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.