Are there any key media ingredients that boost productivity?
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Plasmid DNA Production for Cell and Gene Therapy
Job Title: Associate Director, EMEA Segment Development for Cell and Gene Therapy
Several parameters can affect the bacterial productivity like the master cell bank selection, the growth rate, the culture medium, the feeding rate as well as the appropriate growth conditions and parameters like pH, osmolarity, optical density (DO) and temperature.
The general media composition consists of a carbon source, a nitrogen source, magnesium sulfate, di- and monopotassium phosphate, and minerals. Both minimal and semi-defined media can be used. With minimal media, highly reproducible batches can be obtained. On the other hand, semi-defined media can support higher cell densities as the complex components (e.g. yeast extract) supply growth factors, amino acids, purines and pyrimidines.
As carbon source, glucose is conventionally used as it is efficiently metabolized and inexpensive. However, glucose may cause over production of acetate (Crabtree effect). This can be avoided by (partially) substituting glucose for glycerol. Glycerol can also aid in supporting reduced maximum specific growth rates.
Nitrogen is typically supplied by adding complex components such as yeast extract, casamino acids, or peptones.
Magnesium sulfate and potassium phosphates are added in order to provide a magnesium, sulfur, potassium and a phosphorous source (the phosphates also function as buffering agent). Other trace minerals are present in the complex components of the medium or can be added via specific trace mineral solutions.
The plasmid yield is largely affected by the C:N ratio. It is advised to test different ratios e.g. from 2:1 up to 8:1. The optimal ratio is different for each media type.