Fabrication of electrospun ion exchanger adsorbents with morphologies designed for the separation of proteins and plasmid DNA.

Electrospun cellulose adsorbents are an emergent class of materials applied to a variety of bioprocess separations as an analogue to conventional packed bed chromatography. Electrospun adsorbents have proven to be effective as rapid cycling media, enabling high throughput separation of proteins and viral vectors without compromising selectivity and recovery. However, there is a current lack of knowledge in relation to the manipulation and control of electrospun adsorbent structure with function and performance to cater to the separation needs of emerging, diverse biological products. In this study, a series of electrospun cellulose adsorbents were fabricated by adjusting their manufacturing conditions. A range of fiber diameters (400 to 600 nm) was created by changing the electrospinning polymer solution. Additionally, a range of porosities (0.4 to 0.7 v/v) was achieved by varying the laminating pressures on the electrospun sheets. The adsorbents were functionalized with different degrees of quaternary amine ligand density to create 18 prototype anion exchangers. Their morphology was characterized by BET nitrogen adsorption surface area, X-ray computed tomography, capillary flow porometry and scanning electron microscopy measurements. The physical characteristics of the adsorbents were used in an adapted semi-empirical model and compared to measured permeability data. Permeabilities of prototypes ranged from 10-2 to 10-4 mDarcy. The measured data showed good adherence to modelled data with possible improvements in acquiring wet adsorbent characteristics instead of dried material. Finally, the electrospun adsorbents were characterized for their binding capacity of model proteins of different sizes (diameters of 3.5 nm and 8.9 nm) and plasmid DNA. Static binding capacities ranged from 5 mg/ml to 25 mg/ml for the proteins and plasmid DNA and showed <20 % deviation from monolayer coverage based on BET surface area. Therefore, it was concluded that the electrospun adsorbents most likely adsorb monolayers of proteins and plasmid DNA on the surface with minimal steric hindrance.

Assessment of cytoprotective and genoprotective effects of Moringa oleifera and Tinospora cordifolia extracts in vitro.

Background: Moringa oleifera and Tinospora cordifolia is extensively used as an ingredient of food and in traditional medicine for the management of a variety of diseases. Material and methods: The extracts of leaf of Moringa oleifera and stem of Tinospora cordifolia were assessed to examine their ability to inhibit the oxidative DNA damage (by DNA protection assay), cytoprotective and genoprotective potential (by Comet assay) in V79 cells individually and in combinations. Result: It was found that these extracts could significantly inhibit the OH-dependent damage of pUC18 plasmid DNA. M. oleifera extract (160 and 320 µg/mL) and Tinospora cordifolia extract (640, 1,280 and 2,560 µg/mL) individually showed higher DNA protection activity. M. oleifera (1,280 µg/mL) combined with Tinospora cordifolia (640 µg/mL) showed best cytoprotective and genoprotective activities among different concentration combinations and various concentrations of individual plants in V79 cell line against hydrogen peroxide induced cytotoxicity and genotoxicity. Conclusion: This study demonstrates the cytoprotective and genoprotective activity of M. oleifera and Tinospora cordifolia individually or in combination.

Escherichia coli grown in inexpensive conical flat-bottom polypropylene tubes produce a high level of pUC vector.

The pUC-derived plasmid yield from E. coli using polypropylene tubes (PP) was compared among round and conical tubes. The yield from cells grown in a cheaper conical-PP with flat-bottom was 1.5-fold higher (p < 0.001) than other PP. The use of the conical-PP can save research budgets in the current inflationary environment.

Supercoiled DNA percentage: A key in-process control of linear DNA template for mRNA drug substance manufacturing.

The development of messenger RNA (mRNA) vaccines and therapeutics necessitates the production of high-quality in vitro-transcribed mRNA drug substance with specific critical quality attributes (CQAs), which are closely tied to the uniformity of linear DNA template. The supercoiled plasmid DNA is the precursor to the linear DNA template, and the supercoiled DNA percentage is commonly regarded as a key in-process control (IPC) during the manufacturing of linear DNA template. In this study, we investigate the influence of supercoiled DNA percentage on key mRNA CQAs, including purity, capping efficiency, double-stranded RNA (dsRNA), and distribution of poly(A) tail. Our findings reveal a significant impact of supercoiled DNA percentage on mRNA purity and in vitro transcription yield. Notably, we observe that the impact on mRNA purity can be mitigated through oligo-dT chromatography, alleviating the tight range of DNA supercoiled percentage to some extent. Overall, this study provides valuable insights into IPC strategies for DNA template chemistry, manufacturing, and controls (CMC) and process development for mRNA drug substance.

