Asymmetric flow field-flow fractionation coupled to surface plasmon resonance detection for analysis of therapeutic proteins in blood serum

Abstract

Coupling of surface plasmon resonance (SPR) detection to asymmetric flow field-flow fractionation (AF4) offers the possibility to study active fractions of bio-separations on real samples, such as serum and saliva, including the assessment of activity of possibly aggregated species. The coupling of SPR with AF4 requires the possibility to select fractions from a fractogram and redirect them to the SPR. The combination of SPR with chromatography-like methods also requires a mechanism for regeneration of the receptor immobilised onto the SPR sensor surface. In recent work, the combination of size exclusion chromatography (SEC) with SPR was pioneered as a successful methodology for identification, characterisation and quantification of active biocomponents in biological samples. In this study, the approach using AF4 is evaluated for the antibody trastuzumab in buffer and serum. The particular object of this study was to test the feasibility of using AF4 in combination with SPR to detect and quantify proteins and aggregates in complex samples such as blood serum. Also, in the investigation, three different immobilisation methods for the receptor HER-2 were compared, which involved (1) direct binding via EDC/NHS, the standard approach; (2) immobilisation via NTA-Ni-Histag complexation; and (3) biotin/avidin-linked chemistry using a regenerable form of avidin. The highest specific activity was obtained for the biotin-avidin method, while the lowest specific activity was observed for the NTA-Ni-Histag linkage. The data show that AF4 can separate trastuzumab monomers and aggregates in blood serum and that SPR has the ability to selectively monitor the elution. This is an encouraging result for automated analysis of complex biological samples using AF4-SPR.

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

“…The plasma fractogram contains a peak with a peak maximum at approximately 4 min dominated by human serum albumin (HSA) and a second peak at approximately 7 min dominated by immunoglobulin G (IgG), as shown by comparison with protein standards (Fig. S2, Supplementary Information), and previously by Leeman et al [13,26]. The fractogram also contains a population of larger analytes that start to elute at approximately 10 min, corresponding to larger components such as the larger plasma proteins, fibrinogen, IgM, and α-2-macroglobulin, or complexes of proteins [43,44].…”

Asymmetric flow field-flow fractionation as a multifunctional technique for the characterization of polymeric nanocarriers

“…While the SPR sensor can specifically detect trastuzumab, the coupling with AF4 also allows to discriminate between monomers and aggregates of the monoclonal antibody. On the downside, SPR is limited by its own specificity, as it requires the immobilization of a suitable receptor (in this case, human epidermal growth factor receptor 2) on the surface of the sensor [139].…”

Label-Free Isolation of Exosomes Using Microfluidic Technologies

“…Field flow fractionation (FFF) is a flow-based sorting method applicable for separation of particles ranging from a few nanometers to about 100 μm in diameter. − This technique has been widely utilized to isolate and characterize biological and nonbiological particles. − To separate particles, a liquid suspension containing particles traveling (laminar flow with a parabolic profile) in a narrow channel (height of 50–500 μm) while being subjected to an externally applied field (liquid flows, centrifugal forces, gravity or electrical fields) perpendicular to the direction of suspension flow (Figure A). − The separation mechanism is based on the differences in density and hydrodynamic properties of particles that define particle mobility under the effect of forces perpendicular to each other; the forward force exerted by laminar channel flow and vertical forces exerted by the externally applied field and diffusion. External field forces drive the accumulation of particles at the bottom wall of the channel, whereas Brownian motion of the particles results in their diffusion toward the channel center. − Depending on their equilibrium position from the wall of the microchannel, particles travel at different speeds and thereby are separated as they move down the microchannel (Figure A).…”