Application Note

Cell Harvest, Lysis, Neutralization & Clarification Of Plasmid DNA

DNA

Plasmid DNA (pDNA) is typically produced via fermentation using a microbial source. Following E. coli fermentation, the primary downstream purification begins with harvesting of the cells, lysis, and clarification. During cell harvest, cells are concentrated, and the fermentation broth is removed via centrifugation or microfiltration tangential flow filtration (MF-TFF).

For MF-TFF, open-channel, flat-sheet TFF devices such as ProstakTM cassettes with Durapore® 0.1 or 0.2 μm microfiltration membranes or Pellicon® cassettes with Durapore® V screen or Biomax® 1,000 kD V screen ultrafiltration (UF) membranes are recommended.

The harvested E. coli cells are then disrupted to release the plasmid DNA. Lysis is most often performed via an alkaline method. Alkaline lysis with 0.1–0.5 N NaOH with 0.1–0.2% SDS or Triton® X-100 is commonly used. Lysis time and mixing should be optimized.

Precipitation/flocculation is the first step to separate the supercoiled pDNA by selectively precipitating and removing impurities (high molecular weight RNA and genomic DNA, proteins and endotoxins) typically by use of 0.7–3 M potassium acetate with or without CaCl(1.0–1.5%), pH range 5.0–7.5.

Lysate can be clarified using depth filtration, such as Clarisolve® filter or Millistak+® HC and Millistak+® HC Pro filters, to achieve high filtration capacity and yield. These filters are available in a wide range of formats with sizes from 0.014 m2 to 1.1 m2. Preclarification/ pretreatment significantly affects the capacity of the depth filter and process development should therefore be carefully considered for optimization of the step. Yield from the clarification step is generally >90%.

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