Investigation of native and aggregated therapeutic proteins in human plasma with asymmetrical flow field-flow fractionation and mass spectrometry

Abstract

Physiochemical degradation of therapeutic proteins in vivo during plasma circulation after administration can have a detrimental effect on their efficacy and safety profile. During drug product development, in vivo animal studies are necessary to explore in vivo protein behaviour. However, these studies are very demanding and expensive, and the industry is working to decrease the number of in vivo studies. Consequently, there is considerable interest in the development of methods to pre-screen the behaviour of therapeutic proteins in vivo using in vitro analysis. In this work, asymmetrical flow field-flow fractionation (AF4) and liquid chromatography–mass spectrometry (LC-MS) were combined to develop a novel analytical methodology for predicting the behaviour of therapeutic proteins in vivo. The method was tested with two proteins, a monoclonal antibody and a serum albumin binding affibody. After incubation of the proteins in plasma, the method was successfully used to investigate and quantify serum albumin binding, analyse changes in monoclonal antibody size, and identify and quantify monoclonal antibody aggregates. Graphical abstract

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

“…As the AF4 channel is void of stationary phase, the shear forces and the interacting surface area of the fractionation device are much lower, compared to SEC. The surface area of a SEC column is approximately 10 3 –10 4 than that of the AF4 separation channel and the shear forces are in the order of 10 3 higher in SEC [ 14 ]. In SEC, the risk of interaction with the column close packed material, and adsorption with the risk of column blockage, is often reduced by pH adjustments, or by applying higher salt concentrations or modifiers to the mobile phase.…”