Bi-layered chromatography matrices for the purification of biological nanoplexes

The preparations of SEC/IEC supports from commercially available, underivatised base matrices by AGE activation-partial bromination technique via two different approaches; (i) viscosity enhanced-reaction diffusion (VE-RD) and (ii) microwave-assisted reaction diffusion, were studied and optimised. Selected supports produced by both approaches were further evaluated by applying to packed bed chromatography system in order to purify target pDNA from neutralized E.coli cleared lysates.For VE-RD approach, viscosity enhancement by sucrose (< 0.032 Pa.s) was found to greatly aid the creation of thin inert outer layer. The optimum condition for SEC/IEC Sepharose CL-6B production observed was 10% single bromination at room temperature in 64% (w/v) aqueous sucrose without sodium acetate addition. The effects of different base matrices, conductivities, linear flow rate, target pDNA sizes and support preparations on chromatography performance were investigated. Microwave irradiation heated up the reaction in a rapid controlled manner compared to conventional heating. SEC/IEC Sepharose CL-6B produced via microwave-assisted reaction diffusion approach at 80oC with 10% partial bromination showed almost complete surface charge elimination with the highest SI value of 57.4. This support showed the high core binding capacity. However, a delayed pDNA breakthrough was also observed. It was noted that the plasmid forms remain unchanged after SEC/IEC column purification