Realizing the AF4-UV-SAXS on-line coupling on protein and antibodies using high flux synchrotron radiation at the CoSAXS beamline, MAX IV

Abstract

In this paper, we demonstrate the coupling of synchrotron small angle X-ray scattering (SAXS) to asymmetrical flow-field flow fractionation (AF4) for protein characterization. To the best of our knowledge, this is the first time AF4 is successfully coupled to a synchrotron for on-line measurements on proteins. This coupling has potentially high impact, as it opens the possibility to characterize individual constituents of sensitive and/or complex samples, not suited for separation using other techniques, and for low electron density samples where high X-ray flux is required, e.g., biomolecules and biologics. AF4 fractionates complex samples in native or close to native environment, with low shear forces and system surface area. Many orders of magnitude in size can be fractionated in one measurement, without having to reconfigure the experimental setup. We report AF4 fractionations with correlated UV and statistically adequate SAXS data of bovine serum albumin and a monoclonal antibody and evaluate SAXS data recorded for the two protein systems. Graphical Abstract

Quick assessment of a Small Scattering data using Guinier-Porod model: Introducing the correlative size

“…Those procedures are usually done in the laboratory before measurement or with in-line equipment attached to the SAXS equipment/beamline like exclusion chromatography [19], asymmetric flow field-flow fractionation [20], dialysis, etc. On the other hand, colloids could form aggregates that affect the scattering signal, usually as a small upturn in the low scattering region.…”