Advancing therapeutic monoclonal antibody analysis: A sequential injection lab-on-valve approach

Abstract

Advancements in automated flow-based techniques have significantly improved precision, efficiency, and sustainability in the analytical field. Among these, sequential injection lab-on-valve (SI-LOV) offer a miniaturized and resource-efficient approach for high-throughput analysis. In this work, the feasibility of a fully automated SI-LOV system coupled with a miniaturized spectrometer was investigated for therapeutic monoclonal antibodies (mAbs) assessment. Six mAbs, including Rituximab (RTX), Trastuzumab, Cetuximab, Nivolumab, Panitumumab, and Ramucirumab, were analyzed. Quantification of mAbs, particularly RTX, was achieved with high accuracy, linearity (0.25–1.25 mg mL⁻¹, R > 0.996), and low limit of detection (0.09 mg mL⁻¹). Second-order derivative spectroscopy (245–300 nm) enabled qualitative assessment via well-resolved peaks, mainly related to phenylalanine, tyrosine, and tryptophan present in mAb structures. Mass-based calibration, ensuring consistent quantification regardless of calibration by sample concentration or injection volume, was possible for sample mass > 5 µg. The study of SI-LOV parameters ensured good repeatability (1.8 %), reproducibility (2.3 %), and rapid analysis (ca. 110 s per determination). RTX concentration was estimated for a surrogate cell culture supernatant with good accuracy (> 96 %), demonstrating its potential for application in biopharmaceutical workflows for high throughput mAb analysis. These findings highlight the SI-LOV system's effectiveness as a robust and automated platform for mAb analysis, offering significant potential for routine quality control, high-throughput analysis, and process monitoring in the biopharmaceutical industry.