Achieving low-endotoxin to endotoxin-free plasmid DNA from automated Maxi, Mega, and GigaPrep scales

Clean plasmid DNA preparations are fundamental for many downstream applications in life science workflows. Processes such as transient transfection for production of antibodies and AAV production, generation of single-stranded DNA templates for mRNA production and producing plasmids to screen as potential therapeutic leads all require pure plasmid DNA. To ensure consistent and reliable results, contaminants - including endotoxin, RNA, salts, and other process-related impurities - must be effectively removed.

For applications such as transient transfection, efficiency is further enhanced when the plasmid DNA is predominantly in the supercoiled form. However, while workflows for construct design and screening have been optimized, the throughput of manual plasmid preparation has not kept pace with the growing demand.

The Biotage® PhyPrep system addresses this bottleneck by enabling automated plasmid purification at the Maxi, Mega, and GigaPrep scale, producing up to 1 mg, 5 mg, and 10 mg of plasmid DNA, respectively. By following the Biotage® PhyPrep kit protocols outlined in this application note, researchers can produce high-quality plasmids that are low endotoxin, contaminant-free, and with DNA predominantly in the supercoiled form, making them well-suited for advanced applications in therapeutic development and molecular biology.

Introduction

Plasmid DNA is a workhorse reagent in life science research. In the early days of molecular biology, plasmids were primarily used for cloning and sequencing, later serving as tools for protein expression in bacterial systems. Modern day workflows have expanded their applications dramatically: plasmids are essential in mammalian expression systems, production of AAVs, templates for in vitro mRNA transcription, and even as potential therapeutic agents. Despite their central role in life science workflows, technologies for plasmid production and subsequent purification have remained largely stagnant. Modern assays often require large-scale quantities of plasmid DNA (1-10 mg), placing a heavy burden on researchers to prepare large scale volumes of plasmids with time-consuming manual kits. Options for plasmid prep kits are limited, especially given the stringent cleanliness requirements for plasmids. Attempts to automate large-scale preps have struggled:

• Silica-based technology, are unsuitable due to endotoxin contamination issues.
• Magnetic bead systems fail to deliver clean plasmids because of the inability to remove beads from the final elution.
• Both silica-based technology and magnetic beads are constrained by chemistry and capacity limitations, rarely yielding greater than 1mg of pure plasmid DNA and unable to process cell pellet wet weights greater than a few grams.

The Biotage® PhyPrep system overcomes these challenges, offering the most flexible solution for automated plasmid preparations on the market. With the ability to process a diverse cell pellet load from rich media such as TB, 2xYT, and Plasmid +, the system can consistently generate up to 1 mg, 5 mg, and 10 mg of pure plasmid DNA. There are two kits associated with the Biotage® PhyPrep system. The Växel purification kits for plasmid purification are based on ion-exchange chemistry and are optimized to deliver the high yields, low-endotoxin to endotoxin-free levels depending on growth conditions, and pure plasmids free of small molecular weight DNA contaminants required for modern workflows. The concentration columns further process the pure plasmids using high-capacity Silica chemistry to achieve high concentrations, small elution volumes, and eliminate salt in the final elution.

The procedures described in this technical note will enable plasmid production at concentrations up to 1mg/mL, free of endotoxin and contaminants, and predominantly in the supercoiled form.

Equally important, as the purification kit are the growth conditions used to produce the plasmids, ensuring cell pellets retain a high plasmid copy number while minimizing cell death.

Methods

Bacteria culture growth conditions

Transformation – quality plasmids from fresh transformations are preferred:

1. A water bath was set to 42°C degrees
2. SOC media was warmed by placing the bottle into a 37°C incubator
3. Two LB agar, carbenicillin plates (Teknova, L1010) were removed from the refrigerator and placed right side up on the bench
4. The desired plasmid for transformation was diluted to a working concentration of 10 ng/µL
   1. 1000 ng of plasmid was transferred to a microcentrifuge tube
   2. The dilution was created by bringing volume up to 100 µL with sterile, nuclease free water (Growcells, CCPW-020Q)
5. The competent cells and plasmid DNA were mixed
   1. A fresh tube of competent cells (Invitrogen, 18265017) was thawed on ice
      1. Care was taken not to let it thaw too long
   2. 1µL of the 10ng/µL plasmid DNA (Novalife, PVT10754) was transferred to a new microcentrifuge tube
   3. 50µL of competent cells was added to the microfuge tube to form the transformation reaction mixture
   4. The cell and plasmid mixture was incubated on ice for 30 minutes
6. The cells were heat shocked by placing the transformation reaction mixture in a 42°C water bath for 20 seconds
7. The cells were allowed to recover by placing the transformation reaction on ice for 2 minutes
8. 950 µL of prewarmed SOC medium from step 2 was added to the transformation reaction tube
9. The transformation reaction was incubated at 37°C for 1 hour with shaking at 225 rpm (New Brunswick I26)
10. 20 µL and 200 µL of the transformation reaction was transferred to two different LB agar, carbenicillin plates and were spread using sterile technique
11. The plates were placed upside down in a 37°C incubator overnight (16-24 hours)
12. Single colonies were selected and were restreak onto fresh LB agar, carbenicillin plates and were incubated upside down at 37 °C overnight
13. Single colonies were used to inoculate starter cultures
    
