Purification of plasmid DNA from clarified and non-clarified Escherichia coli lysates by berenil pseudo-affinity chromatography

Abstract

In this study, berenil was tested as a ligand, specifically to purify plasmids of different sizes pVAX1-LacZ (6.05 Kbp) and pCAMBIA-1303 (12.361 Kbp) from clarified Escherichia coli alkaline lysates. For this purpose, chromatographic experiments were performed using Sepharose derivatized with berenil. The results showed that both pDNA molecules are completely purified using lower amounts of salt in the eluent than those previously reported for other pseudo-affinity and hydrophobic interaction chromatography based processes. Total retention of all lysate components was achieved with 1.3M ammonium sulphate in the eluent buffer and pDNA elution was obtained by decreasing the salt concentration to 0.55 M. All impurities were eluted after decreasing the concentration to 0M. The recovery yield for pCAMBIA-1303 (45%) was lower than that obtained for pVAX1-LacZ (85%), however the larger pDNA showed a higher purity level. Purification of pVAX1-LacZ was also performed using non-clarified E. coli process streams, replacing the clarification step with a second chromatographic run on the berenil-Sepharose. Using the same binding and elution conditions as before, a pure plasmid sample was obtained with a 33% yield and with all host impurity levels in accordance with the requirements established by the regulatory agencies. These results suggest that this chromatographic method is a promising alternative to purify pDNA for therapeutic use.

Influenza DNA vaccine purification using pHEMA cryogel support

“…Dynamic binding capacity (DBC) is defined as the amount of target molecules that bind to the matrix under standard flow conditions and should be determined under specific flow and load characteristics [11]. DBC experiments were performed with 0.01, 0.05 and 0.1 mg/mL of NTC7482-41H-VA2 HA plasmid solution, at flow-rate of 1 mL/min.…”

“…Affinity chromatography is the most powerful technology for the purification of sc pDNA both in the analytical and large scale. Moreover, affinity chromatography has several advantages related to the nature of stationary phases and composition of mobile phases in comparison with other types of chromatography [10,11]. The process is also more effective regarding the time and number of steps necessary to purify sc pDNA with success.…”

Plasmid purification by using a new naphthalene tripodal support

“…The naphthalene tripodal ligand was designed to allow multiple non-covalent interactions (hydrophobic, electrostatic and hydrogen bonds) with pDNA, as reported for other similar ligands such as DAPP [10], berenil [11,12], L-tryptophan [9] and L-tyrosine [8]. DAPP is a groove binder, and the interaction with pDNA occurs mainly through p-p stacking and hydrophobic interactions [10].…”

“…Aromatic ligands are promisor molecules in sc pDNA purification due to the unique features derived from the presence of the aromatic ring [7]. Recently, several studies report the use of aromatic amino acids (L-tyrosine [8] and L-tryptophan [9]) and synthetized aromatic compounds (DAPP [10] and berenil [11,12]) for pDNA purification.…”

Chemotherapeutic Drug‐Conjugated Microbeads Demonstrate Preferential Binding to Methylated Plasmid DNA

“…In addition, membrane‐based approaches have also been investigated for purifying DNA . Affinity chromatography approaches exploit selective biomolecular interactions between plasmid DNA molecules and surface‐immobilized affinity or pseudo‐affinity ligands for purification of pDNA from culture broths . Ligands including arginine, histidine, berenil, zinc fingers, LacI‐LacZ moieties, and triple‐helix forming nucleotide sequences have been investigated for purifying plasmid DNA using affinity chromatography .…”