Poly(beta-amino ester)s as high-yield transfection reagents for recombinant protein productionKathryn M. Luly1,2, Stephen J. Lee2,3, Huilin Yang2,3, Wentao Wang1,2, Seth D. Ludwig2,3, Haley E. Tarbox4, David R. Wilson1,2,5, Jordan J. Green1,2,3,5,6,7,8,9,10*, Jamie B. Spangler1,2,3,6,7,8*1Department of Biomedical Engineering, Johns Hopkins University School of Medicine, 2Translational Tissue Engineering Center, Johns Hopkins University School of Medicine,3Department of Chemical & Biomolecular Engineering, Johns Hopkins University, 4Department of Chemistry, Johns Hopkins University, 5Institute for Nanobiotechnology, Johns Hopkins University,6Department of Oncology, Johns Hopkins University School of Medicine, 7Bloomberg~Kimmel Institute for Cancer Immunotherapy, Johns Hopkins University School of Medicine, 8Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine,9Departments of Neurosurgery and Ophthalmology, Johns Hopkins University School of Medicine, 10Department of Materials Science & Engineering, Johns Hopkins University*Correspondence should be addressed to:Jamie B. Spangler400 N Broadway, Smith 5011, Baltimore, MD 21231(443) email@example.comJordan J. Green400 N Broadway, Smith 5017, Baltimore, MD 21231(410) firstname.lastname@example.orgGrant numbers: R01EY031097, R01EB029455, R01CA228133, P41EB028239, R01CA240339Transient transfection is an essential tool for recombinant protein production, as rapid screening for expression is conducted without stable integration of genetic material into a target cell genome. Poly(ethylenimine) (PEI) is the current gold standard for transient gene transfer, but transfection efficiency and resulting protein yield are limited by the polymer’s toxicity. This study investigated the use of an alternative class of cationic polymers, poly(beta-amino ester)s (PBAEs), for transient transfection of human embryonic kidney 293F (HEK) and Chinese hamster ovary-S (CHO) cell suspensions. In both HEK and CHO cells, several PBAEs demonstrated superior transfection efficiency and production of a cytosolic reporter compared to PEI. This result extended to secreted proteins, as a model PBAE increased the production of three secreted antibodies compared to PEI at scales ranging from 20-2,000 mL. In particular, non-viral gene transfer using the lead PBAE/plasmid DNA nanoparticles led to robust transfection of mammalian cells across different constructs, doses, volumes, and cell types. These results show that PBAEs enhance transfection efficiency and increase protein yield compared to a widespread commercially available reagent, making them attractive candidates as reagents for use in recombinant protein production.Keywords: protein production, transient transfection, poly(beta-amino ester)s, transfection reagentsCurrent research into chemical-based transfection methods focuses largely on optimizing agents for use in the development and production of recombinant proteins. Transient transfection, in which introduced genetic material is not incorporated into the host genome, is especially useful during the high-throughput design and screening of proteins (e.g., candidate biologics) wherein stable expression is not needed. While culture conditions and plasmid design have been popular targets for optimization in transient transfection workflows (Backliwal et al., 2008; Galbraith, Tait, Racher, Birch, & James, 2006), further research into improved transfection reagents has even greater potential for boosting protein yields. Chemical-based transient transfection relies on condensation and encapsulation of plasmid DNA by a biocompatible material into particles which are taken up by target cells; differences in particle size can affect the method of cellular uptake, leading to differences in transfection efficiency (Kim, Sunshine, & Green, 2014). Particles must then escape the endosome and the encapsulating material must degrade to allow for DNA release, nuclear translocation, transcription and subsequent export, and finally translation and processing into fully formed protein (Karlsson, Rhodes, Green, & Tzeng, 2020).Transfection reagent structure and buffering capacity have been demonstrated to influence DNA uptake and escape, making these properties particularly consequential in reagents for transient transfection workflows (Sunshine, Peng, & Green, 2012). Maximizing protonability, for example, facilitates endosomal swelling and consequent rupture via the “proton sponge” effect (Boussif et al., 1995; Bus, Traeger, & Schubert, 2018). Cationic polymers have typically been among the most promising transfection reagents; their charge-based association with DNA into particles offers protection from degradation and offers sufficient buffering capacity to facilitate endosomal escape following cellular uptake (Sunshine et al., 2012).Poly(ethyleneimine) (PEI) is a commercially available cationic polymer used extensively as a transfection reagent that has a high density of protonatable amines, giving rise to high buffering capacity and efficient endosomal escape (Boussif et al., 1995). PEI of average molecular weight 25 kDa is most frequently used in transfection workflows, but its toxicity limits transfection efficiency and, consequently, protein yield (Breunig, Lungwitz, Liebl, & Goepferich, 2007; Yang, Li, Goh, & Li, 2007). Previously, PEI has been conjugated to polyethylene glycol (Petersen et al., 2002) and arginine modified oligo(-alkylaminosiloxane) [P(SiDAAr)n] (Morris & Sharma, 2010) to mitigate cytotoxicity.A promising alternative to PEI, poly(beta-amino ester)s (PBAEs) are a class of cationic polymers used to facilitate efficient gene transferin vitro (Bishop, Kozielski, & Green, 2015). PBAEs are composed of an acrylate base monomer, an amine sidechain, and a terminal end-capping group, each of which can be varied to create a vast library of materials (Akinc, Lynn, Anderson, & Langer, 2003). Hydrolyzable ester linkages allow for degradation of the PBAEs in transfection conditions which allows for use of the