    

Restreaking transformations for single colonies:

1. One isolated colony was selected from the transformation plate (must be less than 1 week old) using a sterile inoculation loop.
2. The colony was transformed to a fresh LB agar plate containing carbenicillin. The loop was streaked with the colony across a small section of the plate as shown in the diagram below:

3. Additional lines were streaked that overlap the previous section to dilute the cells and produce well-isolated colonies
4. The plate was placed in an incubator for 20-24 hours. Care was taken not to leave the plate longer than 24 hours.
5. The plate was wrapped in parafilm and stored at 4°C upside down

Starter culture:

1. (Using sterile technique) 10mL of TB (Teknova, T7060) was added to a 50mL Conical Tube
2. 10µL of 100mg/mL carbenicillin (Teknova, C2135) was added to the 50mL Conical Tube
3. Using a sterile inoculation loop, a single colony was picked from a re-streaked transformation plate that is less than 1 week old.
4. The inoculation loop was dipped and swirled in the 50mL conical tube.
5. The cap of the conical tube was loosely secured with a lab tape, so it stayed in position. A loose cap was required to allow air flow so that the culture grows under aerobic conditions.
6. The tube was placed in a snug-fitting rack. If the rack was too wide, paper towels were used to narrow the rack diameter so that the tube stayed fixed in place. This positioning ensured optimal aeration of the culture.
7. The rack containing the culture was placed into the shaking incubator. The cultures were shaken for 8 hours at 350 rpm 38°C.

Overnight culture protocol:

1. Care was taken to maintain sterile technique: A flame was used to handle all materials aseptically.
2. The growth flask was prepared: a brand new, sterile 2.5L, single-use baffled flask (Thomson, 931136-B) was used. 500mL of TB broth was added using the flask’s graduations.
3. Antibiotic was added: 500µL of 100mg/mL carbenicillin was added to the flask
4. The starter culture was inoculated: 500µL of starter culture was added to flask
5. The culture was incubated overnight: the flask was placed in a shaking incubator at 350 rpm, 38°C for 16 hours.
6. The culture was transferred: After 16 hours of incubation, the culture was poured into centrifuge flask – either 250 mL (ThermoFisher Scientific, 001-0303) or 1 L (ThermoFisher Scientific, 010-1491).
7. The cells were centrifuged: The culture was centrifuged for 15 minutes at 6,000 RPM, 4°C using the Sorval Lynx 4000.
8. The supernatant was removed: The spent media was carefully decanted and discarded. The flask was inverted onto a paper towel to drain residual media.
9. The cells were labeled and stored: The flask was marked clearly. The cells were used immediately or stored at -20°C for up to 1 month.

Plasmid purification procedures

Biotage® PhyPrep Växel purification and concentration kits

Plasmid purification on the Biotage® PhyPrep was performed usings the Växel purification column kit and subsequent concentration column kit.

• The Växel purification column kit included ion exchange columns and reagents that delivered endotoxin-free plasmids under the specified conditions.
• The appropriate kit was used depending on cell pellet weight:
  • MaxiPrep Växel purification kit: 2-5g
  • MegaPrep Växel purification kit: 4.5-6g
  • GigaPrep Växel purification kit: 7-15g
• Biotage® PhyPrep method software guided users on where to place reagents and consumables.
• After sequential purification, plasmids were concentrated with the Concentration Column Kit using automated, on-column alcohol precipitation. This yielded plasmids at ≥ 1 mg/mL.

Note: The concentration column does not remove endotoxins. To maintain low endotoxin or endotoxin-free plasmid preparation, handle samples under standard operating conditions with minimal user intervention.

