Antibiotics will not be used at any stage during the production of the MCB, MVB or of 
purified recombinant virus for clinical use to ensure the highest quality material free of 
contaminating pathogenic organisms. 
2.4.4. 1 Facilities for Virus Production: Production of recombinant AvlCF2 vector for 
preclinical and clinical use is done under Good Laboratory Practices" (GLP) in a facility 
dedicated for this purpose. The room is set up for biosafety level 2 + containment. Specifically, 
the facility (Figure 13) has an outer room for changing into protective outerware and a inner 
room where viral production occurs. The production room contains a HEPA filtering system. 
Protective clothing consists of a "bunny" suit, gloves, booties, haircap and a mask. 
2. 4. 4. 2 Drug Masterfile and Standard Operating Procedures: A drug masterfile is being 
prepared for AvlCF2 for submission to the Food and Drug Administration (FDA) concurrent 
with this protocol. Details of the production, quality assessment, packaging, labeling and 
storage of the recombinant adenovirus AvVlCF2 intended for clinical use are contained in the 
drug masterfile and will be on file with the FDA. 
2. 4. 4. 3 Master Cell Bank: The Master Cell Bank (MCB) will be evaluated as outlined in 
recommendations from the FDA’s "Points To Consider" for use of mammalian cell lines. The 
MCB is required to pass all tests and will be discarded if any test is failed. Many of the tests 
will by conducted in the laboratories at Microbiological Associates (M.A.) Inc. 
Karyology Assay; M.A. Inc, Protocol No. 1516.018: This test is designed to confirm the 
species identity of cultured cells by means of isozyme and cytogenetic analysis. 
Tumorigenicity Assay; M.A. Inc, Protocol No. 1514.001: This study utilizes athymic nude 
mice which are inoculated subcutaneously with cells. Animals are observed for clinical signs, 
palpated bi-weekly, and submitted for histopathology evaluation of multiple tissues. Any animal 
which shows a progressing nodule is sacrificed and submitted for histopathologic examination. 
Soft Agarose; M.A. Inc, Protocol No. P1516.029: This assay is designed to assess the 
capacity of cells to form colonies in soft agarose. 
Sterility Test; M.A. Inc, Protocol No. 1514.510: This test exceeds 21 CFT 610.12 in 
utilizing three different broth media and one agar medium for detection of both aerobic and 
anaerobic bacterial as well as fungal contaminants. 
Mycoplasma; M.A. Inc, Protocol No. 1514.102003: This protocol reflects the 1987 
"Points to Consider" attachment and US FDA expectations as to mycoplasma testing. The study 
includes direct inoculation of test article on two types of agar and in the semi-solid broth 
medium. Broth inoculated with test article is subcultured at three intervals, and incubated 
aerobically and anaerobically. In addition to the agar and broth cultivation, direct inoculation 
of the test article on Vero indicator cells and staining with Hoechst stain permits microscopic 
examination for the presence of mycoplasma. 
Electron Microscopy; M.A. Inc, Protocol No. P1516.013: This protocol is designed to 
evaluated cell cultures for the presence of virus by examination of fixed cell pellets using 
transmission electron microscopy. 
In Vitro Virus Assay; M.A. Inc, Protocol No. 1514.003: A broad spectrum of viral 
contaminants are detected using an in vitro assay with sensitive indicator cells. Indicator cells; 
including the test article cells, MRC-5 cell line, and Vero cells. Up to three additional cell lines 
may be required to comply with European regulatory guidelines. Test procedures include direct 
inoculation of test article as well as blind passage of supernatant fluids at 14 days. Indicator 
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