After optimization of the crossflow, channel height, and laser power, the AF4-MALLS785nm-DLS-RI setup was able to accurately characterize the PS particle standards. For PS-15 and PS-50 an average Rg,avg of 13 and 43 nm, and an average Rh,avg of 15 and 49 nm was obtained, respectively (Figure and Table ). Moreover, correct shape factors of  = 0.79 for PS-15 and 0.85 for PS-50 were obtained (Table ). In the next step, LNPs were subjected to the AF4-MALLS785nm-DLS-RI system. By using MALLS detection an accurate determination of the LNPs particle size (Rg), i.e., an Rg,avg= 85, 115, 84, and 107 nm for LNP-1, LNP-2, LNP-3, and LNP-4 was achieved, respectively (Table and Figure ). Although the average particle sizes differ to some degree (Table ), the particle size distributions overlap quite well when comparing offline DLS with the online MALLS approach (Figure ). The obtained average particle size was comparably larger in offline DLS for LNPs that contained aggregates (i.e., LNP-2, LNP-3 and LNP-4), which highlights their impact on the measurements. In DLS, the intensity-based determination of particle size emphasizes large particles, thus the presence of large particles or aggregates could skew the average towards higher values in offline measurements. Here, the particles are separated according to size before being analyzed by light scattering. This provides particle fractions of uniform size to be analyzed and thus more accurate values. Moreover, AF4-MALLS785nm-RI is able to separate samples with broad distributions and aggregates, i.e., LNP-4 (Figure ).