One-Step Formation Method of Plasmid DNA-Loaded, Extracellular Vesicles-Mimicking Lipid Nanoparticles Based on Nucleic Acids Dilution-Induced Assembly.

We propose a nucleic acids dilution-induced assembly (NADIA) method for the preparation of lipid nanoparticles. In the conventional method, water-soluble polymers such as nucleic acids and proteins are mixed in the aqueous phase. In contrast, the NADIA method, in which self-assembly is triggered upon dilution, requires dispersion in an alcohol phase without precipitation. We then investigated several alcohols and discovered that propylene glycol combined with sodium chloride enabled the dispersion of plasmid DNA and protamine sulfate in the alcohol phase. The streamlined characteristics of the NADIA method enable the preparation of extracellular vesicles-mimicking lipid nanoparticles (ELNPs). Among the mixing methods using a micropipette, a syringe pump, and a microfluidic device, the lattermost was the best for decreasing batch-to-batch differences in size, polydispersity index, and transfection efficiency in HepG2 cells. Although ELNPs possessed negative ζ-potentials and did not have surface antigens, their transfection efficiency was comparable to that of cationic lipoplexes. We observed that lipid raft-mediated endocytosis and macropinocytosis contributed to the transfection of ELNPs. Our strategy may overcome the hurdles linked to supply and quality owing to the low abundance and heterogeneity in cell-based extracellular vesicles production, making it a reliable and scalable method for the pharmaceutical manufacture of such complex formulations.

Plasmid DNA Complexes in Powder Form Studied by Spectroscopic and Diffraction Methods.

Currently, new functional materials are being created with a strong emphasis on their ecological aspect. Materials and devices based on DNA biopolymers, being environmentally friendly, are therefore very interesting from the point of view of applications. In this paper, we present the results of research on complexes in the powder form based on plasmid DNA (pDNA) and three surfactants with aliphatic chains containing 16 carbon atoms (cetyltrimethylammonium chloride, benzyldimethylhexadecylammonium chloride and hexadecylpyridinium chloride). The X-ray diffraction results indicate a local hexagonal packing of DNA helices in plasmid DNA complexes, resembling the packing for corresponding complexes based on linear DNA. Based on the Fourier-transform infrared spectroscopy results, the DNA conformation in all three complexes was determined as predominantly of A-type. The two relaxation processes revealed by dielectric spectroscopy for all the studied complexes are connected with two different contributions to total conductivity (crystallite part and grain boundaries). The crystallite part (grain interior) was interpreted as an oscillation of the polar surfactant head groups and is dependent on the conformation of the surfactant chain. The influence of the DNA type on the properties of the complexes is discussed, taking into account our previous results for complexes based on linear DNA. We showed that the type of DNA has an impact on the properties of the complexes, which has not been demonstrated so far. It was also found that the layer of pDNA-surfactant complexes can be used as a layer with variable specific electric conductivity by selecting the frequency, which is interesting from an application point of view.

From Bioreactor to Bulk Rheology: Achieving Scalable Production of Highly Concentrated Circular DNA.

DNA serves as a model system in polymer physics due to its ability to be obtained as a uniform polymer with controllable topology and nonequilibrium behavior. Currently, a major obstacle in the widespread adoption of DNA is obtaining it on a scale and cost basis that accommodates bulk rheology and high-throughput screening. To address this, recent advancements in bioreactor-based plasmid DNA production is coupled with anion exchange chromatography producing a unified approach to generating gram-scale quantities of monodisperse DNA. With this method, 1.1 grams of DNA is obtained per batch to generate solutions with concentrations up to 116 mg mL-1. This solution of uniform supercoiled and relaxed circular plasmid DNA, is roughly 69 times greater than the overlap concentration. The utility of this method is demonstrated by performing bulk rheology measurements at sample volumes up to 1 mL on DNA of different lengths, topologies, and concentrations. The measured elastic moduli are orders of magnitude larger than those previously reported for DNA and allowed for the construction of a time-concentration superposition curve that spans 12 decades of frequency. Ultimately, these results can provide important insights into the dynamics of ring polymers and the nature of highly condensed DNA dynamics.

Blood pressure reduction through brain delivery of nanoparticles loaded with plasmid DNA encoding angiotensin receptor shRNA.

