Alternatives for the intermediate recovery of plasmid DNA: performance, economic viability and environmental impact.
Robust cGMP manufacturing is required to produce high-quality plasmid DNA (pDNA). Three established techniques, isopropanol and ammonium sulfate (AS) precipitation (PP), tangential flow filtration (TFF) and aqueous two-phase systems (ATPS) with PEG600/AS, were tested as alternatives to recover pDNA from alkaline lysates. Yield and purity data were used to evaluate the economic and environmental impact of each option. Although pDNA yields > or = 90% were always obtained, ATPS delivered the highest HPLC purity (59%), followed by PP (48%) and TFF (18%). However, the ability of ATPS to concentrate pDNA was very poor when compared with PP or TFF. Processes were also implemented by coupling TFF with ATPS or AS-PP. Process simulations indicate that all options require large amounts of water (100-200 tons/kg pDNA) and that the ATPS process uses large amounts of mass separating agents (65 tons/kg pDNA). Estimates indicate that operating costs of the ATPS process are 2.5-fold larger when compared with the PP and TFF processes. The most significant contributions to the costs in the PP, TFF and ATPS processes came from operators (59%), consumables (75%) and raw materials (84%), respectively. The ATPS process presented the highest environmental impact, whereas the impact of the TFF process was negligible.
Freitas
Sindelia
S
IBB-Institute for Biotechnology and Bioengineering, Cente for Biological and Chemical Engineering, Instituto Superior Técnico, Lisbon, Portugal.
Canário
Sónia
S
Santos
José A L
JA
Prazeres
Duarte M F
DM
D005591:Chemical Fractionation / Q000379:methods*; D004777:Environment; D004787:Environmental Pollution / Q000191:economics* / Q000517:prevention & control*; D010957:Plasmids / Q000191:economics* / Q000302:isolation & purification*; D011174:Portugal; D014462:Ultrafiltration / Q000191:economics* / Q000379:methods*