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

Unraveling the mysteries of modern size exclusion chromatography - the way to achieve confident characterization of therapeutic proteins

“…Hydrophobic interactions can become critical on some sub-2 µm columns, particularly with ADCs, which are often highly hydrophobic, due to the high lipophilicity of the drug and linker attached to the mAb [6,10]. As reported elsewhere, two sub-2 µm UHP-SEC columns with a diol bonding were not suitable for the analysis of hydrophobic ADC products when using purely aqueous mobile phase, while the columns packed with 2.0 µm (diol bonding) and 2.7 µm particles (alternative proprietary bonding) allowed a baseline resolution between the HMWS and the main peak of two FDA-and EMAapproved ADCs [18]. Interestingly, the last generation of TSK UP-SW3000 columns packed with 2 µm particles did not require the addition of organic solvent (which is often used to limit or eliminate undesired hydrophobic interactions) in the mobile phase, contrary to the former generation of TSK 3000SW XL columns packed with 5 µm particles, meaning that column chemistry has also been improved by the provider [6,10].…”

“…Lipophilic linker-payload related species were characterized, but the use of 20 % acetonitrile in the mobile phase (to elute the lipophilic payload) may denature non-covalent aggregates. Therefore, an alternative approach was recently proposed to separate the HMWS from the ADC main peak under non-denaturing conditions prior elution of the free payload-related species by applying an acetonitrile gradient ( Figure 11) [18]. This allowed the simultaneous determination of two CQAs, using a standard 1D LC instrumentation.…”

Cutting-edge multi-level analytical and structural characterization of antibody-drug conjugates: present and future

“…Similarly, if charged payloads [49] are conjugated on the antibodyeven through cysteine residues -then the use of IEX would make sense. Probably, its separation quality would be [51].…”

“…As an alternative, a multiple heart-cutting SEC-RPLC-UV method was developed for the simultaneous separation of HMWS from the monomeric ADC and for the separation of free payloads from the free linkers and another type of small molecules impurities [49]. For this purpose, the protein species (i.e., HMWS, monomeric ADC, and LMWS) were eluted with less than 5% acetonitrile.…”

“…By using a suitable switching valve, different fractions can be collected, corresponding to the small molecules elution region of the SEC chromatogram. This is illustrated in [49], where numerous small molecular weight species were chromatographically quantified with UV detection, with also the possible identification by MS detection (only volatile salts were used in the RPLC dimension). In the end, the "multiple heart cutting" method significantly increases the peak capacity compared to previously published single heart cutting SEC-RPLC methods.…”