6.3.2 RPMI media and 1.5 ml of processed autologous plasma are added to bring the 
total volume in the bag to 150 ml. The entire 3.0 mg vial of antibody is added 
to the bag. The cells are incubated at room temperature for 25 minutes. 
6.3.3 The cells are washed with one liter of PBS and brought back to a total volume 
of 300 ml with PBS. Samples are removed for counting and analysis. 
6.4 CEPRATE Column Set Up and Run 
6.4. 1 During the antibody incubation the CEPRATE™ SC Stem Cell Concentrator is 
set up and primed. 
6.4.2 Transfer packs, Reagent bags and Collection bags are prepared and labelled per 
manufacturers instructions. 1% autologous plasma is used for the PBS/Protein 
bag. 
Reagent Bags: 
PBS 
300 ml PBS 
PBS/PROTEIN 
200 ml PBS/2ml plasma 
PBS 
1 liter 
Collection Bags: 
STEM CELL BAG 
5 ml plasma in 150ml ba 
WASH 
empty 300 ml bag 
UNABSORBED 
empty 1 liter bag 
PRIME 
empty 600 ml bag 
6.4.3 The CEPRATE instrument is turned on and the computer performs the 
electrical check. The sterile tubing sets, prefilter, precolumn and column are 
installed via the LOAD REAGENT, SET UP, and RUN menus. The PRIME 
menu is then run to allow the instrument to automatically remove any air from 
the system. 
6.4.4 The bag containing the antibody-treated cells is installed on the CEPRATE 
instrument. The RUN PROCESS menu is run and the cells flow through the 
filter and columns automatically. The CD34+ antibody coated cells bind to the 
avidin conjugated beads in the column, while the unlabelled cells pass through 
into the "unabsorbed" bag. The bound cells are then removed from the column 
by gentle agitation and collected automatically in the "stem cell" bag. The 
entire procedure should be monitored using the System Functions Table. 
6.4.5 At the end of the run, instructions on the screen are followed to remove the 
"stem cell", "wash" and "unabsorbed" bags from the machine. 
6.5 Volume Reduction 
6.5.1 The stem cell bag will contain an approximate volume of 100 ml. Heparin will 
be added to a final concentration of 10 units/ml. 
6.5.2 The stem cell bag contents will be transferred to 50 cc conical tubes coated with 
a small volume of autologous plasma, and the bag will be rinsed with 
heparinized (10 units/ml) TC199 media. Aliquots will be removed for counting 
and analysis. 
6.5.3 The tubes will be centrifuged at 1200 rpm (420 g) for 5-10 minutes. 
6.6 Transduction of the CD34-Enriched Bone Marrow and Peripheral Blood Stem Cells 
6.6.1 Clinical-grade, fully certified viral supernatant obtained from Genetic Therapy, 
Inc. will be used to transduce CD34 immunoselected cells from bone marrow 
and peripheral blood. These clinical grade supernatants, derived from the 
G1MD-C5 producer clone, will contain between 2 and 20 x 10 5 infectious viral 
particles per ml. 
6.6.2 The final concentration of CD34-enriched cells at the initiation of transduction 
will be 1 X 105/ml. Sufficient supernatant for the transduction will be thawed 
at 37 degrees in a water bath. Preservative free protamine sulfate (i.v. grade, 
Elkins-Sinn Inc., 10,000 ug/ml) will be added to the supernatant to reach a 
final concentration of 4 ug/ml. Human recombinant interleukin 3 will be 
added to the supernatant to reach a final concentration of 20 ng/ml. Human 
recombinant stem cell factor will be added to the supernatant to reach a final 
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