Figure Legends
Figure 1: Principle of PTSelect™ technology. a. PTSelect™-siRNA design consisting of BART-10 siRNA precursor (90bp) flanked by 100bp of its native genomic sequence on both 5’ and 3’ flanks. This construct is situated within sequence taken from the human β-globin (HBG) intron (486bp). The entire cassette is cloned upstream of the GOI. b.Expected sequences of processed PTSelect™-siRNAs of siRNA1 (3P segment) and siRNA2 (3P segment) in the context of precursor stem loop sequences.c. PTSelect™-siRNA is processed within cells that contain GOI. When CD4/siRNA mRNA is introduced by electroporation into cells containing the GOI, PTSelect™-siRNAs bind to the complementary sequences on CD4/siRNA mRNA and degrade the mRNA, eliminating exogenous cell-surface CD4 expression (core concept of PTSelect™ technology).d. Depletion is possible due to the variable expression of CD4 on the cell surface (present on cells that do not contain GOI and absent from the cell surface in cells that contain GOI), enabling the separation of cells with GOI from cells without GOI (steps 1 through 4).
Figure 2: Establishment of GFP-stable pool population as a proof of concept: a. Map of EGFP-siRNA1 plasmid with the GOI and the PTSelect™-siRNA1 cassette. b. In each depletion experiment, CD4/siRNA mRNA and dTomato/siRNA1 mRNA are introduced into stock cells by electroporation. Both mRNAs contain sequences complementary to the PTSelect™-siRNA1. At each depletion, cells retained by the magnetic beads (isolate, no EGFP), cells in the supernatant (expressing EGFP) and cells not subjected to separation (stock) were analyzed by flow cytometry analysis c. Flow cytometry analysis of depletion experiments performed on days 3, 10, and 19. The dTomato (PE) signals are suppressed by the PTSelect™-siRNA1, which is expressed by cells that contain EGFP (supernatant) and not by cells that do not contain EGFP (isolate). Top panel shows the dTomato (PE) signals from the supernatant (cells with EGFP and PTSelect™-siRNA), in which the dTomato (PE) signal is suppressed by the PTSelect™-siRNA and from parental CHO cells (control), in which the dTomato (PE) signal remains unaffected. On day 10, the cell population has stabilized. It is possible to sort the fraction of cells with low dTomato signals (circled) and proceed with limited dilution cloning. Bottom panel shows the dTomato (PE) signal in the isolate fraction, in which there is no suppression of dTomato (PE) signal).
Figure 3: Comparison of EpoFc clones generated by PTSelect™ and conventional technologies. a. EpoFc-ELISA of clones three weeks after limited dilution cloning. Significantly more EpoFc-positive clones were generated by PTSelect™, compared to clones generated by puromycin selection or neomycin/MTX amplification. First two columns in each plate are ELISA standards in duplicate. b. Productivity of EpoFc clones (mIU*/cell/day) generated using, PTSelect™ (EpoFc_siRNA1), puromycin selection (EpoFc_siRNA1_Puro) or neomycin selection followed by MTX amplification (EpoFc_DHFR_Neo). Each point represents an individual clone selected as indicated by the legend. c.Productivity of EpoFc clones generated by varying the amount of CD4 mRNA (CD4-500ng; depletion on day 11, CD4-500ng and 1000ng; depletion on day 21). Each point represents an individual clone selected as indicated by the legend. (*mlU definition: The standards in the EPO kit were assays against WHO standard and the conversion between IU (International Units) and mass is ~125 IU/µg).
Figure 4 : Productivity over 12 weeks of top 6 EpoFc-producing clones generated by PTSelect™. a. Clones ES24 (top 1), ES25 (top 2) and ES22 (top 6) dropped below 70% of their initial productivity, while the ES11 (top 3), ES4 (top 4) and ES21 (top 5) maintained productivity. b. Productivity over 12 weeks of top six EpoFc clones generated by neomycin selection followed by MTX amplification. Clones DEN43 (top 1), DEN44 (top 2), DEN30 (top 3), DEN39 (TOP 5) and DEN53 (top 6) dropped below 70% of their initial productivity, while the DEN31 (top 4) maintained productivity.
Figure 5: Comparison of adalimumab clones generated by PTSelect™ and conventional technologies. a. HC­_siRNA1-LC_siRNA2_GS plasmid expressing adalimumab was generated with siRNA1 under the control of the same promoter as HC and siRNA2 under the control of the same promoter as LC. In addition, glutamine synthetase was cloned into this vector to enable stable cell generation by MSX selection. The presence of siRNA1 and siRNA2 along with GS enables this plasmid to be used for both conventional (MSX-selection) and PTSelect™ technologies. b.Productivity of adalimumab clones generated by PTSelect™ (mAb-500 and mAb-1000) using 500 ng or 1000 ng of CD4/siRNA1 mRNA and CD4/siRNA2 mRNA or MSX (25mM) selection (mAb-MSX-25mM). Each point represents an individual clone selected as indicated by the legend.
Figure 6: Monitoring clonal productivity by measuring silencing activity of PTSelect™-siRNA. a. Two high, medium, and lower productivity EpoFc clones generated by PTSelect™ were electroporated with GFP/siRNA1 mRNA with sequences complementary to PTSelect™-siRNA1. The number of cells with high suppression of GFP (FITC) signals (100 on x-axis) correlated with the productivity.b. PTSelect™-generated EpoFc clones that passed (ES4 & ES21) and failed the stability assay (ES24) were electroporated with dTomato/siRNA1 mRNA and GFP-mRNA (transfection control). For control, parental CHO-K1 cells were transfected with either dTomato/siRNA1 or GFP-mRNA. Clones that passed the stability test show significant suppression of dTomato (PE) signals (100 on x-axis) even during week 12, correlating with high expression of EpoFc, while this is absent in the clone that failed the test (clone 24). c.Enrichment of clone 15 for high producers by cell sorting. Left panel, EpoFc-clone-15 showed three different populations (Clone 15-pre-sorted) when electroporated with dTomato/siRNA1 mRNA, based on the inhibition exerted by PTSelect™-siRNA1. From this clone, the three distinct populations were selected by sorting (low, medium and high PE corresponding to high, medium and low expression of EpoFc, respectively). Right panel, three weeks after expansion, sorted Low-PE clone was transfected with GFP-mRNA (transfection control) and dTomato/siRNA1 mRNA. The appearance of population 2 and 3 indicates variation and/or drift in the sorted population. Since GFP was used, signals were compensated in 6b and 6c.