Work

Direct infusion intact mass spectrometry for rapid and sensitive characterization of antibody therapeutics in complex matrices

Year: 2026

Type: article

Abstract: Native intact mass spectrometry (MS) is essential for resolving the structural heterogeneity of complex biotherapeutics. Traditionally, intact MS combines online chromatography (e.g. ion-exchange chromatography or size-exclusion chromatography). Here, we report an MS-centric, column-free direct infusion MS workflow that combines high-throughput off-line 96-well affinity purification with rapid and sensitive direct infusion MS. This approach achieves a sampling rate of ~1 min/sample with minimal sample consumption (<1 µL) and high sensitivity (detection of antibody drugs in the low µg/mL range). The method demonstrates excellent linearity (R2 ≥ 0.99) in the 1.953–500 µg/mL range, enabling relative quantitation and relative quality ranking across samples based on the deconvoluted MS signal. The utility of this direct infusion MS workflow is demonstrated through two case studies: 1) analysis of antibody-drug conjugate (ADC) stability and biotransformation in mouse serum, where direct infusion MS successfully tracked changes in drug-load distribution and average drug-to-antibody ratio; and 2) sensitive, high-throughput MS screening during early-stage bispecific antibody clone development. The workflow allows early detection of high-quality bsAb clones, providing results that show potential as an early indicator of drug quality in expanded fed-batch cultures. The serum stability study illustrates how this direct infusion MS workflow – despite being column free – can reveal critical product changes in highly complex sample matrices using a robust, rapid, and sensitive workflow. The established workflow uses commercially available instrumentation, reagents, and software, enabling easy implementation in biopharmaceutical analytical development and quality control laboratories.

Source: mAbs

DOI: 10.1080/19420862.2026.2761105