Protocol ID93-008 
Page 7 
3.1.4 Incompatibility 
Reconstitution or dilution with Bacteriostatic Water for injection, USP or 0.9% Sodium 
Chloride injection. USP should be avoided because of precipitation (see 5.4 for special 
consideration for use of rlL-2 with TIL). Proleukin for injection should not be mixed 
with other drugs. 
Studies of bioactivity after dilution of reconstituted Proleukin in diluents of 5% 
Dextrose injection. USP containing varying percentages of Albumin Human. USP have 
been performed. A final concentration of 0.1% Albumin Human. USP. in 5% Dextrose 
Injection. USP. has been found to be optimal for full recovery of Proleukin. rlL-2 
biological activity. Some loss of bioactivity has been observed with increasing 
percentages of Albumin Human. USP. Dilution of reconstituted Proleukin rIL-2 with 
2% Albumin Human. USP in 5% Dextrose injection produced solution with 
approximately 90% of the bioactivity measured with 0.1% Albumin in the diluent. 
It is recommended that in-line filters not be used for delivery of Proleukin in 
administration devices. Bioassays have shown significant loss of Proleukin when filters 
are used. 
Note: Parenteral drug products should be inspected visually for particulate matter and 
discoloration prior to administration, whenever solution and container permit. 
3.1.5 Sterility 
Although reconstituted Proleukin is stable, the product does not contain a preservative 
and accidental microbial contamination may occur. Use of water containing 
preservatives (benzyl alcohol or parabens) to reconstitute Proleukin results in the 
formation of a precipitate. 
Vials should be entered once only for reconstitution and once only to remove an aliquot 
for further dilution in order to minimize the chance of contamination. Sterility cannot 
be guaranteed after the vial has been entered. We recommend prompt administration 
to patient following reconstitution. Any unused portion should be discarded. 
3.2 Collection. Ebcpansion and Administration of TIL 
Cell suspensions of TIL and tumor cells are prepared from peritoneal fluids or solid 
tumor metastases. excised from patients with ovarian cancer as we have described 
previously (17^. 
(a) Suspensions of TIL and tumor cells from peritoneal fluid or washings are washed tvace 
and resuspended at 5x10^ cells/ml in AIM V serum-free medium (Gibco. Grand Island. 
NY) with 600 lU/ml rll^2 in 24 well Costar plates. Cells are expanded to approximately 
1x10® in plastic bags (PL732 plastic Fenwal Labs. Deerfield. IL), and cultured at 37°C 
with 5% CO 2 . Approximately every 5 days, new AIM V medium with rIL-2 is added, and 
the cell density is maintained at < 2x10® TIL/ml during the enUre culture. 
(b) Enzyme digestion procedure for solid specimens 
Transfer tissue (1 to 4 grams) to 100 mm sterile glass petri dish. Add 10 ml AIM V 
medium (Gibco). Using sterile scalpels mince the tissue to 1 mm size pieces. Transfer 
minced tissue along with medium to sterile spinner flask. Add 2.5 ml of 3% collagenase 
type I (Sigma C-0130) in AIM V medium and 5 ml of 0.02% DNase I, type II (Sigma D- 
4527) in AIM V medium. Place on stir plate at medium speed in 37°C incubator (CO 2 
not necessary) for 16 hours or overnight. Less than 1 gram of tissue may take a shorter 
time. Transfer contents of spinner flask to a 50 cc centrifuge tube. Spin at 450xg for 5 
minutes. Wash once with AIM V. Resuspend pellet in AIM V culture medium and rIU-2 
as above in Section 3.2(a). 
Recombinant DNA Research, Volume 19 [^65] 
