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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.

With the aim of exploring the potential of ultra-fast chiral chromatography for high-throughput analysis, the new sub-2 micron Whelk-O 1 chiral stationary phase (CSP) has been employed in supercritical fluid conditions to screen 129 racemates, mainly of pharmaceutical interest. By using a 5-cm long column (0.46cm internal diameter), a single co-solvent (MeOH) and a 7-min gradient elution, 85% of acidic and neutral analytes considered in this work have been successfully resolved, with resolution (Rs) larger than 2 in more than 65% of cases. Moreover, almost a half of basic samples that, for their own characteristics, are known to be difficult to separate on Whelk-O 1 CSP, have shown Rs greater than 0.3. The screening of the entire library could be accomplished in less than 24h (single run) with 63% of positive score. For well-resolved enantiomers (Rs roughly included between 1 and 3), we show that method transfer from gradient to isocratic conditions is straightforward. In many cases, isocratic ultra-fast separations (with analysis time smaller than 60s) have been achieved by simply employing, as isocratic mobile phase, the eluent composition at which the second enantiomer was eluted in gradient mode. By considering the extension and variety of the library in terms of chemico-physical and structural properties of compounds and numerousness, we believe that this work demonstrates the real potential of the technique for high-throughput enantioselective screening.