Extending the limits of size exclusion chromatography: Simultaneous separation of free payloads and related species from antibody drug conjugates and their aggregates

Size exclusion chromatography (SEC) is commonly performed in isocratic conditions to separate partially excluded molecules from the pores of the stationary phase, based on their difference in hydrodynamic volume. In this work, a baseline resolution was obtained between the monomeric antibody drug conjugate (ADC) and high molecular weight species (HMWS). Besides HMWS, small free payloads, linkers and linker-payloads of ADCs, which would not be discriminated solely based on their size (MW < 1.5 kDa), were also separated on the same SEC column by applying sequentially an acetonitrile gradient after the elution of the largest species. Such an approach allowed a simultaneous i) measurement of the HMWS amount under native conditions, and ii) quantitation of the free payloads, within one generic SEC run. For this purpose, a state-of-the-art 150 × 4.6 mm SEC column packed with 2.0 μm particles and 250 Å pore size, was selected to achieve fast separations of the species within 10 min. A second dimension (RPLC) was also developed to further extend the possibility offered by this experimental setup. The SECxRPLC multiple heart cutting mode was operated by using a modern 2D-LC instrument containing twelve 120 μL sampling loops. Repeatabilities (0.01% < RSD < 3.68%) and recoveries (between 82% and 107%) were found to be suitable with both approaches (SEC and SECxRPLC), whereas the LOQs remain similar. Finally, the SEC method was applied for the screening of ADC crude reaction mixtures, whereas the SEC x RPLC method facilitated separating some additional impurities. The streamlined methodology will further support the development and characterization of ADC products.