Rapid and selective characterization of antibody-drug conjugates in complex sample matrices by native affinity liquid chromatography-mass spectrometry.

Antibody-drug conjugates (ADCs) and other biopharmaceuticals require robust analytical methods to assess biotransformation in biological matrices. Current approaches often require off-line enrichment and extensive chromatographic separation, limiting throughput and complicating data processing. We developed a native affinity liquid chromatography-mass spectrometry (aLC-MS) method using POROS CaptureSelect FcXL columns combined with optimized solvents and MS parameters for direct analysis (1D aLC-MS) of ADCs and other antibody-derived formats in complex sample matrices, such as serum. The method was evaluated using stability studies and concentration series in mouse serum. Direct analysis enabled accurate determination of drug-antibody ratio (DAR), drug-load distribution (DLD) and relative drug abundance across samples without chromatographic peak integration. Stability studies revealed distinct ADC biotransformation profiles in serum versus PBS, including maleimide hydrolysis and disulfide exchange at under-conjugated cysteine sites. The aLC-MS method achieved excellent linearity (R2 = 0.99) over 125-2000 µg/mL in serum and demonstrated sensitivity to 31.25 µg/mL. This rapid, selective aLC-MS method enables high-throughput monitoring of ADC quality attributes in complex matrices with minimal sample preparation, supporting biopharmaceutical product development and bioanalysis applications. The method is exclusively based on MS results, which makes data processing and reporting fast and easy to automate.