Extending the limits of size exclusion chromatography: Simultaneousseparation of free payloads and related species from antibody drugconjugates and their aggregates

tSize exclusion chromatography (SEC) is commonly performed in isocratic conditions to separate partiallyexcluded molecules from the pores of the stationary phase, based on their difference in hydrodynamicvolume. 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 alsoseparated on the same SEC column by applying sequentially an acetonitrile gradient after the elutionof the largest species. Such an approach allowed a simultaneous i) measurement of the HMWS amountunder native conditions, and ii) quantitation of the free payloads, within one generic SEC run. For thispurpose, a state-of-the-art 150 × 4.6 mm SEC column packed with 2.0 m particles and 250 Å pore size,was selected to achieve fast separations of the species within 10 min. A second dimension (RPLC) was alsodeveloped to further extend the possibility offered by this experimental setup. The SECxRPLC multipleheart cutting mode was operated by using a modern 2D-LC instrument containing twelve 120 L samplingloops. Repeatabilities (0.01% < RSD < 3.68%) and recoveries (between 82% and 107%) were found to besuitable with both approaches (SEC and SECxRPLC), whereas the LOQs remain similar. Finally, the SECmethod was applied for the screening of ADC crude reaction mixtures, whereas the SEC x RPLC methodfacilitated separating some additional impurities. The streamlined methodology will further support thedevelopment and characterization of ADC product