UV-derived scattering as a high-throughput readout for adeno-associated virus-like particle separation screenings

Virus-like particles (VLPs), including those derived from adeno-associated virus (AAV), are increasingly investigated for vaccination, capsid engineering, and gene-delivery applications. Although precipitation and aqueous two-phase separation (ATPS) represent promising alternatives to adsorption-based capture, their optimization is limited by the lack of rapid analytical methods suitable for complex process samples. Here, VP3-only AAV5 VLPs produced in Escherichia coli were used as a model system to evaluate UV-derived light scattering as a high-throughput separation readout. Blank-corrected absorbance at 320 nm was used as a relative descriptor of particulate-associated scattering, while scattering-corrected absorbance at 280 nm monitored the soluble biomolecular fraction. During PEG precipitation, A320 showed condition-dependent agreement with anti-AAV5 ELISA, with the closest correspondence for PEG4000 and increasing divergence at lower PEG molecular weights. Capsid-spiking experiments further showed that dispersed intact capsids alone contributed only marginally to A320, indicating that the signal primarily reflects larger scattering-active structures accompanying capsid depletion. ... mehrThe workflow enabled screening and analysis of 170 precipitation and PEG/phosphate ATPS conditions in biological duplicate within 3 days. Low-molecular-weight PEG systems provided stronger separation between soluble biomolecular material and particulate-associated scattering. In PEG400 ATPS, pH altered the distribution of scattering-active material and capsids between the liquid phases and interphase, whereas selected precipitation conditions showed higher recovery of assembled capsids after resolubilization than ATPS, at the expense of lower impurity removal. Overall, UV-derived scattering provides a rapid, scalable first-line readout for comparing separation conditions and reducing the number of samples requiring capsid-specific and structural characterization.