Presented at The Workshop on Simian Virus 40 (SV40): A Possible Human Polyomavirus Workshop - January 1997

     Good afternoon. I'm Bonnie Brock from Wyeth Lederle. I've been asked to provide a brief overview regarding the quality control testing of the oral polio vaccine. I'd like to start by providing you with some product background on OPV.
     The oral polio vaccine is a trivalent preparation of attenuated Sabin strains of poliovirus types 1, 2, and 3 in an oral dosage form. The vaccine induces an immune response comparable to the natural disease. The vaccine is credited with the eradication and control of wild type polio in the United States.
     Lederle Laboratories has distributed over 650 million doses since the licensure of Orimune in 1963. The viral content of the vaccine is specified by FDA regulations. The individual three polio virus types are combined in specific ratios to assure that all three stains immunize effectively.
     The manufacture and testing of Orimune is a multi-stage process that's closely monitored by the FDA following explicit protocols and requires extensive quality control testing.
     I'd like to describe cell culture preparation. Preparation of the cell substrate is in primary monkey kidney cells obtained from Green Monkeys that do not harbor the SV40 virus. The monkeys used as a source of kidney tissue are purpose-bred in isolated breeding colonies. They're tested for tuberculosis and vial antibodies. They're held in isolation quarantine under strict veterinary supervision.
     A kidney perfusion process is performed under aseptic conditions which liberates kidney cells in preparation for cell culturating. Perfused kidneys are then delivered to the cell culture laboratory.
     The cells are disbursed into monocellular suspensions under aseptic conditions. The cells are diluted into a growth media containing the nutrients necessary for growth and replication. Cells are planted into roller bottles and incubated to form a cell monolayer.
     Cells are grown and observed for at least 11 days in the cell culture laboratory. After cell growth is completed, 75 percent of the roller bottles are sent to the virus production laboratory for polio virus inoculation. The remaining 25 percent of the roller bottles are sent to quality control for testing.
     Fluids from all the roller bottles are tested to detect the presence of any transmissible, microbial agent by inoculation into four cells lines - cercopithecus monkey kidney cells, CMK cells - for an initial 14 days, followed by a 14-day subculture, again in CMK; Rhesus monkey kidney cells for at least 14 days; rabbit kidney cells for at least 14 days; and BSC-1 cells for at least 14 days.
     The 25 percent of all the cell culture bottles that are sent to quality control are then observed in their original control bottles for at least 14 more days, followed by a test to detect hemabsorptive viruses.
     At day-4 of the quality control observation period, fluids are removed from the original bottles and again tested in the same cell systems I previously described. Again, to detect the presence of any transmissible microbial agent. We always include that additional 14-day subculture on CMK.
     Again, at day-14 of the quality control observation period, fluids are again removed from the original bottles and again tested in those same cell systems, including a 14-day subculture in CMK. Therefore, every individual cell batch is observed for a total of more than 50 days in culture. The appearance of any sign of contamination at any stage of testing results in rejection of the cell batch.
     I'd like to move on to virus production. One of the Sabin attenuated strains is prepared to inoculate production bottles. Master polio virus seed stocks are maintained in a viable state in liquid nitrogen storage.
     Master viral strains have been prepared in the presence of SV40 virus neutralizing antiserum. All subsequent working seed strains have been prepared in CMK tissue and screened to assure they're free of SV40 virus.
     The same level of virus is used for each group of bottles inoculated. Production bottles are examined and records checked. Only one polio virus type is processed at a time and incubated. At the appropriate time, post-polio virus infection fluids from infected tissue which contain polio virus are harvested.
     I'd like to describe viral harvest testing now. Viral harvest samples are sent to the quality control laboratory for evaluation and the rest of the harvested fluids are stored frozen until testing is completed. Fluids from these bottles are again tested to detect the presence of any transmissible microbial agent in CMK for 14 days, followed by a subculture in CMK for another 14 days.
     Viral harvest fluids are also tested again in Rhesus monkey kidney cells, rabbit kidney cells, and BSC-1 cells, all for 14 days. Samples are also tested to demonstrate the absence of microplasma.
     Quality assurance releases a virus harvest for further processing when all testing has been completed with satisfactory results - for the original cell culture, the cell culture fluid testing and subcultures, and the viral harvest samples.
     In summary over 4,000 individual cell culture observations are made during the quality control testing of a single trivalent bulk lot.
      Any product contamination observed at any point, results in rejection.
     When the appropriate number of harvests for a single polio virus type are released by quality assurance, they are thawed and combined to form a monopool. Samples from an unfiltered, prorata monopool are tested to ensure freedom from adventitious agents in rabbits, guinea pigs, adult mice, and newborn mice.
     The production monopool is then passed through a .22 micron filter. Samples are taken for monopool testing by quality control to include testing for potency, testing for polio neurovirulence, testing for markers of attenuation. The appearance of any adventitious agent at any stage of testing results in rejection of the monopool. This process is repeated for each monopool virus type.
     A document is then prepared containing the production history and test results on the monopool by quality assurance. This document is submitted to FDA Center for Biologics, Evaluation and Research, along with monopool samples for testing. The FDA reviews the manufacture's test results, performs tests as appropriate, and provides notification of the release of the monopool for further manufacture.
     Released monopools, one for each type, are combined with diluent to make a trivalent vaccine bulk preparation. Samples are tested by quality control for potency and sterility. The vaccine is aseptically filled into a single dose final containers. Samples are tested for quality control, for potency, identity, and safety. Final container samples are also sent to the FDA with a final protocol for the release of the final filled container vaccine for distribution.
     And that completes my talk. Thank you for your attention.

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