Comparison of stable adalimumab clones generated by PTSelect™ and
conventional methods
We generated plasmid mAb_siRNA1_siRNA2_GS that expresses both
adalimumab heavy chain (HC) and light chain (LC) with siRNA1 and siRNA2
cassettes, respectively, cloned upstream. The glutamine synthetase
(GS ) gene was also cloned into the same plasmid (Figure 5a) to
test whether the presence of GS had any effect on PTSelect™
depletion. For this study, stable clones were established and compared
as shown in Supplementary Table 1, comparison 3. Plasmid with
mAb_siRNA1_siRNA2_GS was introduced into CHO-K1 cells by
electroporation; a fraction of cells was taken for MSX selection, and
the rest were selected using PTSelect™ technology. Depletion was done on
days 3, 10, and 14 with 150 ng of each of CD4/siRNA1 mRNA and CD4/siRNA2
mRNA and on day 19 with 250 ng of each mRNA (mAb-500) or 500 ng of each
mRNA (mAb-1000). LDC for both depleted samples was performed on day 19.
For the traditional selection method, cells were selected with 25 mM MSX
in glutamine-free medium. Selection was completed on day 24
(Supplementary Figure 7) and LDC was performed. After three weeks, the
number of clones producing mAb was 4 ± 2 out of 80 for MSX-generated
clones and 9 ± 6 out of 80 for PTSelect™-generated clones (data not
shown). Among the forty positive clones from both methods tested for
productivity, the highest producer was generated by the conventional
method (1.69 pg/cell/day), while the top producers from PTSelect™ had
specific productivities of ~ 1 pg/cell/day (Figure 5b,
Supplementary Table 2). Interestingly, there was no difference in the
average doubling times (Supplementary Table 2) between the forty clones
generated by both methods.