Viral Inactivation Process Monitoring by APIES (Absorbance, Polarized Intrinsic Emission, and Light Scattering): Simultaneous Protein Aggregation, Concentration, and Stability Analysis.

The manufacture of monoclonal antibody (mAb) therapeutics relies on stringent downstream processing, where low-pH viral inactivation (VIN) safeguards against enveloped viruses. Here we introduce simultaneous absorbance, polarized intrinsic emission, and scattering (APIES) measurements as a rapid (<1 min), robust analytical platform for real-time assessment of protein aggregation, concentration, and tertiary structural integrity during VIN. Dual excitation wavelengths (280 and 350 nm), multichannel detection, and parallel polarizers provide high sensitivity for aggregation monitoring. APIES provides three orthogonal readouts: protein concentration via A280, aggregation by an aggregation index (AggIndex = (A350/(A280 - A350)) × 100), Rayleigh-to-fluorescence ratios (IR/IF), and large tertiary structure changes (e.g., unfolding) via an intrinsic emission ratio (I350/I330). We evaluated APIES using a bovine IgG model (∼1 mg mL-1) undergoing simulated VIN at pH 3.4, 3.6, and 3.8 for 8 h. Measurements were acquired hourly using APIES, dynamic light scattering (DLS), and absorbance spectroscopy, with offline size exclusion chromatography (SEC) after sample neutralization. Under all pH conditions, DLS and APIES confirmed extensive increases of in-vessel aggregation, with AggIndex and IR/IF strongly correlating with DLS determined aggregate content (R2 > 0.9). However, SEC only showed an increase in irreversible aggregates (of ∼5% after 8 h) at pH 3.4, with no increase for pH 3.6 or 3.8. This highlights a limitation of SEC for in-process monitoring and shows that APIES better monitors in-vessel aggregation. Furthermore, APIES, via AggIndex and IR/IF ratio scatter plots, potentially distinguishes between reversible from irreversible aggregation pathways. Repeating these experiments using a flowcell over 3 h, with 1 min APIES measurement sampling intervals, confirmed that APIES was suitable for online aggregation monitoring. Overall, APIES is a simple, fast, and sensitive process analytical technology for real-time protein aggregation monitoring during industrial processes like VIN.