Galpin/DA/N2 IIIBenv 
length and processed gpl60 and gpl20 HIV-1 env proteins are expressed in the 
transduced cells. Densitometric analysis of the autoradiograph was used to 
determine the amount of HIV-1 env protein expressed per cell (Figure 3). The 
level of HIV-1 env expression correlated with multiplicity of infection of 
HIV-IT (V). Human, chimpanzee, Rhesus monkey, and mouse primary and 
transformed ceil lines can be transduced with the N2 IIIBenv vector. Figure 4 
shows expression of HIV-1 gpl60 and gpl20 in the N2 IIIBenv transduced cells. 
2.3.2 Growth Conditions of the Producer Cell Line and Purification of 
HIV-IT (V) 
HIV-IT(V) is prepared from the DA/N2 IIIBenv. 2C3 producer cell line grown 
in a bioreactor. The producer cell line is grown in media consisting of DMEM (Irvine 
Scientific) supplemented with glucose, IX non-essential amino-acids, lOmM Hepes 
and 10% FBS (gamma irradiated, Hyclone) in a 5L bioreactor (New Brunswick 
Scientific) on microcarrier beads. Currently, cell densities of 1.5 X 10 6 /ml are 
routinely attained, yielding unprocessed vector titers (neo r colony forming units, cfu) 
of approximately 5 X 10 6 cfu/ml. 
The vector preparation is removed from the bioreactor, and purified by a 
proprietary process. The vector is formulated and stored frozen at -80°C. After 
purification, HIV-IT(V) titers of greater than 1 x 10 7 cfu/ml are usually attained. 
2.3.3 Safety Testing of Producer Cell Line 
The DA/N2 IIIBenv. 2C3 producer cell line has been characterized and subjected 
to numerous quality control analyses. The testing is described in Figure 5. This cell 
line is the subject of a master file submitted to the FDA. 
2.3.4 Safety Testing of the Vector, HIV-IT (V) 
In addition to comprehensive testing of the producer cell line, HIV-IT (V) has 
also been extensively tested to ensure safety and biological activity. Tests for 
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Recombinant DNA Research, Volume 17 
