Optimization of isopropanol and ammonium sulfate precipitation steps in the purification of plasmid DNA.
Large-scale processes used to manufacture grams of plasmid DNA (pDNA) should be cGMP compliant, economically feasible, and environmentally friendly. Alcohol and salt precipitation techniques are frequently used in plasmid DNA (pDNA) downstream processing, as concentration and prepurification steps, respectively. This work describes a study of a standard 2-propanol (IsopOH; 0.7 v/v) and ammonium sulfate (AS; 2.5 M) precipitation. When inserted in a full process, this tandem precipitation scheme represents a high economic and environmental impact due to the large amounts of the two precipitant agents and their environmental relevance. Thus, major goals of the study were the minimization of precipitants and the selection of the best operating conditions for high pDNA recovery and purity. The pDNA concentration in the starting Escherichia coli alkaline lysate strongly affected the efficiency of IsopOH precipitation as a concentration step. The results showed that although an IsopOH concentration of at least 0.6 (v/v) was required to maximize recovery when using lysates with less than 80 microg pDNA/mL, concentrations as low as 0.4 v/v could be used with more concentrated lysates (170 microg pDNA/mL). Following resuspension of pDNA pellets generated by 0.6 v/v IsopOH, precipitation at 4 degrees C with 2.4 M AS consistently resulted in recoveries higher than 80% and in removal of more than 90% of the impurities (essentially RNA). An experimental design further indicated that AS concentrations could be reduced down to 2.0 M, resulting in an acceptable purity (21-23%) without compromising recovery (84-86%). Plasmid recovery and purity after the sequential IsopOH/AS precipitation could be further improved by increasing the concentration factor (CF) upon IsopOH precipitation from 2 up to 25. Under these conditions, IsopOH and AS concentrations of 0.60 v/v and 1.6 M resulted in high recovery (approximately 100%) and purity (32%). In conclusion, it is possible to reduce substantially the mass of precipitation agents used without affecting recovery, if a small concession is made regarding purity. This directly translates into an improvement of the process economics and in a reduction of the environmental impact of the process.
Freitas
S S
SS
Centro de Engenharia Biológica e Química, Instituto Superior Técnico, Lisbon, Portugal.
Santos
J A L
JA
Prazeres
D M F
DM
D019840:2-Propanol / Q000737:chemistry*; D000645:Ammonium Sulfate / Q000737:chemistry*; D011232:Chemical Precipitation; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000302:isolation & purification*; D005590:Fractional Precipitation; D057927:Hydrophobic and Hydrophilic Interactions; D010957:Plasmids / Q000737:chemistry / Q000302:isolation & purification*
D004247:DNA; D019840:2-Propanol; D000645:Ammonium Sulfate