Elevated brain angiotensin II activity plays a key role in the development of neurogenic hypertension. While blood pressure (BP) control in neurogenic hypertension has been successfully demonstrated by regulating central angiotensin II activity, current techniques involving cerebrovascular injections of potential therapeutic agents are not suitable for clinical translation. To address this gap, we present the synthesis of dual-functionalized liposomes functionalized with targeting ligand and cell-penetrating peptide. Functionalized liposomes were synthesized using the thin film hydration technique and loaded with plasmid DNA encoding short hairpin RNA targeted toward angiotensin II receptors (PEAS), via the post-insertion method. The synthesized liposomes had a cationic surface charge, an average size of 150 nm, and effectively entrapped more than 89% of loaded PEAS. These liposomes loaded with PEAS demonstrated biocompatibility and efficient delivery to brain-derived cell lines, resulting in a remarkable reduction of more than 70% in receptor expression within 7 days. To assess the therapeutic potential, spontaneously hypertensive rats were administered intravenous injections of functionalized liposomes loaded with PEAS, and the changes in mean arterial pressure were monitored for 45 days. Remarkably, this treatment led to a significant (p < 0.001) decrease in BP of more than 30 mm Hg compared with saline-treated rats.

Producing Plasmid DNA Template for Clinical Grade RNA Vaccine Manufacture.

A plasmid production process has been established to manufacture plasmid DNA at a large scale in High-Quality grade. This is used as a starting material to produce mRNA vaccines for clinical trials. Recently, the World Health Organization (WHO) has released regulatory guidelines related to the quality, safety, and efficacy for DNA- as well as for mRNA-based vaccines. Following an extraordinary year of scientific, regulatory, and manufacturing developments, the scientific community today stands considerably better equipped to deal with urgent production requirements in large scale for nucleic acid-based vaccinations and therapies. Going forward, work needs to be done in better coordinating the supply and logistics of essential raw materials for biological manufacturing, especially under emergency conditions.

Intracellular Delivery of Plasmid DNA Using Amphipathic Helical Cell-Penetrating Peptides Containing Dipropylglycine.

Cell-penetrating peptides (CPPs) serve as potent vehicles for delivering membrane-impermeable compounds, including nucleic acids, into cells. In a previous study, we reported the successful intracellular delivery of small interfering RNAs (siRNAs) with negligible cytotoxicity using a peptide containing an unnatural amino acid (dipropylglycine). In the present study, we employed the same seven peptides as the previous study to evaluate their efficacy in delivering plasmid DNA (pDNA) intracellularly. Although pDNA and siRNA are nucleic acids, they differ in size and biological function, which may influence the optimal peptide sequences for their delivery. Herein, three peptides demonstrated effective pDNA transfection abilities. Notably, only one of the three peptides previously exhibited efficient gene-silencing effect with siRNA. These findings validate our hypothesis and offer insights for the personalized design of CPPs for the delivery of pDNA and siRNA.

Characterization of peptide-fused protein assemblies in living cells.

Assembly of de novo peptides designed from scratch is in a semi-rational manner and creates artificial supramolecular structures with unique properties. Considering that the functions of various proteins in living cells are highly regulated by their assemblies, building artificial assemblies within cells holds the potential to simulate the functions of natural protein assemblies and engineer cellular activities for controlled manipulation. How can we evaluate the self-assembly of designed peptides in cells? The most effective approach involves the genetic fusion of fluorescent proteins (FPs). Expressing a self-assembling peptide fused with an FP within cells allows for evaluating assemblies through fluorescence signal. When µm-scale assemblies such as condensates are formed, the peptide assemblies can be directly observed by imaging. For sub-µm-scale assemblies, fluorescence correlation spectroscopy analysis is more practical. Additionally, the fluorescence resonance energy transfer (FRET) signal between FPs is valuable evidence of proximity. The decrease in fluorescence anisotropy associated with homo-FRET reveals the properties of self-assembly. Furthermore, by combining two FPs, one acting as a donor and the other as an acceptor, the heteromeric interaction between two different components can be studied through the FRET signal. In this chapter, we provide detailed protocols, from designing and constructing plasmid DNA expressing the peptide-fused protein to analysis of self-assembly in living cells.

Gene Transfection Efficiency Improvement with Lipid Conjugated Cationic Carbon Dots.

