## 2 Materials and Methods
### HF5-Multidetection  
HF5 analyses were performed using an Agilent 1200 HPLC system (Agilent Technologies, Santa Clara, CA, USA) consisting of a degasser, an isocratic pump, with an Agilent 1100 DAD UV/Vis spectrophotometer combined with an Eclipse(r) DUALTEC separation system (Wyatt Technology Europe, Dernbach, Germany). The HF5 channels used for the experimental section were standard cartridges containing a 17 cm long hollow fiber, commercially available. The fiber was a type FUS 0181 polyethersulfone (PES) fiber, available from Microdyn-Nadir (Wiesbaden, Germany) with the following characteristics: 0.8 mm inner diameter, 1.3 mm outer diameter, and 10 kDa Mw cut-off, corresponding to an average pore diameter of 5 nm. Channel pressure limit was set at 20 bar. The ChemStation version B.04.02 (Agilent Technologies, Santa Clara, CA, USA) data system for Agilent instrumentation was used to set and control the instrumentation and method parameters. An 18-angle light scattering detector model DAWN HELEOS (Wyatt Technology Corporation, Santa Barbara, CA, USA), operating at 658 nm wavelength, was used to measure the light scattering signal from particles in solution. ASTRA(r) software version 6.1.7 (Wyatt Technology Corporation, Santa Barbara, CA, USA) was used to handle signals from the detectors (MALS and UV) and to compute the sample root mean square radius of gyration (R\({}_{\text{g}}\)) values. An HF5 method is composed of four steps: focus, focus-injection, elution and elution-injection. During focus the mobile phase enters from both inlet and outlet and stabilizes; during focus-injection, the flow settings remain unvaried while the sample is introduced into the channel through the inlet and focused in a narrow band. Then, in the elution step, the flow of mobile phase enters the channel inlet and part of it comes out transversely (cross-flow, Vx), while the rest (channel flow, Vc) reaches the detectors; lastly, during elution-injection, no cross-flow is applied allowing for any remaining sample inside the channel to be released; also, the flow is redirected in the injection line as well to clean it before the next injection. A non-separative method can be used to evaluate sample recovery. In particular, a Flow Injection Analyis (FIA), i.e., a run with zero focus flow, zero crossflow, allows for total recovery of the sample, including molecules and particles smaller than the membrane's cutoff. By comparing areas between a FIA and a separation method, recovery % is obtained. Analyses were carried out in isotonic, pH 7.4 PBS as mobile phase. The flow rate program of the separation method was set up as follows: inject flow of 0.20 mL/min, a focus flow of 0.80 mL/min (4 min), an initial crossflow of 0.40 mL/min decreasing to 0.2 (16 min), an isocratic step (6 min) and a detector flow of 0.40 mL/min. MALS analysis allows for the determination of particle R\({}_{\text{g}}\) by the mass distribution within the particle. Knowing the absorptivity (\(\epsilon\)) and the specific refractive index increment (dn/dc) for the eluted sample (\(\epsilon\) values were set as 0.667 mL/(mg-cm) for BSA and 1.37 for IgGs, while a generic 1 valuewas used for later eluted proteins [54]; dn/dc was set as 0.185 mL/g), molar mass can be calculated from \(\mathrm{R_{g}}\). Molar mass and \(\mathrm{R_{g}}\) distributions can be combined into a scaling exponent v, i.e., the slope in a double logarithmic logMM vs. \(\mathrm{logR_{g}}\) plot, which describes the conformation of the molecules in solution. It is theoretically defined as v = 0.33 for spheres, v = 0.5-0.6 for random-coils, and v \(\sim\)1 for rod-like structures [48]. The total method duration was 28 min. Method precision was assessed both on retention times and on signal intensity by performing three independent replicates for serum and protein standards. The limit of quantification (LOQ) was calculated as the injected amount (pmol) at which the signal is 10 times the signal noise of the baseline. All graphs were elaborated in GraphPad Prism.