Protein adsorption and transport in dextran-modified ion-exchange media. I: Adsorption

Adsorption behavior is compared on a traditional agarose-based ion-exchange resin and on two dextran-modified resins, using three proteins to examine the effect of protein size. The latter resins typically exhibit higher static capacities at low ionic strengths and electron microscopy provides direct visual evidence supporting the view that the higher static capacities are due to the larger available binding volume afforded by the dextran. However, isocratic retention experiments reveal that the larger proteins can be almost completely excluded from the dextran layer at high ionic strengths, potentially leading to significant losses in static capacity at relevant column loading conditions. Knowledge of resin and protein properties is used to estimate physical limits on the static capacities of the resins in order to provide a meaningful interpretation of the observed static capacities. Results of such estimates are consistent with the expectation that available surface area is limiting for traditional resins. In dextran-modified media, however, the volume of the dextran layer appears to limit adsorption when the protein charge is low relative to the resin charge, but the protein–resin electroneutrality may be limiting when the protein charge is relatively high. Such analyses may prove useful for semiquantitative prediction of maximum static capacities and selection of operating conditions when combined with protein transport information.     

Hydrophobic interaction chromatography of proteins. IV. Protein adsorption capacity and transport in preparative mode

The adsorption isotherms of four model proteins (lysozyme, α-lactalbumin, ovalbumin, and BSA) on eight commercial phenyl hydrophobic interaction chromatography media were measured. The isotherms were softer than those usually seen in ion-exchange chromatography of proteins, and the static capacities of the media were lower, ranging from 30 to 110 mg/mL, depending on the ammonium sulfate concentration and the protein and adsorbent types. The protein-accessible surface area appears to be the main factor determining the binding capacity, and little correlation was seen with the protein affinities of the adsorbents. Breakthrough experiments showed that the dynamic capacities of the adsorbents at 10% breakthrough were 20-80% of the static capacities, depending on adsorbent type. Protein diffusivities in the adsorbents were estimated from batch uptake experiments using the pore diffusion and homogeneous diffusion models. Protein transport was affected by the adsorbent pore structures. Apparent diffusivities were higher at lower salt concentrations and column loadings, suggesting that adsorbed proteins may retard intraparticle protein transport. The diffusivities estimated from the batch uptake experiments were used to predict column breakthrough behavior. Analytical solutions developed for ion-exchange systems were able to provide accurate predictions for lysozyme breakthrough but not for ovalbumin. Impurities in the ovalbumin solutions used for the breakthrough experiments may have affected the ovalbumin uptake and led to the discrepancies between the predictions and the experimental results.     

Protein adsorption and transport in dextran-modified ion-exchange media. II. Intraparticle uptake and column breakthrough

Protein transport behavior was compared for the traditional SP Sepharose Fast Flow and the dextran-modified SP Sepharose XL and Capto S resins. Examination of the dynamic binding capacities (DBCs) revealed a fundamental difference in the balance between transport and equilibrium capacity limitations when comparing the two resin classes, as reflected by differences in the locations of the maximum DBCs as a function of salt. In order to quantitatively compare transport behavior, confocal microscopy and batch uptake experiments were used to obtain estimates of intraparticle protein diffusivities. For the traditional particle, such diffusivity estimates could be used to predict column breakthrough behavior accurately. However, for the dextran-modified media, neither the pore- nor the homogeneous-diffusion model was adequate, as experimental dynamic binding capacities were consistently lower than predicted. In examining the shapes of breakthrough curves, it was apparent that the model predictions failed to capture two features observed for the dextran-modified media, but never seen for the traditional resin. Comparison of estimated effective pore diffusivities from confocal microscopy and batch uptake experiments revealed a discrepancy that led to the hypothesis that protein uptake in the dextran-modified resins could occur with a shrinking-core-like sharp uptake front, but with incomplete saturation. The reason for the incomplete saturation is speculated to be that protein initially fills the dextran layer with inefficient packing, but can rearrange over time to accommodate more protein. A conceptual model was developed to account for the partial shrinking-core uptake to test whether the physical intuition led to predictions consistent with experimental behavior. The model could correctly reproduce the two unique features of the breakthrough curves and, in sample applications, parameters found from the fit of one breakthrough curve could be used to adequately match breakthrough at a different flow rate or batch uptake behavior.     

