Large-volume methacrylate monolith for plasmid purification. Process engineering approach to synthesis and application.
The extent of exothermicity associated with the construction of large-volume methacrylate monolithic columns has somewhat obstructed the realisation of large-scale rapid biomolecule purification especially for plasmid-based products which have proven to herald future trends in biotechnology. A novel synthesis technique via a heat expulsion mechanism was employed to prepare a 40 mL methacrylate monolith with a homogeneous radial pore structure along its thickness. Radial temperature gradient was recorded to be only 1.8 degrees C. Maximum radial temperature recorded at the centre of the monolith was 62.3 degrees C, which was only 2.3 degrees C higher than the actual polymerisation temperature. Pore characterisation of the monolithic polymer showed unimodal pore size distributions at different radial positions with an identical modal pore size of 400 nm. Chromatographic characterisation of the polymer after functionalisation with amino groups displayed a persistent dynamic binding capacity of 15.5 mg of plasmid DNA/mL. The maximum pressure drop recorded was only 0.12 MPa at a flow rate of 10 mL/min. The polymer demonstrated rapid separation ability by fractionating Escherichia coli DH5alpha-pUC19 clarified lysate in only 3 min after loading. The plasmid sample collected after the fast purification process was tested to be a homogeneous supercoiled plasmid with DNA electrophoresis and restriction analysis.
Danquah
Michael K
MK
BEL (Bio Engineering Laboratory), Department of Chemical Engineering, Monash University, Wellington Road, Melbourne 3800, Australia. michael.danquah@eng.monash.edu.au
Forde
Gareth M
GM
D001709:Biotechnology / Q000379:methods*; D004247:DNA / Q000302:isolation & purification; D004587:Electrophoresis, Agar Gel; D004926:Escherichia coli; D008689:Methacrylates / Q000737:chemistry*; D008855:Microscopy, Electron, Scanning; D010957:Plasmids / Q000302:isolation & purification*; D011108:Polymers / Q000737:chemistry; D016062:Porosity; D011312:Pressure; D013696:Temperature; D013997:Time Factors
D008689:Methacrylates; D011108:Polymers; D004247:DNA