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

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.