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Figure 1. Protein production workflow and particle characterization. (A)
Schematic of transient transfection workflow for cytosolic and secreted
proteins. Plasmid DNA and polymer were combined to allow for
nanoparticle self-assembly. Cells were transfected with plasmid DNA
encoding a fluorescent reporter or a secreted protein. (B) PBAE and PEI
monomer structures. Backbone monomers B4 and B5, side chain monomers
S3-S5, endcap monomers E6, E7, and E39 used to synthesize PBAE polymers,
and PEI 25 kDa. (C) Structure of B4S4E6 (4-4-6) polymer. Additional
polymer structures are shown in Figure S1. (D) Nanoparticle size
determined via dynamic light scattering (DLS) in HEK media (blue) or CHO
media (red). (E) Zeta potential of nanoparticles in HEK media (blue) or
CHO media (red). Error bars represent SD.
Figure 2. Comparison of polymers for transient transfection of
cytosolic mCherry in HEK and CHO cells. (A) HEK cells were transfected
with 2 µg/mL mCherry-encoding DNA via PBAE or PEI nanoparticles(n =5). In HEK cells, PEI was used at a 2:1 polymer:DNA w/w
ratio, whereas in CHO cells, PEI was used at a 3:1 polymer:DNA w/w
ratio. All PBAEs were used at a 60:1 polymer:DNA w/w ratio in both cell
lines. mCherry fluorescence was assessed via plate reader each day, and
significance was calculated on day 5. (B) mCherry transfection
efficiency was determined via flow cytometry 5 days following transient
transfection of HEK cells with 4 µg mCherry-encoding DNA via PBAE or PEI
nanoparticles (n =5). (C) CHO cells were transfected with 3 µg/mL
mCherry-encoding DNA via PBAE or PEI nanoparticles (n =5).mCherry fluorescence was assessed via plate reader each day, and
significance was calculated on day 5. (D) mCherry transfection
efficiency determined by flow cytometry 5 days following transient
transfection of CHO cells with 6 µg mCherry-encoding DNA via PBAE and
PEI nanoparticles (n =5). (E) Representative fluorescence
microscopy images of HEK and CHO cells 5 days following transient
transfection of mCherry-encoding DNA (4 µg for HEK cells; 6 µg for CHO
cells) with 4-4-6 or PEI nanoparticles. Scale bars are 200 µm. For all
panels, error bars represent SD. Significance on day 5 was calculated
using one-way ANOVA with Dunnett post-test, comparing all conditions to
treatment with PEI. Increases relative to PEI are designated: **p
< 0.01, ***p < 0.001, ****p < 0.0001.
Figure 3. Comparison of 4-4-6 and PEI for transient transfection and
secretion of two recombinant antibodies at various scales in HEK cells.
All transfections used 1 µg/mL DNA at a 60:1 polymer:DNA w/w ratio for
4-4-6 or a 2:1 polymer:DNA w/w ratio for PEI. (A) Comparative yields
from scaled transfections of the 10H2 monoclonal antibody utilizing
4-4-6 or PEI nanoparticles. (B) Comparative yields from transient
transfections of 10H2 antibody utilizing 4-4-6 or PEI nanoparticles at 2
L scale. (C) Comparative yields from transient transfections of the BS2
bispecific antibody utilizing 4-4-6 or PEI nanoparticles at 2 L scale.
Significance was determined by unpaired Student’s t -test (**p
< 0.01). Error bars represent SD.
Figure S1. PBAE synthesis and resulting polymer structures. (A)
Synthesis of 4-4-6 PBAE. Acrylate and amine monomers react for 24h at
85C, followed by an end-capping reaction, resulting in a linear capped
polymer. (B) Resulting structures of 4-4-6, 4-5-7, 4-5-39, and 5-3-6
polymers. (C) Dynamic light scattering measurements and electrophoretic
mobility for PBAE and PEI nanoparticles in HEK or CHO media.
Measurements represent mean values from n=3 individually prepared
replicates.
Figure S2. 4-4-6 and PEI DNA dose optimization in HEK and CHO cells. In
HEK cells, PEI was used at a 2:1 polymer:DNA w/w ratio, whereas in CHO
cells, PEI was used at a 3:1 polymer:DNA w/w ratio. 4-4-6 was used at a
60:1 polymer:DNA w/w ratio in both cell lines. (A) DNA dose optimization
in HEK and CHO cells over 5-day time courses. Cells were transfected
with varying amounts of mCherry DNA and fluorescence was assessed via
plate reader on each day (n =1). (B) Viability was assessed via
MTS assay 24 h following transfection with 2 µg/mL mCherry DNA for HEK
cells or 4 µg/mL mCherry DNA for CHO cells using 4-4-6 or PEI
nanoparticles (n =5). Error bars represent SD. (C) mCherry
transfection efficiency determined via flow cytometry 5 days following
transfection with 2 µg/mL mCherry DNA for HEK cells or 4 µg/mL mCherry
DNA for CHO cells using PBAE or PEI nanoparticles.
mCherry+ cells were gated on live cells.
Representative plots are presented (n =5). (D) Representative
fluorescence microscopy images (n =5) of HEK and CHO cells 5 days
following transfection with 2 µg/mL mCherry DNA for HEK cells or 4µg/mL
mCherry DNA for CHO cells using PBAE or PEI nanoparticles. Scale bars
are 200 µm.
Figure S3. DNA dose optimization for transient transfection of secreted
recombinant antibodies. In HEK cells, PEI was used at a 2:1 polymer:DNA
w/w ratio, whereas in CHO cells, PEI was used at a 3:1 polymer:DNA w/w
ratio. 4-4-6 was used at a 60:1 polymer:DNA w/w ratio in both cell
lines. (A) Reducing SDS-PAGE analysis showing expression of the 10H2
monoclonal antibody following transient transfection of HEK cells with
the indicated doses of DNA encapsulated in PEI (+) or
4-4-6 nanoparticles. (B) Quantification of 10H2 expression from (A),
presented as fold improvement over PEI. (C) Reducing SDS-PAGE analysis
showing expression of the BS2 bispecific antibody following transient
transfection of HEK cells with the indicated doses of DNA encapsulated
in PEI (+) or 4-4-6 nanoparticles. (D) Quantification
of BS2 expression from (C), presented as fold improvement over PEI.
(E) Reducing SDS-PAGE analysis showing expression of the 602 monoclonal
antibody following transient transfection of CHO cells with the
indicated doses of DNA encapsulated in PEI (+) or
4-4-6 nanoparticles. (F) Quantification of 602 expression from (E),
presented as fold improvement over PEI. (G) Comparative yield (pre-FPLC)
from transient transfection of CHO cells with the 602 monoclonal
antibody utilizing 4-4-6 or PEI DNA-containing nanoparticles at 50 mL
scale (4 µg/mL DNA dose). HC, heavy chain; LC, light chain.