A cell banking process was established for rFIX that initially involved adapting the rFIX-producing CHO cells to a serum-free medium, with recombinant human insulin as the only protein component. Stable cell lines producing the highest levels of rFIX were used to establish a master cell bank (MCB). A working cell bank (WCB) is then established in the same medium as the MCB. Cells from the WCB are used to prepare individual lots of rFIX by initiating the cell culture process and the associated purification steps. An extended cell bank for postprocess analysis is also established. The MCB, WCB, and the end-of-production (EOP) cells used to synthesize rFIX undergo extensive testing for purity, viral safety, and cellular productivity .
The MCB, WCB, and EOP cells are routinely screened for the presence of mycoplasma, bacterial and fungal contaminants, and for the presence of viral contaminants such as porcine and bovine parvovirus, murine viruses, and infectious retroviruses. The CHO cells used in rFIX production have been shown to be poor substrates for the growth of many types of viruses, including adventitious human viruses [65,66]. Electron microscopic examination of the MCB and EOP cells has revealed that they contain A- and C-type retroviral particles, as observed with all CHO cells used in recombinant protein production. These particles are not associated with any evidence of infectivity and have been shown by sequencing and cloning studies to be defective .
Additional analyses of the plasmid encoding rFIX, pMT2-1X, and the plasmid encoding PACE-SOL, pEA-PACE-SOL, reveals that the copy number of both plasmids remains the same from rFIX WCB through to rFIX EOP cells. Furthermore, both plasmids remain stably integrated and maintained in the genome of the CHO cell line during inoculum buildup and full-scale production [59,65].
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