M.J. Welsh and A.E. Smith, RAC Application 
5. Medium and Serum 
The 293 cells are grown in 925 medium. This is a proprietary protein-free medium 
developed at Genzyme for the growth of recombinant cells and has been used to produce a 
variety of recombinant proteins, two of which are currently in human clinical trials. The 
serum used is 10% Donor Calf Serum (DCS) obtained from vendors which utilize only 
non-European sources. The DCS is certified by the vendor to be free of adventitious viral 
agents, including bovine diarrhea virus, parainfluenza 3, infectious bovine rhinotracheitis, 
and other agents capable of causing cytopathic effects. Porcine trypsin used in the cell 
culture process is also certified by the vendor to be free of porcine parvovirus and 
mycoplasma. 
6. Growth of Virus Seed 
To generate a Master Viral Seed Stock (MVSS), a sample of an RVSS shown to contain the 
required recombinant is propagated on a fresh batch of human 293 cells obtained from 
ATCC at the same time as, and of the same lot as used to initiate the MCB. Details of the 
conditions for growth of virus are given below. 
7. Tests for Master Viral Seed Stock (to be performed by Microbiological Associates) 
The MVSS will be characterized by Microbiological Associates, Rockville, MD, and 
Genzyme, Framingham, MA. The test to be performed by Microbiological Associates are 
listed below. Summaries of these tests are listed above in the Master Cell Bank 
characterization section. 
Sterility 
Mycoplasma 
Retrovirus-Electron Microscopy 
In Vitro Virus 
Human Viruses 
EBV 
CMV 
HIV 
Parvoviruses: AAV, B 19 
8. Tests for Master Viral Seed Stock (to be performed by Genzyme) 
Identity - Restriction maps of DNA from the viral seed stock will be compared to 
restriction fragment sizes predicted by DNA sequence of the viral vector. SDS 
polyacrylamide gel electrophoresis for identification of major viral proteins will also be 
performed. 
Purity - The absence of wild-type adenovirus will be assessed by PCR analysis of the viral 
seed stock. For PCR screening three sets of primers are used. The first hybridizes to 
nucleotides 3076 to 3092 and 4487 to 4482 and produces a 1.4 kd band specific for wild- 
type Ad2 and Ad5. The primers of this set map in Elb and sequences 3' to early region 1. 
In consequence, a signal is not obtained from El deleted recombinant viruses nor from the 
El region integrated in 293 cells. The second set is specific for Ela and hybridizes to 
nucleotides 771 to 788 and 1144 to 1129 of Ad2 to produce a 373 bp band. This primer set 
will also detect Ad5 and the integrated sequences in 293 cells. A third set hybridizes to the 
viral E4 at nucleotides 33178 to 33196 and 34032 to 34065 to generate an approximately 
900 bp band. 
Recombinant DNA Research, Volume 16 
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