Plasmid DNA Purity grades

Researchers in the fields of cell & gene therapy and other advanced therapeutics are well aware of the problems that endotoxins pose. Endotoxins, that are bacterial toxins, are the major source of impurities in nucleic acid and recombinant protein purification processes. In downstream applications, these contaminations can affect optimal cell culture conditions and can cause unexpected and unwanted side effects. Therefore, significant effort is dedicated to the development of endotoxin free workflows in plasmid preparations.

It is noteworthy, that not all cell lines nor applications are affected by endotoxins. Depending on the cell line and the downstream application, different amounts of endotoxins are still adequate. For molecular biology applications, standard plasmid isolation kits with > 50 EU/µg are reported to not cause significant issues. For transfection methods endotoxin levels of 0.1 – 10.0 EU/ µg are typically suitable, and for highly sensitive applications, endotoxin levels of ≤ 0.1 EU/µg, often referred as “endotoxin-free”, are required. Even lower levels of endotoxins are required for pharmaceutical applications. The plasmid purification product that best fits your needs will depend on your downstream applications.

MACHEREY-NAGEL offers various kits for different endotoxin-thresholds.

What are endotoxins?

Endotoxins are main components of the outer-membrane of gram-negative bacteria providing structural integrity (Fig 1A & B). Chemically, most endotoxins are lipopolysaccharides (LPS) that consist of a hydrophobic lipid A tail and a hydrophilic long complex polysaccharide tail (Fig 1 C). The later chain can itself be divided into a core polysaccharide chain and the O-specific polysaccharide side chains (O-Antigen). Although the term endotoxins is commonly used as a synonym for LPS, there are a few endotoxins that are no LPS.

Endotoxins are typically released in small amounts during bacterial growth and division, and in large amounts during cell death, lysis and mechanical damage. The lipid A tail, that anchors the LPS to the bacterial membrane when intact, is responsible for the toxicity of LPS upon release. In contrast to the bacteria they derive from, LPS are heat-stable molecules.

Schematic illustration of the bacterial cell wall structure from gram-negative bacteria.

Schematic illustration of a gram-negative bacterium. The inner compartment represents the cytosol with bacterial genomic DNA (blue) and Plasmid DNA (yellow). The different outer layers represent the different layers of the bacterial cell wall. Blue box: Zoom-in of the gram-negative bacterial cell wall structure. Green box: Schematic illustration of lipopolysaccharides (LPS).

Why is it important to consider endotoxins?

Endotoxins …

1) … trigger immune responses and inflammation → Can result in altered gene expression, cell death and impaired/false experimental results

2)… induce inflammatory reactions of the mammalian immune system → Fever-inducing (pyrogenic)

3) … are toxic to cells grown in tissue culture → Can trigger endotoxic shock in tissue culture

4) … induce cytokines

How to pick the right product?

The choice of the right product depends on your downstream application.