Integration of Bacteria Capture via Filtration and in Situ Lysis for Recovery of Plasmid DNA under Industry-Compatible Conditions

Abstract

Combining capture and lysis of the bacteria with partial purification of the plasmid DNA is beneficial for the design of efficient plasmid production processes at larger scale. Such an approach is possible when the bacteria are captured by filtration. Taking industrial requirements into account, however, such a capture requires complex filtration mixtures containing retentive additives such as bentonite and polycations. This makes the straightforward transfer of established lysis protocols to in situ lysis difficult. In this contribution, the different steps of such a protocol are designed for complex filter cakes, including fragilization (by lysozyme), lysis (alkaline pH/acidic pH, 70/37 degrees C, urea/NaCl/Triton), and specific elution (pH, NaCl, CaCl2, guanidinium hydrochloride). Results are compared in regard to plasmid quality (topoisomeric form) and quantity (compared to the yield obtained by a commercial miniprep of a small aliquot of the bacteria suspension from the bioreactor). Best results in these terms were obtained by the Triton lysis protocol performed at 37 degrees C (30 min of contact with a lysis buffer composed of 50 mM Tris pH 8, 1% Triton, 1 g/L lysozyme, and 6 M guanidinium hydrochloride) followed by the specific elution of the plasmid DNA in 50 mM Tris buffer pH 8.

Concentration-dependent like-charge pairing of guanidinium ions and effect of guanidinium chloride on the structure and dynamics of water from all-atom molecular dynamics simulation

“…Gdmcl is also used in nucleic acid purification [15] and microfluidic nucleic acid probe assays [16]. Despite its ubiquitous use, the molecular mechanism of guanidinium ion induced protein and nucleic acid denaturation in aqueous solutions remains unsolved and quite elusive.…”

Biomedical application of plasmid DNA in gene therapy: A new challenge for chromatography

“…constant, linear, stepwise, or exponential feeding). Recently a growth-controlled fed-batch process was proposed for plasmid production and yielded up to 250 mg/L of pDNA (O'Mahony et al 2007).…”

A continuous cell alkaline lysis, neutralization, and clarification combination process for production of plasmid pUDK‐HGF

“…Chemical [13][14][15][16], thermal [17][18][19][20], and mechanical [21] methods can be used; the most common method is alkaline lysis based on the work of Birnboim and Doly [22] using 0.2 N NaOH and 1% sodium dodecyl sulfate (SDS) as lysis reagents. In this lysis process, local extreme pH and shear force decreased the plasmid purity and yield.…”