Rapid monoclonal antibody adsorption on dextran-grafted agarose media for ion-exchange chromatography

The binding capacity and adsorption kinetics of a monoclonal antibody (mAb) are measured for experimental cation exchangers obtained by grafting dextran polymers to agarose beads and compared with measurements for two commercial agarose-based cation exchangers with and without dextran grafts. Introduction of charged dextran polymers results in enhanced adsorption kinetics despite a dramatic reduction of the accessible pore size as determined by inverse size-exclusion chromatography. Incorporation of neutral dextran polymers in a charged agarose bead results instead in substantially lower binding capacities. The effective pore diffusivities obtained from batch uptake curves increase substantially as the protein concentration is reduced for the resins containing charged dextran grafts, but are much less dependent on protein concentration for the resins with no dextran or uncharged dextran grafts. The batch uptake results are corroborated by microscopic observations of transient adsorption in individual particles. In all cases studied, the adsorption kinetics is characterized by a sharp adsorption front consistent with a shell-progressive, diffusion limited mechanism. Greatly enhanced transport rates are obtained with an experimental resin containing charged dextran grafts with effective pore diffusivities that are 1-9 times larger than the free solution diffusivity and adsorption capacity approaching 300 mg/cm3 of particle volume.     

Effect of polysaccharide structure on protein adsorption

Using X-ray photoelectron spectroscopy for quantification, the adsorption has been studied of chicken egg lysozyme, human serum albumin (HSA), bovine colostrum lactoferrin, and γ-globulin (IgG) from single solutions onto surface-immobilised polysaccharide coatings, which were produced by the covalent attachment of a series of carboxymethyldextrans (CMDs) onto aminated fluoropolymer surfaces. CMDs with differing degrees of carboxymethyl substitution were synthesized by the reaction of dextran with bromoacetic acid under different reactant ratios. Substantial amounts of protein adsorption onto these coatings were observed with the majority of the coating/protein combinations. On the most extensively substituted CMD (1 carboxyl group per 2 dextran units), lysozyme and lactoferrin adsorbed to approximately monolayer amounts whereas there was minimal adsorption of HSA, indicating the importance of electrostatic interfacial interactions. CMD 1:14 was similar whereas the least substituted, least dense coating, from CMD 1:30, adsorbed less lysozyme and lactoferrin but more HSA. Adsorption of the large multidomain protein IgG varied little with the coating. Grazing angle XPS data indicated that for the CMD 1:30 coating there occurred significant in-diffusion of the lower molecular weight proteins. The data suggest that elimination of adsorption of a broad spectrum of proteins is not straightforward with negatively charged polysaccharide coatings; elimination of protein accumulation onto/into such coatings may not be achievable solely with a balance of electrostatic and steric–entropic interfacial forces.     

Protein adsorption and transport in agarose and dextran-grafted agarose media for ion exchange chromatography

This work examines the relationship between the physical properties of agarose and dextran-grafted agarose cation exchangers and protein adsorption equilibrium and rates. Four different sulfopropyl (SP) matrices were synthesized using a neutral agarose base material--two based on a short ligand chemistry and two obtained by grafting 10 and 40kDa dextran polymers. The pore accessibility, determined by inverse size exclusion chromatography (iSEC) with dextran probes, decreases dramatically as a result of the combined effects of crosslinking, dextran grafting, and the introduction of ionic ligands, with pore radii decreasing from 19nm for the base matrix to 6.1nm for the 40kDa dextran-grafted SP-matrix. In spite of this reduction, while the adsorption isotherms were similar, protein uptake rates were greatly increased with the dextran-grafted SP-matrices, compared to SP-matrices based on the short ligand chemistry. The effective pore diffusivities were 4-10 times higher than free solution diffusivity for the dextran-grafted matrices, indicating that the charged dextran grafts result in enhanced protein mass transfer rates.     