An ideal vehicle with a high transfection efficiency is crucial for gene delivery. In this study, a type of cationic carbon dot (CCD) known as APCDs were first prepared with arginine (Arg) and pentaethylenehexamine (PEHA) as precursors and conjugated with oleic acid (OA) for gene delivery. By tuning the mass ratio of APCDs to OA, APCDs-OA conjugates, namely, APCDs-0.5OA, APCDs-1.0OA, and APCDs-1.5OA were synthesized. All three amphiphilic APCDs-OA conjugates show high affinity to DNA through electrostatic interactions. APCDs-0.5OA exhibit strong binding with small interfering RNA (siRNA). After being internalized by Human Embryonic Kidney (HEK 293) and osteosarcoma (U2OS) cells, they could distribute in both the cytoplasm and the nucleus. With APCDs-OA conjugates as gene delivery vehicles, plasmid DNA (pDNA) that encodes the gene for the green fluorescence protein (GFP) can be successfully delivered in both HEK 293 and U2OS cells. The GFP expression levels mediated by APCDs-0.5OA and APCDs-1.0OA are ten times greater than that of PEI in HEK 293 cells. Furthermore, APCDs-0.5OA show prominent siRNA transfection efficiency, which is proven by the significantly downregulated expression of FANCA and FANCD2 proteins upon delivery of FANCA siRNA and FANCD2 siRNA into U2OS cells. In conclusion, our work demonstrates that conjugation of CCDs with a lipid structure such as OA significantly improves the gene transfection efficiency, providing a new idea about the designation of nonviral carriers in gene delivery systems.

Electrophoretic Microfluidic Characterization of mRNA- and pDNA-Loaded Lipid Nanoparticles.

Lipid nanoparticles (LNPs) are clinically advanced nonviral gene delivery vehicles with a demonstrated ability to address viral, oncological, and genetic diseases. However, the further development of LNP therapies requires rapid analytical techniques to support their development and manufacturing. The method developed and described in this paper presents an approach to rapidly and accurately analyze LNPs for optimized therapeutic loading by utilizing an electrophoresis microfluidic platform to analyze the composition of LNPs with different clinical lipid compositions (Onpattro, Comirnaty, and Spikevax) and nucleic acid (plasmid DNA (pDNA) and messenger RNA (mRNA)) formulations. This method enables the high-throughput screening of LNPs using a 96- or 384-well plate with approximate times of 2-4 min per sample using a total volume of 11 µL. The lipid analysis requires concentrations approximately between 109 and 1010 particles/mL and has an average precision error of 10.4% and a prediction error of 19.1% when compared to using a NanoSight, while the nucleic acid analysis requires low concentrations of 1.17 ng/µL for pDNA and 0.17 ng/µL for mRNA and has an average precision error of 4.8% and a prediction error of 9.4% when compared to using a PicoGreen and RiboGreen assay. In addition, our method quantifies the relative concentration of nucleic acid per LNP. Utilizing this approach, we observed an average of 263 ± 62.2 mRNA per LNP and 126.3 ± 21.2 pDNA per LNP for the LNP formulations used in this study, where the accuracy of these estimations is dependent on reference standards. We foresee the utility of this technique in the high-throughput characterization of LNPs during manufacturing and formulation research and development.

Interaction of γ-Polyglutamic Acid/Polyethyleneimine/Plasmid DNA Ternary Complexes with Serum Components Plays a Crucial Role in Transfection in Mice.

Typical examples of non-viral vectors are binary complexes of plasmid DNA with cationic polymers such as polyethyleneimine (PEI). However, problems such as cytotoxicity and hemagglutination, owing to their positively charged surfaces, hinder their in vivo use. Coating binary complexes with anionic polymers, such as γ-polyglutamic acid (γ-PGA), can prevent cytotoxicity and hemagglutination. However, the role of interactions between these complexes and serum components in in vivo gene transfer remains unclear. In this study, we analyzed the contribution of serum components to in vivo gene transfer using PEI/plasmid DNA binary complexes and γ-PGA/PEI/plasmid DNA ternary complexes. In binary complexes, heat-labile components in the serum greatly contribute to the hepatic and splenic gene expression of the luciferase gene. In contrast, serum albumin and salts affected the hepatic and splenic gene expression in the ternary complexes. Changes in physicochemical characteristics, such as increased particle size and decreased absolute values of ζ-potential, might be involved in the enhanced gene expression. These findings would contribute to a better understanding of in vivo non-viral gene transfer using polymers, such as PEI and γ-PGA.

Delivery of DNA-Based Therapeutics for Treatment of Chronic Diseases.

