9. Drug Supply Product 
G-CSF (Amgen) is obtained from the bacterial fermentation of a strain of E. coli bearing a 
genetically engineered plasmid containing the human G-CSF gene. 
A. Drug Formulation 
1. Preparation of G-CSF 
G-CSF is formulated in a preservative-free solution for injection in vials 
containing 0.80 mg of protein per viaL The composition of the lyophilizate 
consists of 800 /zg of protein and approved standard excipient. The vials may be 
stored securely in refrigeration at 2°C-8°C. 
Dilution of product for intravenous infusion is accomplished by withdrawing the 
desired dose to be given for that day as per protocol with a glass tuberculin 
syringe and diluting in normal saline 50 ml. 
2. Stability of Reconstituted Drug 
The drug is stable for 6 hours at room temperature. 
10. Procedure of Peripheral Blood Nucleated Cell Collection 
Before receiving the 5th dose of G-CSF, approximately 20 hours after the 4th dose of G-CSF 
. the first of 4 consecutive peripheral blood nucleated cell collections will begin. Collections will take 
place in the FHCRC Pheresis Unit and use the Fenwal CS3000, Cobe Spectra, Cobe 2997 machine. A 
short double lumen catheter is necessary for peripheral blood collections. Collections will be performed 
for 4 hours on 4 successive days... Peripheral blood cells will be cryopreserved in the marrow processing 
and cryopreservation laboratory under the supervision of Dr. Rowley. 
11. Procedure of CD34 Cell Surface Antigen Analysis and Cell Culture 
Ten daily samples of blood (10 oc in a green top tube) will be drawn for analysis of CD34 
antigen expression and CFU-GM culture. The first sample will be drawn just prior to the first dose of 
G-CSF. During the period of peripheral blood cell harvesting samples will be drawn just prior to each 
collection. Once collections of peripheral blood cells are completed, samples will be drawn for the 
following 3 days. Analysis and culture will be done at the FHCRC with the FACS in Dr. Bensinger’s 
laboratory. After the first 3 patients have been studied, additional samples of the PBMC harvest will be 
analyzed for CD34 + /rhodamine dull cells and their in vitro growth properties in long-term marrow 
culture by Dr. Jack Singer. M 
12. Variables to be evaluated 
A. Primary Variables 
1. Percent and absolute number of cells expressing CD34 antigen and concentration 
of CFU-GM. 
2. Number of days from day 0 to an ANC of 100, 500 and 1000/mm 3 . 
3. Number of days from day 0 to platelet transfusion independence (platelets 
> 20,000/mm 3 ). 
4. Number of patients failing to achieve an ANC of 500/mm 3 and platelets of 
20,000/mm 3 . 
5. Number of patients dying before day 22. 
6. Dose of PBMC and PB CFU-GM infused. 
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Recombinant DNA Research, Volume 16 
