Critical evaluation of fast size exclusion chromatographic separations of protein aggregates, applying sub-2 μm...

A new size exclusion chromatography column packed with 1.7 μm particles and possessing 200 Å pore size has been critically evaluated for the determination of proteins and monoclonal antibody aggregates. In a first instance, the kinetic performance of this column was compared with that of a conventional column packed with 5 μm particles and with a recently launched 3 μm material, also possessing 200 Å pore size. In average, 2-5 times lower plate height were achieved on the 1.7 μm packing, compared with the conventional 5 μm particles. It was also demonstrated that elevated mobile phase temperature (up to 50 or 60 °C) allows improving the kinetic efficiency by 20-40% in size exclusion chromatography, compared to 30 °C. On the other hand, the new 3 μm material performed only slightly lower kinetic efficiency than the 1.7 μm one. When considering the upper pressure and temperature limits of these three columns, the 1.7 μm column systematically outperforms the 5 and 3 μm materials in the "practical" plate number range (N<30,000) and analysis times could be cut by 2-4 times. The column packed with 5 μm particles was only beneficial for plate counts beyond 100,000 plates, while the 3 μm packing could be considered as a good compromise between speed, efficiency and pressure. Besides the excellent kinetic performance of 1.7 μm size exclusion material under high temperature conditions, some artifacts were observed when quantifying protein aggregates. Indeed, both high pressure observed with 1.7 μm particles (shear forces, frictional heating) and elevated temperature produce some non negligible amount of on-column additional protein aggregates.