Using the DNA 20 kb Plasmid and Linear kit on the PA 800 Plus system

Fang Wang, Marcia Santos
SCIEX, USA

Abstract
 

This technical note provides instructional details and a comprehensive assessment of assay repeatability using the DNA 20 kb Plasmid and Linear kit on the PA 800 Plus system from SCIEX. The DNA 20 kb Plasmid and Linear kit is engineered to analyze both plasmid and linear double-stranded DNA (dsDNA). As demonstrated in previous studies,^1–3 the single ready-to-use kit facilitates high-quality separation of plasmid topology, which is crucial for monitoring plasmid purity (supercoiled percentage), and delivers high-resolution separation of linear dsDNA, which enables size estimation and pattern identification (Figure 1).

For plasmid topology, tunable resolution can be achieved by adjusting the separation voltage. For linear dsDNA, ranging from 0.1 kb to 20 kb, tunable size resolution can be achieved by selecting different capillary lengths. The adjustable resolution and assay throughput for both plasmid and linear dsDNA, enabled by the flexibility of this workflow, helps ensure the precision and performance required for robust and reliable DNA analysis.

Key features 
 

• Streamlined operation with bare-fused silica (BFS) capillary: Utilizing dynamic coating technology, the DNA 20 kb Plasmid and Linear kit simplifies plasmid and linear dsDNA analysis with a ready-to-use gel and <20 minutes of separation time, ensuring ease and efficiency 
• High-resolution separation of plasmid DNA topologies: This technique distinguishes between different plasmid DNA topology—including supercoiled (SC), SC-multimer, linear and open circular (OC) species—with exceptional clarity 
• Tunable size resolution for wide-range linear dsDNA analysis: The resolution for sizing linear dsDNA is 5%– 10%, showing good repeatability across various capillary lengths

Introduction

Plasmid DNA is a fundamental starting material in biopharmaceutical cell and gene therapy manufacturing processes, including the production of messenger RNA (mRNA) and the generation of viral vectors or gene-of-interest (GOI) plasmids. In addition, plasmid DNA is an indispensable molecular tool for supporting protein production.⁴ Analyzing the presence of various topological forms—such as SC, linear, OC, and others—is critical for the quality control of plasmid DNA. Sizing and purity confirmation of linearized DNA intermediate before in-vitro transcription (IVT) mRNA production is also important in the production process.⁵

Confirmation of the gene mapping of the intended vector via endonuclease digestion at the pertinent restriction sites is recommended by the US Food and Drug Administration (FDA) as an important step during a plasmid identity check.^6–8 A confident comparison of the experimental restriction digestion pattern with the in-silico digestion pattern requires the assay to provide good size measurement and resolution over an extended size range. Both workflows can be achieved using the DNA 20 kb Plasmid and Linear kit on the PA 800 Plus system with good assay repeatability by following the detailed steps in this technical note. Additional information about the kit can be found in the application guide for the DNA 20 kb Plasmid and Linear kit (for the BioPhase 8800 system).⁹ The information in this guide⁹ also applies to using the kit on the PA 800 Plus system, except for instrument setup and operation, which will be detailed here.

Materials

Materials: All materials, unless listed otherwise, were from SCIEX. The DNA 20 kb Plasmid and Linear kit (P/N: 5311708) contains DNA 20 kb Plasmid and Linear gel, DNA 20 kb Plasmid and Linear sample buffer, DNA 20 kb Plasmid test mix, SYBR™ Gold Nucleic Acid gel stain,* DNA 20 kb Plasmid and Linear conditioning solution, acid wash/regenerating solution and CE Grade water. Universal vials (pack of 100, P/N: A62251), universal vial caps (blue, pack of 100, P/N: A62250) and PCR micro vials (200 μL, pack of 100, P/N: 144709) were used to contain reagents and/or samples on the PA 800 Plus system. For high-resolution linear dsDNA separation on a 50-cm capillary, a pre-cut BFS capillary (50 μm ID × 67 cm long, P/N: 338451) and a standard capillary cartridge assembly (P/N: 144738) were used to build the cartridge. For plasmid analysis and linear dsDNA on a 30-cm capillary, a pre-assembled capillary cartridge (P/N: A55625) was used to eliminate the need to cut the capillary and assemble the coolant tubes. Additional information can be found on the SCIEX website.¹⁰ 

Nuclease-free water (P/N: AM9932) and the 1 Kb Plus DNA Ladder (500 ng/μL, P/N: 10787018) were obtained from Thermo Fisher Scientific (Waltham, MA).

Storage of the DNA 20 kb Plasmid and Linear kit: Upon receipt, the acid wash/regenerating solution, CE Grade water and DNA 20 kb Plasmid and Linear sample buffer were stored at room temperature. The DNA 20 kb Plasmid and Linear conditioning solution and the DNA 20 kb Plasmid and Linear gel were refrigerated at 2o C to 8o C. The DNA 20 kb Plasmid test mix and the SYBR™ Gold Nucleic Acid gel stain* were kept at -35°C to -15°C.