polymers at high weight ratios relative to other non-biodegradable materials, maximizing density of buffering amines to facilitate endosomal escape (Sunshine et al., 2012). Their biodegradability also obviates the need for medium replacements or additions, themselves contributors to cell death, which are common where PEI is utilized (Galbraith et al., 2006). These linear polymers are synthesized from inexpensive, commercially available reagents using a two-step polymerization method (Fig. S1A) and are stable long term when stored dry at -20°C (Wilson et al., 2019).Given the high transfection efficacy observed with PBAEs in variousin vitro contexts, we sought to investigate the use of PBAE nanoparticles for transient transfection of suspension cultures in intracellular and secreted protein production workflows (Fig. 1A). We selected four PBAEs with varying base (B), sidechain (S), and end-cap (E) structures to evaluate in comparison with linear 25 kDa PEI: B4-S4-E6 (4-4-6); B4-S5-E7 (4-5-7); B4-S5-E39 (4-5-39); and B5-S3-E6 (5-3-6) (Fig. 1B-C, S1B). Physiochemical characterization of PBAE and PEI nanoparticles in serum-free transfection media indicated that PBAE nanoparticles maintained a smaller size in transfection conditions (approximately 200-350 nm) whereas PEI nanoparticles were prone to aggregation, resulting in sizes over 1 µm (Fig. 1D, S1C). Previous studies indicated that PEI nanoparticles were prone to aggregation in serum-free media due to interactions with salts and a lack of adsorbed proteins that can help stabilize discrete particles and prevent clustering (Ogris et al., 1998; Pezzoli, Giupponi, Mantovani, & Candiani, 2017). Analysis of surface charge revealed that PBAE nanoparticles maintained a positive zeta potential in transfection conditions, whereas PEI nanoparticles exhibited a near neutral surface charge (Fig. 1E, S1C). Shielded surface charge of PEI particles may limit interactions with a charged cell membrane, thus hindering cellular uptake.To determine the optimal DNA dose for production of cytosolic mCherry using various polymer-based transfection agents, we selected a representative PBAE, 2-(3-aminopropylamino)ethanol end-capped poly(1,4-butanediol diacrylate-co-4-amino-1-butanol) (referred to here as 4-4-6), and compared this PBAE to 25 kDa PEI at doses ranging from 0.5 to 4 µg/mL DNA. The polymers were compared in two mammalian cell lines frequently employed for protein expression: human embryonic kidney 293F (HEK) cells and Chinese hamster ovary-S (CHO) cells. Evaluation of mCherry fluorescence over a span of 5 days indicated that peak mCherry expression occurred using the 4-4-6 polymer at 2 µg/mL and 4 µg/mL DNA doses in HEK and CHO cells, respectively (Fig. S1A). Notably, peak mCherry expression in PEI-based transfections was not comparable to that attained by 4-4-6 at any dose. Subsequent time course studies using the optimized DNA dose that compared additional PBAE structures demonstrated significantly increased mCherry expression using 4-4-6, 4-5-7, and 4-5-39 in HEK cells, and using 4-4-6 and 4-5-7 in CHO cells, compared to PEI-mediated transfection (Fig. 2A, 2C). Transfection efficiency, as measured by mCherry-positive cells on day 5, was significantly increased with all PBAEs tested in HEK cells and with 4-4-6, 4-5-7, and 4-5-39 in CHO cells (Fig. 2B, 2D, S2C). Fluorescence microscopy confirmed the increase in mCherry expression (Fig. 2E, S2D). Cell viability, assessed via MTS assay 24 h following transfection, indicated that PBAEs (especially 4-5-39 and 5-3-6) showed greater toxicity than PEI, though notably at a 20- to 30-fold higher weight ratio (Fig. S2B). Importantly, this reduced viability did not result in inferior mCherry expression relative to PEI, with 4-4-6 and 4-5-7 demonstrating superior expression in both HEK and CHO cells (Fig. 2A, C).To demonstrate that the results of these fluorescent protein expression experiments were replicable at scales relevant to the development and production of secreted proteins, we transfected HEK cell cultures of varying volumes with DNA encoding the recombinant antibody 10H2 (Chuntharapai, Lee, Hébert, & Kim, 1994; Patent No. WO/2020/243489, 2020) using either 4-4-6- or PEI-based particles. Based on SDS-PAGE image analysis of small-scale dose titrations (Fig. S3A-D), DNA was dosed at 1 µg/mL for secreted proteins in HEK cells, with polymer weight adjusted accordingly. At volumes ranging from 20-200 mL, transfection with 4-4-6 yielded between 4.5-fold and 8.2-fold more protein than did transfection with PEI (Fig. 3A). To further demonstrate the scalability of enhanced protein expression using PBAEs, 2 L cultures of HEK cells were transfected with DNA encoding either 10H2 or the bispecific antibody BS2 (Patent No. WO/2020/243489, 2020) using either 4-4-6 or PEI. Both the 10H2 and BS2 antibodies were recovered in significantly higher quantities (4.9-fold and 5.6-fold higher, respectively) when 4-4-6 was utilized compared to PEI (Fig. 3B-C). Superiority of PBAE nanoparticles was reproducible across cell lines; transfection of CHO cells with DNA encoding the recombinant antibody 602 (Krieg, Letourneau, Pantaleo, & Boyman, 2010; Létourneau et al., 2010; Patent No. WO/2020/264321, 2020) at an optimized dose of 4 µg/mL resulted in 3.4-fold more protein recovered when 4-4-6 was used compared to PEI (Fig. S3E-G).Taken together, experiments with both cytosolic and secreted proteins demonstrated that PBAEs lead to significantly enhanced protein yields compared to leading commercial reagent PEI in two cell lines that are widely used for protein production. Storage stability and straightforward synthesis from inexpensive chemical monomers further strengthen their attractiveness for use in recombinant protein production across batch scales. Overall, the favorable properties of PBAEs combined with the results herein suggest that these polymers hold promise as superior reagents for transient transfection that can significantly improve protein production workflows.