Protein adsorption on novel acrylamido-based polymeric ion-exchangers. I. Morphology and equilibrium adsorption

The protein uptake equilibrium and particle morphology are determined for novel polymeric ion-exchange media based on acrylamido monomers with a high density of functional groups and a variety of morphological characteristics. The study considers two anion-exchangers and a cation-exchanger. Physical properties determined experimentally include particle density, ion-exchange capacity, particle size distribution, and equilibrium isotherms for model proteins. The pore structure was evaluated using size exclusion chromatography with neutral probe molecules and transmission electron microscopy. For the anion-exchangers, two types of structures were inferred. The first is comprised of particles that contain a low-density gel supported by denser polymer aggregates. This material had a very low size-exclusion limit for neutral probes, but exhibited an extremely high and reversible protein adsorption capacity (280-290 mg BSA/ml). The second structure is comprised of particles with large, open macropores. While the size-exclusion limit was very high, the protein adsorption capacity was low (60 mg BSA/ml). Moreover, the adsorption was nearly irreversible. The physical structure of the cation-exchanger appeared to be intermediate between those of the anion-exchangers, containing both large pores and smaller pores yielding an intermediate, but reversible, protein uptake capacity (120-130 mg alphaCHY/ml). The different behavior of these materials with regards to protein adsorption correlates well with their physical structure. For these ion-exchangers, high protein adsorption capacities are attained when a low-density polymer gel with a high concentration of functional groups is present.     

Effect of dextran layer on protein uptake to dextran-grafted adsorbents for ion-exchange and mixed-mode chromatography

In the current research, a series of dextran-grafted adsorbents were prepared using sulfopropyl and 4-(1H-imidazol-1-yl) aniline as chromatographic ligands for ion-exchange (IEC) and mixed-mode chromatography (MMC) to respectively investigate the influence of dextran layer on adsorption of γ-globulin. Experimental evidences of static adsorption on dextran-grafted IEC adsorbents showed that adsorption capacity of γ-globulin increased with dextran content. It could be attributed to the multilayer adsorption of charged protein in dextran layer and thus further induced a significant electrical potential gradient at the boundary of adsorbed area and its proximity, improving mass transfer in combination with concentration gradient. In contrast to IEC adsorbents, adsorption capacity and effective diffusivity of dextran-grafted MMC adsorbents did not change obviously with dextran grafting. It was considered that hydrophobic ligands immobilized onto dextran-grafted MMC adsorbents were stuck together at pH 8.0, resulting in the collapse of dextran layer. In concert with measured effective porosity for γ-globulin at pH 4.0, it was confirmed that dextran layer in MMC adsorbent was more complicated and influenced significantly by buffer pH. It was also manifested by protein adsorption at different pHs. Thus, it revealed the complexity in intraparticle mass transfer of the protein in dextran-grafted MMC adsorbent.     

Surface extenders and an optimal pore size promote high dynamic binding capacities of antibodies on cation exchange resins

Increased recombinant protein expression yields and a large installed base of manufacturing facilities designed for smaller bulk sizes has led to the need for high capacity chromatographic resins. This work explores the impact of three pore sizes (with dextran distribution coefficients of 0.4, 0.53, and 0.64), dextran surface extender concentration (11–20 mg/mL), and ligand density (77–138 μmol H+/mL resin) of cation exchange resins on the dynamic binding capacity of a therapeutic antibody. An intermediate optimal pore size was identified from three pore sizes examined. Increasing ligand density was shown to increase the critical ionic strength, while increasing dextran content increased dynamic binding capacity mainly at the optimal pore size and lower conductivities. Dynamic binding capacity as high as 200 mg/mL was obtained at the optimum pore size and dextran content.     

