Analytical tests that can be utilized for the characterization of antibody-oligonucleotide complexes (AOCs) include gel exclusion chromatography, SDS-PAGE, mass spectrometry, and capillary electrophoresis. In a 2025 paper, Zhou et al. [Ref 1] describe the application of capillary zone electrophoresis (CZE) as a label-free method for evaluating AOCs with varying stoichiometries.
CZE separates molecules based on their charge-to-mass ratios. Since oligonucleotides carry a high negative charge, AOCs with varying levels of oligonucleotides should display different charge-to-mass ratios and, in principle, be separable by CZE.
CZE is an electrophoretic technique in which both negative and positive ions migrate toward the cathode, influenced by the electric gradient and the movement of the buffer solution itself in the electric field (electro-osmotic flow). Initial optimization in CZE typically involves running samples at various pH values, as this is the most effective method for controlling analyte migration and enhancing separation efficiency.
Zhou et al. investigated the anti-CD20 therapeutic mAb rituximab (RTX) and conjugated it to a 21-mer oligonucleotide using two covalent conjugation methodologies. The resolution of AOCs was poor at pH 3, and analytes were lost at pH 5.5 on capillary surfaces. Some degree of resolution of AOCs was observed at pH 7.5, though the free oligonucleotide was significantly retarded and absent from the CZE electropherogram. Only at pH 9, using a borax/2-hydroxypropyl-β-cyclodextrin/SDS buffer system, were all components of the AOC mixture successfully resolved and detected.
RTX has an isoelectric point of 9.2, which renders it slightly positive at pH 9. In contrast, oligonucleotides and all RTX AOCs carry a net negative charge. The components in the AOC mixture were detected in the following order: RTX > RTX-AOCs > oligonucleotides, with increasing negative charge density leading to longer migration times. Mass spectrometry was employed to confirm that the resolution of the RTX-oligo(n) conjugates in CZE was based on their charge-to-mass ratios.
Zhou et al. subsequently investigated RTX AOCs prepared using a glycan site-directed approach (azide-DBCO) and a random approach (SMCC-thiol) for conjugation. They found that the click-chemistry-based method exhibited higher efficiency and produced RTC-oligo(n) products with a wider distribution of n values. In contrast, the random method yielded a greater percentage of mono-conjugated products, although it also resulted in a substantial amount of free antibody.
In conclusion, CZE appears to be a promising technique for the analysis of AOCs. However, since the critical parameter is the charge-to-mass ratio, it seems unlikely that a single set of conditions would be suitable for all antibodies owing to the variation in isoelectric points and the influence of oligonucleotide length on negative charge. CZE is more likely to be applied to therapeutic antibodies than to the thousands of AOCs used in research applications.
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