Sample preparation 

Sample preparation for the plasmid sample: The DNA 20 kb Plasmid test mix was thawed on ice for about 20 minutes, and 4 µL was mixed with 160 µL of DNA 20 kb Plasmid and Linear sample buffer. Then, 100–150 µL of the prepared sample was transferred into the universal vial with the insert.

For unknown plasmid DNA samples, a final concentration of 1 ng/μL is recommended for most samples. If needed, the optimal sample concentration can be confirmed using a concentration titration curve.² The diluted plasmid sample was then transferred at 100 μL per vial for analysis on the PA 800 Plus system.

Sample preparation for linear dsDNA sample: The 1 Kb Plus DNA Ladder was thawed on ice for about 20 minutes. A total of 2 µL of the linear DNA ladder was mixed with 320 µL of DNA 20 kb Plasmid and Linear sample buffer, and 100–150 µL of the prepared sample was then transferred into the PA 800 Plus sample vial with the insert.

For other linear DNA ladders or digested DNA mixture samples (not analyzed in this technical note), a final concentration of 0.5–1 ng/µL is recommended. For purified linear DNA samples, a concentration of around 0.1 ng/µL is recommended. All linear dsDNA samples should be diluted with DNA 20 kb Plasmid and Linear sample buffer. A total of 100 μL of the diluted sample must be transferred into the universal vial with the insert for analysis on the PA 800 Plus system.

Instrument methods 

General instrument operation details can be found in the methods development guide for controlling the PA 800 Plus system via 32 Karat software.¹¹ If using the PA 800 Plus Empower™ Driver as an instrument controller, refer to the user guide for system operation via Waters Empower™ software.¹² Detailed instrument methods using both 32 Karat software and the PA 800 Plus Empower™ Driver can be found at the end of this technical note in Figures 6–11, with Table 1 serving as reference indexes for these screenshots of instrument settings. With the PA 800 Plus Empower™ Driver, using the Next Inj. Delay feature enabled a 30-minute wait in the conditioning method without triggering an instrument error.

Results and discussion for plasmid analysis

The kit separates plasmids based on their topologies, resolving SC, linear, OC and multiple aggregates of the SC species in a single assay. The instrument methods for plasmid analysis separation can be found in Figures 6, 7 and 8. This allows for reporting on the purity of a given plasmid sample for process control. To demonstrate the repeatability of the kit for plasmid analysis, the plasmid test mix included in the kit was used as a sample. The sample was separated with the plasmid separation method and analyzed using 3 different lots of reagents on a single system with the same capillary. For each reagent lot, 6 injections were performed. The overlay electropherograms of the 18 injections are shown in Figure 1A, and the quantitative analysis is summarized in Table 2. When using the same reagent lot, <1% RSD was achieved. Across 3 reagent lots, <1% RSD in peak migration time and SC% purity were also achieved, providing easy peak identification for automatic data processing. The purity reported for the sample was also consistent when different reagent lots were used.

Results and discussion for linear dsDNA analysis

The same DNA 20 kb Plasmid and Linear kit can be used for size-based linear dsDNA separation. The instrument methods can be found in Figures 9, 10 and 11. The linear dsDNA ladder— containing 18 markers of different sizes ranging from 0.1 kb to 15 kb—was used as the test sample. The resolving power at different size regions (defined by the marker size) with a 50-cm long capillary is shown in Table 3. The resolution was a theoretical extrapolation based on experimental results using the equation shown in Figure 2. The σ (deviation, minutes) values reflect the peak width for the markers, and the value used is the maximum value from 18 replicate injections. The k value used in both ∆t and σ was the slope for the linear fitting between the log (bp, size) and the migration time of the existing marker. The results demonstrate 10% size resolving power with a resolution >1.5 in all size ranges and 5% size resolving power with a resolution >1.5 in the 0.3–7 kb size range.

The assay repeatability for linear dsDNA analysis was also evaluated using 3 different reagent lots with 6 replicate injections from each lot on a single system with the same 50- cm long BFS capillary. The overlay electropherograms are shown in Figure 1B. High-quality linear dsDNA samples generally have a minimum purity of 60%.¹³ In the sample, the % corrected area (%CA) for each marker is <10%, indicating that these values are closer to the number of impurity species in a sample than the main peak. To evaluate the reliability of the assay in reporting linear DNA purity, markers sized 0.1–1 kb are grouped and reported as lower markers (LM) and markers sized 1.5–15 kb are grouped and reported as higher markers (HM). The intra-lot and inter-lot average and %RSD for both groups are calculated and reported in Table 4. Four markers (at the lower and higher marker range) were selected to demonstrate the repeatability of linear DNA on the PA 800 Plus system. The results are shown in Table 5.

