Impact of Host Cell DNA and Chromatin on Virus‐like Particle Analysis by Light Scattering in Asymmetrical Flow Field‐flow Fractionation

The development of virus-like particle (VLP) production processes is often constrained by the extensive number of analytical methods required for their quantification and characterization, as well as the significant labor demands associated with these techniques. Asymmetrical flow field-flow fractionation (AF4) coupled with in-line detectors, such as ultraviolet (UV) and multi-angle light scattering (MALS), presents a promising label-free and rapid approach to simultaneously assess the quantity and quality of VLP samples. While AF4-MALS has been widely applied for bionanoparticle characterization and quantification in final products and process development, the influence of host cell-derived impurities on the outcome of the analysis remains underexplored. This study investigates the impact of host cell-derived impurities, particularly host cell DNA and chromatin, on AF4-MALS-DLS analysis of both unpurified and purified VLP samples, using HIV-1 gag VLPs produced in CHO cells as a model system. Our results demonstrate that DNA, chromatin, and VLPs can co-elute due to their overlapping size distribution, which, if overlooked, may lead to imprecise determination of VLP concentrations in early process samples and inaccurate yield calculations at later stages. Nevertheless, for total particle quantification, AF4-MALS was shown to be a suitable surrogate for nanoparticle tracking analysis, as the 90° light scattering peak area exhibited a strong linear correlation with total particle concentration. This substitution enables faster sample processing and reduces sample volume requirements. Additionally, our findings highlight the importance of particle concentration and method parameter selection, particularly the detector flow rate, when characterizing samples based on hydrodynamic radius (R_hyd). Underestimation of R_hyd due to high detector flow rates was proposed as the possible explanation for the higher-than-expected shape factors obtained for VLPs. These results emphasize the need for further optimization of AF4 methods to improve the separation of VLPs from host cell impurities and to ensure reliable characterization of bionanoparticles in complex mixtures.