Gene therapy and its role in the medical field have evolved drastically in recent decades. Studies aim to define DNA-based medicine as well as encourage innovation and the further development of novel approaches. Gene therapy has been established as an alternative approach to treat a variety of diseases. Its range of mechanistic applicability is wide; gene therapy has the capacity to address the symptoms of disease, the body's ability to fight disease, and in some cases has the ability to cure disease, making it a more attractive intervention than some traditional approaches to treatment (i.e., medicine and surgery). Such versatility also suggests gene therapy has the potential to address a greater number of indications than conventional treatments. Many DNA-based therapies have shown promise in clinical trials, and several have been approved for use in humans. Whereas current treatment regimens for chronic disease often require frequent dosing, DNA-based therapies can produce robust and durable expression of therapeutic genes with fewer treatments. This benefit encourages the application of DNA-based gene therapy to manage chronic diseases, an area where improving efficiency of current treatments is urgent. Here, we provide an overview of two DNA-based gene therapies as well as their delivery methods: adeno associated virus (AAV)-based gene therapy and plasmid DNA (pDNA)-based gene therapy. We will focus on how these therapies have already been utilized to improve treatment of chronic disease, as well as how current literature supports the expansion of these therapies to treat additional chronic indications in the future.

Enhancing In Vivo Electroporation Efficiency through Hyaluronidase: Insights into Plasmid Distribution and Optimization Strategies.

Electroporation (EP) stands out as a promising non-viral plasmid delivery strategy, although achieving optimal transfection efficiency in vivo remains a challenge. A noteworthy advancement in the field of in vivo EP is the application of hyaluronidase, an enzyme with the capacity to degrade hyaluronic acid in the extracellular matrix, which thereby enhances DNA transfer efficiency by 2- to 3-fold. This paper focuses on elucidating the mechanism of hyaluronidase's impact on transfection efficiency. We demonstrate that hyaluronidase promotes a more uniform distribution of plasmid DNA (pDNA) within skeletal muscle. Additionally, our study investigates the effect of the timing of hyaluronidase pretreatment on EP efficiency by including time intervals of 0, 5, and 30 min between hyaluronidase treatment and the application of pulses. Serum levels of the pDNA-encoded transgene reveal a minimal influence of the hyaluronidase pretreatment time on the final serum protein levels following delivery in both mice and rabbit models. Leveraging bioimpedance measurements, we capture morphological changes in muscle induced by hyaluronidase treatment, which result in a varied pDNA distribution. Subsequently, these findings are employed to optimize EP electrical parameters following hyaluronidase treatment in animal models. This paper offers novel insights into the potential of hyaluronidase in enhancing the effectiveness of in vivo EP, as well as guides optimized electroporation strategies following hyaluronidase use.

Products of Selenite/Thiols Interaction Have Reducing Properties, Cleave Plasmid DNA and Decrease Rat Blood Pressure and Tension of Rat Mesenteric Artery.

Selenium compounds exert their antioxidant activity mostly when the selenium atom is incorporated into selenoproteins. In our work, we tested the possibility that selenite itself interacts with thiols to form active species that have reducing properties. Therefore, we studied the reduction of 2-(4-carboxyphenyl)-4,5-dihydro-4,4,5,5-tetramethyl-1H-imidazol-1-yloxy-3-oxide radical (•cPTIO), damage of plasmid DNA (pDNA), modulation of rat hemodynamic parameters and tension of isolated arteries induced by products of interaction of selenite with thiols. We found that the products of selenite interaction with thiols had significant reducing properties that could be attributed mainly to the selenide and that selenite had catalytic properties in the access of thiols. The potency of thiols to reduce •cPTIO in the interaction with selenite was cysteine > homocysteine > glutathione reduced > N-acetylcysteine. Thiol/selenite products cleaved pDNA, with superoxide dismutase enhancing these effects suggesting a positive involvement of superoxide anion in the process. The observed •cPTIO reduction and pDNA cleavage were significantly lower when selenomethionine was used instead of selenite. The products of glutathione/selenite interaction affected several hemodynamic parameters including rat blood pressure decrease. Notably, the products relaxed isolated mesenteric artery, which may explain the observed decrease in rat blood pressure. In conclusion, we found that the thiol/selenite interaction products exhibited significant reducing properties which can be used in further studies of the treatment of pathological conditions caused by oxidative stress. The results of decreased rat blood pressure and the tension of mesenteric artery may be perspective in studies focused on cardiovascular disease and their prevention.

Optimization of lipid nanoparticles for gene editing of the liver via intraduodenal delivery.