Dextran-grafted cation exchanger based on superporous agarose gel: Adsorption isotherms,uptake kinetics and dynamic protein adsorption performance

A novel chromatographic medium for high-capacity protein adsorption was fabricated by grafting dextran (40 kDa) onto the pore surfaces of superporous agarose (SA) beads. The bead was denoted as D-SA. D-SA, SA and homogeneous agarose (HA) beads were modified with sulfopropyl (SP) group to prepare cation exchangers, and the adsorption and uptake of lysozyme on all three cation-exchange chromatographic beads (SP-HA, SP-SA and SP-D-SA) were investigated at salt concentrations of 6–50 mmol/L. Static adsorption experiments showed that the adsorption capacity of SP-D-SA (2.24 mmol/g) was 78% higher than that of SP-SA (1.26 mmol/g) and 54% higher than that of SP-HA (1.45 mmol/g) at a salt concentration of 6 mmol/L. Moreover, salt concentration had less influence on the adsorption capacity and dissociation constant of SP-D-SA than it did on SP-HA, suggesting that dextran-grafted superporous bead is a more potent architecture for chromatographic beads. In the dynamic uptake of lysozyme to the three cation-exchange beads, the D_e/D₀ (the ratio of effective pore diffusivity to free solution diffusivity) values of 1.6–2.0 were obtained in SA-D-SA, indicating that effective pore diffusivities of SP-D-SA were about two times higher than free solution diffusivity for lysozyme. At 6 mmol/L NaCl, the D_e value in SA-D-SA (22.0 × 10⁻¹¹ m²/s) was 14.4-fold greater than that in SP-HA. Due to the superior uptake kinetics in SA-D-SA, the highest dynamic binding capacity (DBC) and adsorption efficiency (the ratio of DBC to static adsorption capacity) was likewise found in SP-D-SA. It is thus confirmed that SP-D-SA has combined the advantages of superporous matrix structure and drafted ligand chemistry in mass transport and offers a new opportunity for the development of high-performance protein chromatography.     

大孔PMVBS树脂对茶多酚的吸附研究

茶叶在我国具有丰富的自然资源,饮茶及茶文化在我国已有数千年的历史.茶不仅具有提神解渴的功效,而且富含具有保健、药效功能的茶多酚(Tea Polyphenols TP).茶多酚,又名茶单宁,是一类多羟基酚类有机物.     

Protein adsorption and transport in agarose and dextran-grafted agarose media for ion exchange chromatography: Effect of ionic strength and protein characteristics

This work investigates the effects of ionic strength and protein characteristics on adsorption and transport of lysozyme, BSA, and IgG in agarose-based cation exchangers with short ligand chemistry and with charged dextran grafts. In all cases, the adsorption equilibrium capacity decreased with increasing salt. However, the adsorption kinetics was strongly influenced by the adsorbent structure and protein characteristics. For the smaller and positively charged lysozyme, the effective pore diffusivity was only weakly dependent on salt for the dextran-free media, but declined sharply with salt for the dextran-grafted materials. For this protein, the dextran grafts enhanced the adsorption kinetics at low salt, but the enhancement vanished at higher salt concentrations. For BSA, which was near its isoelectric point for the experimental conditions studied, the effective diffusivity was low for all materials and almost independent of salt. Finally, for the larger and positively charged IgG, the effective diffusivity varied with salt, reaching an apparent maximum at intermediate concentrations for both dextran-free and dextran-grafted media with the kinetics substantially enhanced by the dextran grafts for these conditions. Microscopic observations of the particles during protein adsorption at low ionic strengths showed transient patterns characterized by sharp adsorption fronts for all materials. A theory taking into account surface or adsorbed phase diffusion with electrostatic coupling of diffusion fluxes is introduced to explain the mechanism for the enhanced adsorption kinetics observed for the positively charged proteins.     

