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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.