Lipid nanoparticles (LNPs) have recently emerged as successful gene delivery platforms for a diverse array of disease treatments. Efforts to optimize their design for common administration methods such as intravenous injection, intramuscular injection, or inhalation, revolve primarily around the addition of targeting ligands or the choice of ionizable lipid. Here, we employed a multi-step screening method to optimize the type of helper lipid and component ratios in a plasmid DNA (pDNA) LNP library to efficiently deliver pDNA through intraduodenal delivery as an indicative route for oral administration. By addressing different physiological barriers in a stepwise manner, we down-selected effective LNP candidates from a library of over 1000 formulations. Beyond reporter protein expression, we assessed the efficiency in non-viral gene editing in mouse liver mediated by LNPs to knockdown PCSK9 and ANGPTL3 expression, thereby lowering low-density lipoprotein (LDL) cholesterol levels. Utilizing an all-in-one pDNA construct with Strep. pyogenes Cas9 and gRNAs, our results showcased that intraduodenal administration of selected LNPs facilitated targeted gene knockdown in the liver, resulting in a 27% reduction in the serum LDL cholesterol level. This LNP-based all-in-one pDNA-mediated gene editing strategy highlights its potential as an oral therapeutic approach for hypercholesterolemia, opening up new possibilities for DNA-based gene medicine applications.

Engineering pH-sensitive dissolution of lipid-polymer nanoparticles by Eudragit integration impacts plasmid DNA (pDNA) transfection.

Lipid-polymer nanoparticles offer a promising strategy for improving gene nanomedicines by combining the benefits of biocompatibility and stability associated with the individual systems. However, research to date has focused on poly-lactic-co-glycolic acid (PLGA) and resulted in inefficient transfection. In this study, biocompatible Eudragit constructs E100 and RS100 were formulated as lipid-polymer nanoparticles loaded with pDNA expressing red fluorescent protein (RFP) as a model therapeutic. Using a facile nanoprecipitation technique, a core-shell structure stabilised by lipid-polyethylene glycol (PEG) surfactant was produced and displayed resistance to ultracentrifugation. Both cationic polymers E100 (pH-sensitive dissolution at 5) and RS100 (pH-insensitive dissolution) produced 150-200 nm sized particles with a small positive surface charge (+3-5 mV) and high pDNA encapsulation efficiencies (EE) of 75-90%. The dissolution properties of the Eudragit polymers significantly impacted the biological performance in human embryonic kidney cells (HEK293T). Nanoparticles composed of polymer RS100 resulted in consistently high cell viability (80-100%), whereas polymer E100 demonstrated dose-dependent behaviour (20-90% cell viability). The low dissolution of polymer RS100 over the full pH range and the resulting nanoparticles failed to induce RFP expression in HEK293T cells. In contrast, polymer E100-constructed nanoparticles resulted in reproducible and gradually increasing RFP expression of 26-42% at 48-72 h. Intraperitoneal (IP) injection of the polymer E100-based nanoparticles in C57BL/6 mice resulted in targeted RFP expression in mouse testes with favourable biocompatibility one-week post-administration. These findings predicate Eudragit based lipid-polymer nanoparticles as a novel and effective carrier for nucleic acids, which could facilitate pre-clinical evaluation and translation of gene nanomedicines.

Assessment of pDNA isoforms using microfluidic electrophoresis.

The recent rise in nucleic acid-based vaccines and therapies has resulted in an increased demand for plasmid DNA (pDNA). As a result, there is added pressure to streamline the manufacturing of these vectors, particularly their design and construction, which is currently considered a bottleneck. A significant challenge in optimizing pDNA production is the lack of high-throughput and rapid analytical methods to support the numerous samples produced during the iterative plasmid construction step and for batch-to-batch purity monitoring. pDNA is generally present as one of three isoforms: supercoiled, linear, or open circular. Depending on the ultimate use, the desired isoform may be supercoiled in the initial stages for cell transfection or linear in the case of mRNA synthesis. Here, we present a high-throughput microfluidic electrophoresis method capable of detecting the three pDNA isoforms and determining the size and concentration of the predominant supercoiled and linear isoforms from 2 to 7 kb. The limit of detection of the method is 0.1 ng/µL for the supercoiled and linear isoforms and 0.5 ng/µL for the open circular isoform, with a maximum loading capacity of 10-15 ng/µL. The turnaround time is 1 min/sample, and the volume requirement is 10 µL, making the method suitable for process optimization and batch-to-batch analysis. The results presented in this study will enhance the understanding of electrophoretic transport in microscale systems dependent on molecular conformations and potentially aid technological advances in diverse areas relevant to microfluidic devices.