Unified superresolution experiments and stochastic theory provide mechanistic insight into protein ion-exchange adsorptive separations

Abstract

Chromatographic protein separations, immunoassays, and biosensing all typically involve the adsorption of proteins to surfaces decorated with charged, hydrophobic, or affinity ligands. Despite increasingly widespread use throughout the pharmaceutical industry, mechanistic detail about the interactions of proteins with individual chromatographic adsorbent sites is available only via inference from ensemble measurements such as binding isotherms, calorimetry, and chromatography. In this work, we present the direct superresolution mapping and kinetic characterization of functional sites on ion-exchange ligands based on agarose, a support matrix routinely used in protein chromatography. By quantifying the interactions of single proteins with individual charged ligands, we demonstrate that clusters of charges are necessary to create detectable adsorption sites and that even chemically identical ligands create adsorption sites of varying kinetic properties that depend on steric availability at the interface. Additionally, we relate experimental results to the stochastic theory of chromatography. Simulated elution profiles calculated from the molecular-scale data suggest that, if it were possible to engineer uniform optimal interactions into ion-exchange systems, separation efficiencies could be improved by as much as a factor of five by deliberately exploiting clustered interactions that currently dominate the ion-exchange process only accidentally.ion-exchange chromatography | single-molecule kinetics | bioseparations | optical nanoscopy T he hundred-billion-dollar global pharmaceutical industry relies increasingly on the painstaking purification of therapeutic biomolecules such as proteins and nucleic acids (1). Separation of biologics is often performed using ion-exchange chromatography on stationary phases supporting singly charged ligands (2, 3) and constitutes an expensive, bottlenecking step in production. Improving bioseparations is thus highly desirable (4, 5); yet, a molecular-scale, mechanistic understanding is lacking, for ionexchange chromatography in particular (6). Mechanistic detail is lost in ensemble analyses, reflecting the inherent heterogeneity of both the adsorbed biomolecules and the porous stationary phase supports (7). Ensemble adsorption isotherms, however, suggest the likelihood that protein and nucleic acid separations in ion-exchange columns may involve random ligand clustering (8-10). Additional support for such an assertion lies in the implementation of stationary phases of very high charge density by polymerization of charged monomers or layer-by-layer deposition (11-13), and in the demonstration that patches of high charge density on proteins often play a disproportionate role in their adsorption (4,6,(14)(15)(16)(17). In this work, we provide direct evidence of the importance of charge clustering in ion-exchange systems by direct observation of individual adsorption sites.Although the role of multivalency is broadly accepted and exploited in a wide range of associative and adsorption ...

Characterization of Porous Materials by Fluorescence Correlation Spectroscopy Super-resolution Optical Fluctuation Imaging

“…fcsSOFI analysis was applied to image heterogeneous pore distribution and diffusion within agarose hydrogels, which are used broadly in cell culture growth, electrophoretic and 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 chromatographic separations, 9,36 diffraction-limited position of emitters that are primarily trapped within pores (Figure 2a-c). In contrast, fcsSOFI analysis revealed the heterogeneous distribution of bright areas where beads are free to diffuse (i.e.…”

High ionic strength narrows the population of sites participating in protein ion-exchange adsorption: A single-molecule study

“…Because adsorption heterogeneity is observed even with uniform adsorbate populations and at very low loadings [20], it is likely that inherent heterogeneity of the adsorbent surface commonly plays the larger role. Adsorbent heterogeneity can arise from a range of potential causes, including steric accessibility and constraint, surface entropy and mobility [13], and the stochastic clustering of ligands on the adsorbent surface [21]. We have found that while adsorbents based on pre-organized, penta-valent clustered-charge ligands show higher protein affinity and capacity than adsorbents of the same total density of charge randomly distributed, isotherms for protein adsorption even on these nominally-homogeneous adsorbents show heterogeneity [12].…”

“…We have found that while adsorbents based on pre-organized, penta-valent clustered-charge ligands show higher protein affinity and capacity than adsorbents of the same total density of charge randomly distributed, isotherms for protein adsorption even on these nominally-homogeneous adsorbents show heterogeneity [12]. This observation further implicates adsorbent steric and surface mobility properties as a ubiquitous source of heterogeneity; especially as our recent single-molecule observations highlighted the role of both ligand clustering and steric availability with the porous support [21] .…”

“…Foundational single molecule work resolved variability present at heterogeneous ion-exchange and reverse-phase stationary phase interfaces using confocal microscopy combined with fluorescence correlation spectroscopy and blip analysis [26-32] and total internal reflection microscopy [33-37]. We have recently extended single molecule techniques to the super-resolution level (< 250 nm, below the diffraction limit of light) to investigate adsorptive separations at the single ligand-single protein level [21]. Super-resolution imaging allows for long-sought direct observations at the nanometer scale of the interfacial interactions that control chromatography.…”

Imaging Fluorescence-Correlation Spectroscopy for Measuring Fast Surface Diffusion at Liquid/Solid Interfaces

“…10,12,13 These strong sites are evidence of heterogeneity in surface interactions, which can be related to tailing of chromategraphic peaks arising from a distribution of residence times on the surface. 12,14 Although FCS is a versatile technique for measuring diffusion at homogeneous interfaces, the technique has limitations for surfaces that exhibit adsorption heterogeneity or mixed relaxation kinetics (diffusion together with adsorption and desorption, for example). In the former case, when long-lived adsorption events occur during a segment of FCS data being used to measure the diffusion coefficient of moving molecules, one observes a long-lived tail in the autocorrelation that obscures the measurement of the surface diffusion coefficient.…”