Safety and preparation

1. A lab coat, gloves and mask were worn when processing samples
2. The Biotage® PhyPrep deck was cleared immediately after preps were completed to maintain a clean system
3. The deck was cleaned: 70% ethanol was sprayed on the deck and wiped with a lint-free laboratory wipe
4. The nozzles were cleaned: A lint-free wipe was misted with 70% ethanol used to wipe the nozzles, both outside and inside. Care was taken to not touch or spray onto the drive screw.
5. The wash buffer reservoir was cleaned: 70% ethanol was sprayed into the empty wash buffer reservoir, then wiped dry with a lint-free lab wipe
6. Deep clean if idle or contaminated: If the Biotage® PhyPrep was left idle for a long period of time or exposed to endotoxin contamination from a spill, a commercial endotoxin cleaning reagent was used followed by rinsing with endotoxin free water prior to steps (3-5).
7. 20mL pipette tips were stored in a closed environment.
8. Biotage® PhyPrep tubes were stored in a closed environment
9. All cell lysates were prepared only in designated areas to avoid contaminating the Biotage® PhyPrep system.
10. The Biotage® PhyPrep User Interface was followed to guide sample processing.

Plasmid analysis

Post purification procedures

1. Minimize handling: Handling of pure samples was limited as little as possible
2. Measure recovery: Elution tubes were weighed to record volume recovery
3. Transfer samples: Pure plasmids were transferred into sterile, endotoxin free, pyrogen-free, nuclease-free 50 mL conical tube
4. Storage: Pure plasmids were capped immediately and stored at 4°C if used the same day or stored at-20°C if processed at a later time.
5. UV absorbance check: Residual sample was used in the Biotage® PhyPrep tube to measure UV absorbance data with a NanoDrop OneC (ThermoFisher). Dated was recorded:
   • Concentration
   • A260/A280 ratio
   • A260/A230 ratio

Endotoxin measurement

1. Work environment: All steps were performed in a laminar flow hood
2. Instrument Setup:
   • The Endoscan nexgen-PTS (Charles River) was turned on
   • After 15 minutes the software was launched , the calibration information was added, the test was dragged into the template, and the sample name was entered.
3. Sterile supplies:
4. Fresh 1mL, 0.2mL, and 0.02mL boxes of pipette tips were used
   • A new bottle of endotoxin-free water (Charles River, W130) was used for the dilutions
   • The ejection levers from pipettes were removed, and the pipette liquid ends were sprayed with 70% alcohol and wiped dry.
5. Sample selection: The first aliquot was used from the 50 mL sample storage conical tube for endotoxin testing to minimize contamination
6. Sample preparation:
   • The sample storage conical tube was vortexed for 1 minute
   • Gloved hands were with alcohol, rubbed in, and allowed let air dry or wiped
   • Sterile technique was used to transfer 396µL of endotoxin-free water into a capped, glass test tube (Charles River, T100).
   • 4µL of plasmid elution was added to the tube with endotoxin free water.
   • The tube was vortexed for 1 minute
   • A 1:100 dilution was prepared: 360 µL aliquot of endotoxin-free water was transferred into a new tube, then 40 µL of the first dilution was added.
7. Cartridge loading:
   • The endotoxin test cartridge (Charles River, PTS5505F) was inserted carefully, touching only the sides or ends with the cups. The cups were oriented to remain upright.
   • When the software recognized the cartridge and prompted the user for samples, 25µL of diluted sample was loaded into each of the 4 cups using sterile technique. Bubbles were avoided.
8. Run the test: Start the EndoScan
   • Data Analysis: After the EndoScan ran, the endotoxin units per microgram (EU/mg) was calculated:

                                               EU/mg = EU/mL ÷ ng/mL

Acceptable value: ≤ 0.1 EU/mg

Gel electrophoresis

Plasmid DNA quality was assessed by gel electrophoresis. 100 ng of plasmid DNA was loaded onto 1% agarose TAE gels containing ethidium bromide and electrophoresed at 70 V for 2 hours. Gels were then imaged using a Gel Doc EZ Imager (Bio-Rad).

Results

Plasmids yield from minimal cell pellets

The Växel purification kit is designed to be highly flexible, allowing it to accommodate a wide range of plasmid production conditions. For high-copy plasmids grown in rich media, cell pellets of 2 g, 4 g, 5 g, and 7g (wet weight) typically yield ~ 1 mg, ~5 mg, and ~10 mg of plasmid DNA, respectively, with final concentrations exceeding 1 mg/mL (Figures 1-3).

Figure 1: Yield and concentration from PhyPrep MaxiPrep plasmid purification. Samples were processed as described. Three replicates of 2g cell pellet wet weight were processed using the MaxiPrep kit and 0.6mg or 1.2mg Concentration Column. Yields are reported (A, B) along with the concentration of the final sample (C,D).

