This article is sponsored by Phenomenex.

As antibody-drug conjugates (ADCs) continue to emerge as a pivotal approach in targeted cancer therapeutics, the demand for precise and efficient characterisation methods is increasing. Determining the average drug-to-antibody ratio (DAR) is a key quality attribute for ADCs and conventional analysis via Hydrophobic Interaction Chromatography (HIC) are insufficient due to the inability of identifying positional DAR isomers which are critical to drug efficacy and safety consistency. In addition, time-consuming fractionation is typically used to isolate individual DAR species prior to offline MS characterisation to ensure MS friendly buffer exchanges; thus further impacting data reliability and reproducibility.  With more than 100 ADCs currently in different stages of clinical development, efficient and robust characterisation methods are critical to making timely drug development decisions.

To address these challenges, Phenomenex has developed the Biozen™ Native RP-1 and RP-5 columns. These non-porous, hydrophilic reversed-phase chromatography columns are designed to transform ADC characterisation through reversed-phase separation under native conditions, allowing online mass spectrometry (MS) detection. The proprietary RP-1 and RP-5 particle technology and surface chemistry allow rapid and robust native reversed-phase chromatographic resolution of all DAR species, including positional isomers, while facilitating faster and more efficient analyses compared to traditional methods.

The newly designed particle allows for reduction in run times and sample manipulation, ensuring that laboratories can achieve higher quality data for ADCs using these advancements in chromatographic columns and methods. An illustration of the general structure, nomenclature and arrangement of drug conjugation sites of a cysteine linked ADC is shown in Figure 1.

Padcev® is an example of a cysteine linked ADC analysed by Biozen Native RP-5 column.  Using this novel native reversed-phase approach to calculate average DAR resulted in a value of 3.6 as demonstrated in the MS and UV chromatograms below (Figures 2a, b), highlighting excellent agreement with the literature average DAR value of 3.8 for Padcev.

The DAR4 species of Padcev elute as two chromatographically resolved peaks. The identical raw mass spectra for these two chromatographically resolved peaks provide strong evidence that they are different positional isomers and the chromatographic resolution of these species suggests the two pairs of isomers have different hydrophobicities. Further work is underway to further characterize these two isomers.

Similar analyses of two other cysteine linked ADCs (Polivy®, Tivdak®) were performed and XIC chromatograms are shown in Figures 3 and 4. The chromatographic profile is similar for all commercial ADCs tested. All DAR species in all ADCs were positively identified with MS mass errors of less than 50ppm.

As demonstrated above, Biozen™ Native RP-1 and RP-5 columns provides robust chromatographic separation of all DAR species (DAR0-DAR8), including positional isomers of ADCs in their native, intact state. The high-resolution MS spectra demonstrate elution as well as positive identification of all major DAR species with excellent sensitivity (less than 50 ppm mass error).

This and future analytical work are poised to further establish native reversed-phase analysis as a paradigm-shifting approach for meeting the growing demands of ADC characterization—powered by the complementary selectivities of Biozen™ Native RP-1 and RP-5 to support an increasingly diverse range of next-generation physicochemically distinct ADCs.

For more information on Biozen™ Native RP-1 and RP-5 chemistries—or to stay updated on the latest advancements in native-RPLC for ADC characterization—visit Biozen Native RP-1, RP-5, Biopharma Industry Hub or contact your local Phenomenex representative.

Reference:

1. Ma F, et al. Novel Native Reversed-Phase Liquid Chromatography (nRPLC)/MS for Antibody-Drug Conjugates (ADCs) Characterization and Drug-Antibody Ratio (DAR) Assessment. Anal. Chem. 2025;97(14):7756-7764.