Comparison of EpoFc-stable clones generated by PTSelect™ and conventional methods
Next, we compared individual clones producing a secreted product generated by PTSelect™ and by conventional methods (antibiotic selection and antibiotic selection with MTX amplification) (Supplementary Table 1, comparison 1) for their productivity, doubling time and stability. For this study, three plasmids were generated – EpoFc_siRNA1 (ES), EpoFc_DHFR_Neo (EDN), and EpoFc_siRNA1_Puro (ESP) (Supplementary Figure 3), and transfected into CHO-K1 cells by electroporation. Cells transfected with EDN were selected with neomycin followed by MTX amplification (Supplementary Figure 4a); limited dilution cloning (LDC) was performed on day 31. Cells transfected with ESP were selected with puromycin (Supplementary Figure 4b); LDC was performed on day 24. Cells transfected with ES were depleted on days 3, 7, 10, 13, 15, 18, 24, and 30 with 1000 ng of CD4/siRNA1 mRNA. LDC was performed on a fraction of depleted cells obtained from days 18, 24 and 30. Clonal selection and expansion were performed as outlined in Supplementary Figure 5 and described in materials and methods.
Three weeks after plating, EpoFc levels in expanded clones were assessed by ELISA (Figure 3a). From 80 clones per plate, PTSelect™ generated more positive clones (55 ± 15) compared to antibiotic selection (ESP) (6 ± 4) and antibiotic/MTX amplification (EDN) (10 ± 8). Productivity was assessed for 40 clones from each stable line (Figure 3b). Although the highest producer was generated by PTSelect™ technology (2,755,721 mlU/mL), the next ten highest producers were obtained from clones generated by traditional methods (Supplementary Table 2, Top 5 clones in comparison 1). The average doubling time was 18.6 ± 1.3h for clones generated by PTSelect™, 19.5 ± 3.9h for ESP clones generated by puromycin selection, and 20.5 ± 2.6h for EDN clones generated by neomycin and MTX amplification. The doubling time in PTSelect™-clones is significantly shorter (p < 0.05) compared to the EDN-clones, which could provide a substantial advantage during scale-up.
Next, we evaluated whether the amount of CD4/siRNA1 mRNA used in depletion had any effect on improving the productivity and doubling time of the generated clones (Supplementary Table 1, comparison 2). EpoFc_siRNA1_Puro was introduced into CHO-K1 cells by electroporation. Having the puromycin N-acetyl-transferase gene in the plasmid also allowed us to evaluate the effects of the antibiotic resistance gene on the depletion process. Cells were depleted on days 3 and 8 using 500 ng of CD4/siRNA1 mRNA. On day 11, cells were depleted with 500 ng of CD4/siRNA1 mRNA, and a fraction of cells obtained was subjected to LDC. The remaining depleted cells were allowed to expand until day 21, depleted with either 500 ng or 1000 ng of CD4/siRNA1 mRNA, and then subjected to LDC. Expanded clones were analyzed by ELISA after three weeks. All three depletion approaches produced a similar number of EpoFc-positive clones (55 ± 10, out of 80, Supplementary Figure 6). Productivity was tested for 25 clones per condition (Figure 3c). The highest producer occurred in cells depleted on day 21 with 500 ng of CD4/siRNA1 mRNA (1,119,926 mlU/mL). Overall, there was no significant difference in productivity of the top clones between groups depleted on day 21 (f -ratio = 0.4; p= 0.67, one-way ANOVA). There was no significant difference in the average doubling time of 25 clones generated in each group (Supplementary Table 2). Thus, the amount of CD4/siRNA1 mRNA used in depletion had no significant effect on productivity or doubling time. Furthermore, stable clones can be established as early as day 11, and the presence of an antibiotic gene does not affect stable cell generation by PTSelect™.
To compare clonal stability, we selected the six highest producing clones generated by both PTSelect™ and neomycin selection with MTX amplification. Clonal stability was assessed for a 12-week period as described in Methods. Based on the titer, 3 out of 6 clones generated by PTSelect™ passed the stability criterion with productivity remaining above 70% of initial productivity after 12 weeks (Figure 4a). However, only 1 out of 6 EDN clones passed the same stability criterion (Figure 4b). Thus, PTSelect™ technology generated clones with greater stability than those generated by antibiotic selection and MTX amplification.