Drug-to-Antibody Ratio Estimation via Proteoform Peak Integration in the Analysis of Antibody–Oligonucleotide Conjugates with Orbitrap Fourier Transform Mass Spectrometry

• Published in: Analytical chemistry (Washington) 2021-09, Vol.93 (38), p.12930-12937
• Main Authors: Nagornov, Konstantin O, Gasilova, Natalia, Kozhinov, Anton N, Virta, Pasi, Holm, Patrik, Menin, Laure, Nesatyy, Victor J, Tsybin, Yury O
• Format: Article
• Language: English
• Subjects: Analytical chemistry; Antibodies; Chemistry; Complexity; Conjugates; Data acquisition; Deglycosylation; Fourier analysis; Fourier transforms; Integration; Mass spectra; Mass spectrometry; Mass spectroscopy; Oligonucleotides; Pharmacokinetics; Quality management; Scientific imaging; Signal processing; Size exclusion chromatography; Spectroscopy; Streamlining; Trastuzumab
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/acs.analchem.1c02247

The therapeutic efficacy and pharmacokinetics of antibody–drug conjugates (ADCs) in general, and antibody–oligonucleotide conjugates (AOCs) in particular, depend on the drug-to-antibody ratio (DAR) distribution and average value. The DAR is considered a critical quality attribute, and information pertaining to it needs to be gathered during ADC/AOC development, production, and storage. However, because of the high structural complexity of ADC/AOC samples, particularly in the initial drug-development stages, the application of the current state-of-the-art mass spectrometric approaches can be limited for DAR analysis. Here, we demonstrate a novel approach for the analysis of complex ADC/AOC samples, following native size-exclusion chromatography Orbitrap Fourier transform mass spectrometry (FTMS). The approach is based on the integration of the proteoform-level mass spectral peaks in order to provide an estimate of the DAR distribution and its average value with less than 10% error. The peak integration is performed via a truncation of the Orbitrap’s unreduced time-domain ion signals (transients) before mass spectra generation via FT processing. Transient recording and processing are undertaken using an external data acquisition system, FTMS Booster X2, coupled to a Q Exactive HF Orbitrap FTMS instrument. This approach has been applied to the analysis of whole and subunit-level trastuzumab conjugates with oligonucleotides. The obtained results indicate that ADC/AOC sample purification or simplification procedures, for example, deglycosylation, could be omitted or minimized prior to the DAR analysis, streamlining the drug-development process.