Surface-functionalized electrospun carbon nanofiber mats as an innovative type of protein adsorption/purification medium with high capacity and high throughput

Due to recent advances in the production of biotherapeutics, high capacity, high throughput adsorption media for efficient and economic separation of these medically important products are in great demand. One option that has been evaluated extensively is membrane/mat adsorption. While these media allow for rapid adsorption (due to the decreased internal diffusion) and high throughput processing (due to the open porous structure), they often suffer from low capacity and poor enrichment factors. Herein, we report the fabrication, characterization, and protein adsorption evaluation of an innovative type of membrane/mat adsorption media based on electrospun carbon nanofibers. By surface-functionalization of these nanofibers with a weak acid cation-exchange ligand, the capacity was doubled for binding a model protein (i.e., lysozyme) compared to commercial products; and the capacity value was over 200 mg lysozyme per gram of adsorption media. Meanwhile, the thin nanofibers (having diameters of ~300 nm) along with open pores among nanofibers in the mats (having sizes of ~10-15 μm) allowed for higher operating flow rates and lower pressure drops. Furthermore, the incorporation of higher ligand density and the addition of a non-ionic surfactant (i.e., Triton X-305) into the adsorption buffer eliminated the non-specific binding of a competing protein (bovine serum albumin). In combination, this study suggested that electrospun carbon nanofiber adsorption media would provide a promising alternative to packed resin beds for bioseparations.     

Enrichment of proteinaceous materials on a strong cation-exchange diol silica restricted access material: protein-protein displacement and interaction effects

A study of size exclusion and enrichment of proteins employing strong cation-exchange diol silica restricted access material (SCX-RAM) under saturation conditions is presented. Experiments were carried out with bacitracin, protamine, ribonuclease, lysozyme and bovine serum albumin as individual proteinaceous analytes as well as comprehensive binary mixtures and with human urine samples. Protein size dependent capacity features of the SCX-RAM column was observed. Bacitracin demonstrated the highest capacity followed by protamine while adsorption capacities of both ribonuclease and lysozyme were found smaller by a factor of 10. Applying binary protein samples occurring displacement effects were apparent: proteins with strong cationic properties displaced those already adsorbed by the bonded cation-exchange ligands. Bacitracin was displaced in all binary mixture experiments in particular by protamine. Furthermore, the binary mixtures displayed increased adsorption for some proteins due to complex formation. Lysozyme and ribonuclease showed double capacity values when paired with bacitracin. Both phenomena, displacement and enhanced adsorption occurred in the saturated state and led to changes in the urine composition during sample preparation. Injecting urine samples the relative proportions of fractions changed from 4 up to more than 20 times, due to the differences of the protein adsorption capacities on the SCX-RAM column. Analysing urine samples the SCX-RAM column provided extensive long-term stability.     

Bioinert surface to protein adsorption with higher generation of dendrimer SAMs

Interactions between proteins and biomaterial surfaces correlate with many important phenomena in biological systems. Such interactions have been used to develop various artificial biomaterials and applications, in which regulation of non-specific protein adsorption has been achieved with bioinert properties. In this research, we investigated the protein adsorption behavior of polymer brushes of dendrimer self-assembled monolayers (SAMs) with other generations. The surface adsorption properties of proteins with different pI values were examined on gold substrates modified with poly(amidoamine) dendrimer SAMs. The amount of fibrinogen adsorption was greater than that of lysozyme, potentially because of the surface electric charge. However, as the generations increased, protein adsorption decreased regardless of the surface charge, suggesting that protein adsorption was also affected by density of terminal group.     