Figure 2: Yield and concentration from PhyPrep MegaPrep plasmid purification. Samples were processed as described. Four replicates of 4.5g cell pellet wet weight were processed using the MegaPrep kit and 3.0mg Concentration Column or 5.5mg concentration column. Yields are reported (A, B) along with the concentration of the final sample (C,D).

Figure 3: Yield and concentration from PhyPrep GigaPrep plasmid purification. Samples were processed as described. Four replicates of 7g cell pellet wet weight were processed using the GigaPrep kit and 9.5mg Concentration Column or 12.0mg Concentration Column. Yields are reported (A, B) along with the concentration of the final sample (C,D).

The choice of concentration column capacity further supports this flexibility:

• Lower-capacity concentration columns (MaxiPrep) achieve higher concentrations, up to 3mg/mL.
• Higher-capacity concentration columns (MegaPrep, GigaPrep) provide greater total yields, while still maintaining concentrations above 1mg/mL. 2g of cell pellet wet weight, the higher capacity Concentration Column yields well above 1mg of pure plasmid DNA.

This choice – higher concentration from lower capacity columns and higher yield from higher-capacity columns – allows researchers to select the best option for their downstream needs

Importantly, each Concentration Column shows a linear relationship between the plasmid mass input and the resulting final concentration (Figure 4). This standard curve enables prediction of the final plasmid concentration based on the input of an unknown plasmid production system. A range of cell pellet samples were processed and the resulting data, delineated as colored markers, fits closely with the standard curve (Figure 4).

Figure 4: Predictable concentration from the full range of plasmid input. Plasmids were spiked into mock samples in duplicate and processed using the six different available Concentration Columns to generate the grey curves. Samples were processed from cell pellets and the results were overlayed on the standard curves using colored markers.

Endotoxin-free plasmid preparations

The concentration column does not incorporate chemistry to remove excess endotoxin, instead relying upon the Växel purification to remove endotoxin. As such preserving the endotoxin-free purification achieved by the Växel kit is required. All plasmid preparations meet the definition of endotoxin-free (<0.1 EU/µg) (Table 1).

Reliability of the endotoxin measurements is confirmed by spike recovery values between 50-200%. meeting the standard criteria. The final purity of the plasmids is further validated by absorbance ratios (Table 1):

• A230/A260 = 1.80-1.95 and
• A260/A280 ratios >2.0

Plasmid DNA retains supercoiled form

Plasmid DNA purified with the Biotage® PhyPrep system and manual Qiagen Endofree kits were analyzed for percent supercoiling. Across all preparation scales - MaxiPrep, MegaPrep, and GigaPrep - the predominant band corresponded to the supercoiled isoform, demonstrating performance comparable to Qiagen preparations (Figure 5).

Figure 5: DNA Gel analysis for retention of supercoiled plasmid. Electrophoresis analysis of plasmid integrity shown on a 1% EtBr agarose gel of three Biotage Maxi, Mega and Giga scale PhyPrep runs. All samples were normalized to 100ng of nucleotide per well from individual purification runs along with a leading competitor as a control in lane 12. The main product is the supercoiled (SC) plasmid with the alternative plasmid diver form.

Supercoiled plasmid DNA is the most efficient form of DNA for successful transient transfections. The high ratio of supercoiled DNA, combined with endotoxin-free, high concentration, and optimal absorbance ratios provided optimal conditions for efficient transient transfection and expression (Figure 6).

Figure 6: Mammalian GFP Expression. Purified plasmids of GFP under the control of the CM promoter were used for transient transfection of expi 293 cells. 1-3: Qiagen Maxi. 4-6: PhyPrep 1.2mg Concentration column. 7-9 Biotage® PhyPrep 0.6mg concentration column. 10-12: Qiagen Mega. 13-15: Biotage® PhyPrep 5.5mg concentration column. 16-18 Biotage® PhyPrep 3.0mg concentration column. 19-21: Qiagen Giga. 22-24: Biotage®PhyPrep 12mg Concentration column. 25-27 Biotage® PhyPrep 9.5mg concentration column.

Summary

The fully automated Biotage® PhyPrep system, together with Växel purification kits and concentration column kits, integrates seamlessly into transient transfection workflows for producing antibodies, AAVs, template DNA, and therapeutic plasmid drug leads. Key advantages include:

• High yields and concentration.
• High purity- optimal absorbance ratios
• Very low-endotoxin to endotoxin-free levels
• High levels of supercoiled DNA

These features directly support high-efficiency transient transfection and expression.

Literature number: AN1023

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