Stability of EPC (End of production cell).pdf

3.2.S.2.3.4.4 Stability of the PPCB 3.2.S.2.3.4.4.1 Stability of EPC Stability of end of production cell (EPC) was tested with cells recovered after culture of WCB. Stability of recombinant cell line, E. coli BL21 (DE3)/pT0191, was tested. Fermentation for the production of drug substance is fed-batch fermentation consisted of three steps of a batch culture, a growth medium feeding fed-batch, and a fed-batch for the production of recombinant protein. Defining criteria of EPC is set as 22 hr (96 generations) of fermentation time during a fed-batch for the production of recombinant protein and optical density (OD600) of more than 160. When the optical density does not increase, the fermentation was stopped. Stability of EPC was tested by the recombinant cell line identification methods such as the phenotype of cell, the analysis of genotype, and the expression analysis of target protein (3.2.S.2.3.4.5), viable cell count, and the presence of microbial contamination (Figure 3.2.S.2.3-11, 12, 13, and 14, Table 3.2.S.2.3-6, 7, and 8). Under the fermentation condition for the production of target product, the recombinant cell line showed the same results with cells of MCB and WCB. When the stability of recombinant microorganisms during fermentation was tested by comparing the colony numbers of LB plate and LB plate containing 200 μg/mL of ampicillin for the antibiotic resistance, the recombinant plasmid-harbouring microorganisms after 22 hr of main culture, which is passed about 96 generations, occupied 95% of total colony number as shown in Table 3.2.S.2.3-6. From these results, it was confirmed that the recombinant cell line has recombinant plasmid under the fermentation condition for the production of target protein. Table 3.2.S.2.3-6 Stability of EPC Test item Colony number Recombinant plasmid harbouring microorganisms (%) LB LB + ampicillin EPC 300 2