FabRICATOR® in a DAR Analysis of Antibody-Oligonucleotide Conjugates

In this paper, scientists at Spectroswiss and Orion Pharma present a novel strategy for DAR analysis of structurally complex antibody-oligonucleotide conjugates using SmartEnzymes™ and a proteoform integration approach combined with native SEC-Orbitrap FTMS.
 

Antibody-oligonucleotide conjugates (AOCs) combine the specificity of monoclonal antibodies with the advantages of adding oligonucleotide sequences to the biomolecule for applications in both imaging and ADC development. The drug-to-antibody ratio (DAR) of antibody-drug conjugates impacts the therapeutic efficacy and pharmacokinetics, and as a critical quality attribute, distribution and average value of the DAR needs to be monitored carefully. However, the structural complexity of AOCs samples can pose challenging for analysis even with current state-of-the-art mass spectrometric approaches.
 
In this work, native SEC-Orbitrap Fourier transform mass spectrometry (FTMS) was used in an approach to integrate proteoform-level mass spectra peaks to estimate both the distribution and average value of the DAR. The method was benchmarked using conjugates obtained by random labeling of trastuzumab with cross-linkers and oligonucleotides, resulting in a heterogenous pool of AOCs with varying DAR.
 
The conjugates were subjected to analysis at both intact and subunit level, the latter assisted by FabRICATOR (IdeS) digestion below the hinge to obtain homogenous F(ab’)2 and Fc/2 subunits. Modifying the AOC sample by enzymatically removing the 50 kDa Fc subunit, carrying most glycosylations, significantly reduced the sample complexity. The results of the experiments presented in this work demonstrate that reduced mass spectral resolution may lead to a straightforward and rapid DAR analysis with minimal sample preparation for streamlining of the drug-development process.
 

Reference

Nagornov et al., 2021. Drug-to-Antibody Ratio Estimation via Proteoform Peak Integration in the Analysis of Antibody–Oligonucleotide Conjugates with Orbitrap Fourier Transform Mass Spectrometry. Analytical Chemistry. doi: 10.1021/acs.analchem.1c02247

FabRICATOR – Below hinge digestion of IgG