Enhancing plasmid DNA (pDNA) production: Innovations in process, manufacturing, and purification techniques

Materials and Methods

Transformation and cultivation

The pCMV3-GFP plasmid (6883 bp) containing an ampicillin resistance gene (Sino Biological) was transformed into NEB 5-alpha competent E. coli cells (New England Biolabs) according to the manufacturer’s protocol. The transformation mix was plated on a selection plate (LB agar with 100 µg/mL ampicillin) and incubated overnight at 37°C. A single colony was chosen from the selection plate and an overnight culture was prepared in a 500 mL unbaffled glass Erlenmeyer flasks containing 50 mL of LB medium (tryptone 10 g/L, Yeast extract 5 g/L, NaCl 10 g/L, pH 7.0 ±0.2) with 100 µg/mL ampicillin. The flask was sealed with aluminum foil and was incubated at 37°C and 180 min^–1 in an Multitron incubator shaker with Sticky Stuff adhesive mats overnight (at least 18 hours). For the main culture, triplicates of 250 mL unbaffled glass Erlenmeyer flasks containing 25 mL of LB with 100 µg/mL ampicillin were prepared, which is considered a standard cultivation design.

Triplicates of 250 mL Ultra Yield® flasks containing 100 mL of Plasmid+® medium with 100 µg/mL ampicillin and 0.02% of Antifoam 204 (Sigma-Aldrich) were prepared. Both the standard cultivation system and Thomson cultivation system were inoculated from the overnight culture, with a starting OD_600nm of 0.2. The Ultra Yield® flasks were sealed with the AirOtop® Enhanced Seal and the Erlenmeyer flasks with aluminum foil. Flasks were incubated at 37°C at 350 min^–1 or 180 min^–1 in the INFORS HT Multitron Incubator Shaker (25 mm shaking throw) with flask clamps. Note that Sticky Stuff adhesive mats are not suitable for 350 min^–1 and the use of clamps is mandatory. Cultures were grown for at least 24 hours and samples were taken at regular intervals to measure OD_600nm over time.

DNA purification

The PureYield™ Plasmid Miniprep System (Promega) was used for plasmid purification. LB cell suspension was used undiluted, Thomson cell suspensions were diluted 9-fold to approximately match the OD value of LB samples. 600 µL was sampled of each cell suspension which was then centrifuged, and the pellet was resuspended in 600 µL nuclease free water. All other subsequent steps were carried out in accordance with the PureYield™ Plasmid Miniprep System quick protocol FB093 for kit A1223 or A1222. The DNA purification was performed by the centrifugation method. The plasmids were eluted with nuclease free water provided with the kit. The DNA concentration and purity of the eluted samples were measured at 230, 260 and 280 nm with a NanoDrop™ 2000/2000c Spectrophotometers (Thermo Fisher Scientific).

Plasmid analysis using HPLC

For plasmid analysis an HPLC 1100 Series (Agilent) with DAD detector 1100 Series (Agilent, G1315B) at 260 nm was used. Samples were measured on a BioPro IEX QF, 100 x 4.6 mm, 5 µm particle size column (YMC, QF00S05-1046WP). 5 µL of each sample was injected and measured in triplicates. The column temperature was set at 35°C. Mobile phase A (20 mM Tris-HCl, pH 7.4) and mobile phase B (20 mM Tris-HCl, 1 M NaCl, pH 7.4) were used to elute the sample in a gradient method with a constant flow of 0.5 mL/min. The gradient was performed from minute 0 until minute 0.7 with mobile phase A (MPA) at 25% and mobile phase B (MPB) at 75%. Starting at minute 0.7 the gradient was applied and the composition from 75% MPB was changed to 100% during 20 minute until minute 20.7 and was kept at 100% until minute 24. At minute 24.01 the composition was changed to 25% MPA and 75% MPB until minute 30 (end of the injection).