Rapid Quantification of Polysorbates in Complex In-process Formulation Buffers Using Variable Pathlength UV Spectrometry with a Slope-Intercept Strategy

We developed a rapid method using variable pathlength UV spectrometry combined with a slope-intercept strategy to quantify polysorbates in complex in-process formulation buffers for biologics manufacturing. By leveraging Beer-Lambert law and pathlength-dependent absorbance measurements, the method decouples polysorbate signals from excipient interference, enabling accurate determination of PS concentrations even in matrices with overlapping UV spectra. Optimal wavelengths were identified (240 nm for PS-20 and 235 nm for PS-80) to balance sensitivity and interference. Calibration curves demonstrated good linearity (R² > 0.99) across spiking and flushing buffer concentrations, and the method achieved near real-time quantification with accuracy within ±10% and faster turnaround time than HPLC method, reducing analysis time from days to minutes. By systematically evaluating excipient interference, optimizing wavelength selection, and separating analyte and background contributions through intercept analysis, this study extends variable pathlength UV spectrometry beyond simple systems and establishes its applicability for near real-time process control in biologics manufacturing.