Results and discussion for method optimization

When analyzing plasmids, the previously discussed method (20 kV) provided baseline resolution for the 3 most known species (SC, linear, and OC). However, this method shows limitations when applied to more complex samples containing additional species, such as SC-multimer. To address this, a second method with reduced separation voltage (9 kV) was adopted, further improving the resolution for additional species in the sample (Figure 3). Although this second method was effective, the tradeoff was a longer runtime. Therefore, a fit-for-purpose choice between these methods depends on the complexity of the samples (lower voltage to resolve more species) and the time constraints of the project (higher voltage to reduce method cycle time). A comparison between the 2 methods for separating the plasmid test mix is shown in Figure 3, with the potential SC-dimer highlighted with a circle in each electropherogram. Another consideration when choosing the separation voltage for a plasmid sample is the size of the plasmid. When the plasmid is <3 kb, a higher separation voltage is recommended as it reduces the peak broadening due to diffusion (data not shown).

When analyzing linear dsDNA samples, if the impurity species and main peak have significant size differences, using a shorter capillary (30 cm) can be advantageous. A shorter capillary can reduce analysis time, enhancing throughput and efficiency.

Using a 30-cm capillary also allows plasmid analysis in the same testing sequence, providing the potential to estimate the size of the linear species in the sample. This approach can streamline the process, allowing for faster turnaround times. The reduced analysis time directly translates into quicker results, which is crucial in high-speed biopharmaceutical development where time is of the essence. A comparison between using the short (30-cm) and long (50-cm) capillary for linear dsDNA analysis is shown in Figure 4. The run time can be 10 minutes less when using the 30-cm capillary

Running the samples with the PA 800 Plus Empower™ Driver 

As mentioned previously, the PA 800 Plus system can be controlled using both 32 Karat software and the PA 800 Plus Empower™ Driver. The PA 800 Plus Empower™ Driver time programs for all the previously mentioned methods are listed in Table 1. An example electropherogram of the plasmid test mix and the linear DNA ladder is shown in Figure 5.

Conclusions
 

• Easy-to-use kits and easy-to-set-up methods: The DNA 20 kb Plasmid and Linear kit coupled with a BFS capillary provides an easy-to-use and flexible solution for both plasmid and linear dsDNA analysis. With customizable peak resolution and assay throughput, it allows for fit-for-purpose analyses with ease and precision. 
• Consistent results and good repeatability: Both peak migration time and sample purity were shown to be <1% in %RSD for both plasmid and linear dsDNA analysis. 
• Flexibility in software control and data processing: The new kit can be used on the single-capillary PA 800 Plus system with both 32 Karat software and the PA 800 Empower™ Driver as control software. Data can be processed with the BioPhase 8800 system or the PA 800 Empower™ Driver. 

References
 

1. Unlock the full potential of the DNA 20 kb Plasmid and Linear kit for comprehensive plasmid topology and linear DNA sizing analysis. SCIEX technical note, MKT32622-A. 
2. A new method for monitoring plasmid purity: seamless method transfer and consistent results across single and multi-capillary systems. SCIEX technical note, MKT32653-A.
3. Intermediate precision study of DNA analysis with the DNA 20 kb Plasmid and Linear kit on the BioPhase 8800 system. SCIEX technical note, MKT-32655-A. 
4. Prazeres DMF, Monteiro GA. Plasmid Biopharmaceuticals. Microbiol Spectr. 2014;2(6). 
5. Chen, Z. et al. Topological and chemical analysis of plasmid DNA by capillary electrophoresis. J. Chromatogr. A. 2020, 1624:461-472. 
6. Kay MA. State-of-the-art gene-based therapies: the road ahead. Nat. Rev. Genet. 2011 May;12(5):316–328. 
7. Samulski RJ, Muzyczka N. AAV-Mediated Gene Therapy for Research and Therapeutic Purposes. Annu. Rev. Virol. 2014 Nov;1(1):427–451.
8. Goyon A et al. Separation of Plasmid DNA Topological Forms, Messenger RNA, and Lipid Nanoparticle Aggregates Using an Ultrawide Pore Size Exclusion Chromatography Column. Anal Chem. 2023 Oct 10;95(40):15017–15024.
9. DNA 20 kb Plasmid and Linear kit for the BioPhase 8800 system. SCIEX application guide, RUO-IDV-05- 15737-A. 
10. DNA 20 kb Plasmid and Linear kit. SCIEX product page. 
11. PA 800 Plus Pharmaceutical Analysis System. SCIEX methods development guide, RUO-IDV-05-5330-A. 
12. PA 800 Plus Empower™ Driver. SCIEX user guide, RUOIDV-03-9721-A.
13. US Food and Drug Administration, Center for Biologics Evaluation and Research (CBER). Considerations for the Design of Early-Phase Clinical Trials of Cellular and Gene Therapy Products: Guidance for Industry. 2015 June, FDA-2013-D-0576.