU and 
5 x 10 7 PFU of purified Ad2/CFTR-1. Assuming a minimum particle to PFU ratio of 500, 
this corresponds to 1 x 10 9 , 1 x 10 10 and 2.5 x 10 10 viral particles, these correspond to a 
dose by mass of 0.25 jig, 2.5 |ig and 6.25 tig assuming a molecular mass for adenovirus of 
150 x 10 6 dal tons. 
The origin of the materials from which a production lot of the purified Ad2/CFTR-1 is 
derived was described in detail above and is illustrated as a flow diagram in Fig. 6. All the 
starting materials from which the purified virus is made (i.e., MCB, and WCB, and the 
MVSS) will be 
extensively tested. Further, the growth medium used will be tested at Genzyme and the 
serum will be from only approved suppliers who will provide test certificates. In this way, 
all the components used to 
generate a production lot will have 
been characterized. Following 
growth, the production lot virus 
will be purified by two rounds of 
CsCl centrifugation, dialyzed, and 
tested. A production lot should 
constitute 1-5 x 10 10 PFU 
Ad2/CFTR-1. 
As described above, to detect any 
contaminating material, aliquots of 
the production lot will be analyzed 
by SDS gel electrophoresis and 
restriction enzyme mapping. 
However, these tests have limited 
sensitivity. Indeed, unlike the 
situation for purified recombinant 
proteins, it is very difficult to 
quantitate the purity of the 
Ad2/CFTR-1 using SDS 
polyacrylamide gel electrophoresis 
(or similar methods). An 
alternative is the immunological 
detection of contaminating proteins 
(IDCP). Such an assay utilizes 
antibodies raised against the 
proteins purified in a mock 
purification run. Development of 
such an assay has not yet been attempted for the CsCl purification scheme for Ad2/CFTR- 
1. However, initially we will use an IDCP assay developed at Genzyme for the detection of 
contaminants in recombinant proteins produced in Chinese hamster ovary (CHO) cells. In 
addition to hamster proteins, these assays detect bovine serum albumin (BSA), transferrin 
and IgG heavy and light chain derived from the serum added to the growth medium. Tests 
using such reagents to examine research batches of Ad2/CFTR-1 by both ELISA and 
Western blots are in progress. 
293 Cells 
(ATCC) 
Ad2 & CF DNA 
Vectors (ATCC) 
293 Cells 
(ATCC) 
Engineered 
DNA Fragments 
Research 
Virus 
Seed Stock 
par 
^ & Serum 
V////////////A 
if7rrrrrr7rr7777rrrrm\ 
V/, _ Virus p 
^^hara^enzatforr 
tj Stage 
\ss/sss/s/ss/ssJ 
Cells/ 
Medium 
DNA/ 
Virus 
D A 
Cell/Virus 
Expansion 
Figure 6. Flow diagram for virus production. 
[882] 
Recombinant DNA Research, Volume 16 