Succinic acid adsorption from fermentation broth and regeneration

More than 25 sorbents were tested for uptake of succinic acid from aqueous solutions. The best resins were then tested for successive loading and regeneration using hotwater. The key desired properties for an ideal sorbent are high capacity, complete stable regenerability, and specificity for the product. The best resins have a stable capacity of about 0.06 g of succinic acid/g of resin at moderate concentrations (1–5 g/L) of succinic acid. Several sorbents were tested more exhaustively for uptake of succinic acid and for successive loading and regeneration using hot water. One resin, XUS 40285, has a good stable isotherm capacity, prefers succinate over glucose, and has good capacities at both acidic and neutral pH. Succinic acid was removed from simulated media containing salts, succinic acid, acetic acid, and sugar using a packed column of sorbent resin, XUS 40285. The fermentation byproduct, acetate, was completely separated from succinate. A simple hot water regeneration successfully concentrated succinate from 10 g/L (inlet) to 40–110 g/L in the effluent. If successful, this would lower separation costs by reducing the need for chemicals for the initial purification step. Despie promising initial results of good capacity (0.06 g of succinic/g of sorbent), 70% recovery using hot water, and a recovered concentration of >100 g/L, this regeneration was not stable over 10 cycles in the column. Alternative regeneration schemes using acid and base were examined. Two (XUS 40285 and XFS-40422) showed both good stable capacities for succinic acid over 10 cycles and >95% recovery in a batch operation using a modified extraction procedure combining acid and hot water washes. These resins showed comparable results with actual broth.     

Kinetic modeling of liquid-phase adsorption of phosphate on dolomite

The adsorption of phosphate from aqueous solution on dolomite was investigated at 20 and 40 degrees C in terms of pseudo-second-order mechanism for chemical adsorption as well as an intraparticle diffusion mechanism process. Adsorption was changed with increased contact time, initial phosphate concentration, temperature, solution pH. A pseudo-second-order model and intraparticle diffusion model have been developed to predict the rate constants of adsorption and equilibrium capacities.The activation energy of adsorption can be evaluated using the pseudo-second-order rate constants. The adsorption of phosphate onto dolomite are an exothermically activated process. A relatively low activation energy and a model highly fitting to intraparticle diffusion suggest that the adsorption of phosphate by dolomite may involve not only physical but also chemisorption. This was likely due to its combined control of chemisorption and intraparticle diffusion. However, for phosphate/dolomite system chemical reaction is important and significant in the rate-controlling step, and for the adsorption of phosphate onto dolomite the pseudo-second-order chemical reaction kinetics provides the best correlation of the experimental data.     

High-speed protein purification by adsorptive cation-exchange hollow-fiber cartridges

Novel cation-exchange adsorptive membranes were assessed according to their protein adsorption capacity and permeation flowrate. Maximum static adsorption capacities for the three main egg-white proteins, lysozyme, ovoalbumin and conalbumin, were 140, 88 and 66 mg/ml, respectively. However, membranes showed an inverse relationship between permeation flowrate and static protein adsorption capacity. Two size cartridges (membrane volume of 0.42 and 3.5 ml) were built using the selected membrane. An adsorptive cross-flow cartridge was tested to recover and purify lysozyme from an egg-white solution. Breakthrough curves developed using a pure lysozyme solution showed a dynamic-to-static capacity ratio of 0.6, which was reduced to 0.4 during lysozyme recovery from egg-white solution in cross-flow mode. Total process cycle for the enzyme recovery and purification was in the range of 10–15 min for both cartridges. In both cases high-purity lysozyme (95%) was recovered with a productivity of 150 g/(l h) and no size-exclusion effect was detected.     

IgG adsorption on a new protein A adsorbent based on macroporous hydrophilic polymers. I. Adsorption equilibrium and kinetics

Experimental determination and modeling of IgG binding on a new protein A adsorbent based on a macroporous resin were performed. The new adsorbent consists of polymeric beads based on hydrophilic acrylamido and vinyl monomers with a pore structure optimized to allow favorable interactions of IgG with recombinant protein A coupled to the resin. The particles have average diameter of 57 μm and a narrow particle size distribution. The IgG adsorption equilibrium capacity is 46 mg/cm³ and the effective pore diffusivity determined from pulse response experiments for non-binding conditions is 8.0 × 10⁻⁸ cm²/s. The IgG adsorption kinetics can be described with the same effective diffusivity by taking into account a heterogeneous binding mechanism with fast binding sites, for which adsorption is completely diffusion controlled, and slow binding sites for which adsorption is controlled by the binding kinetics. As a result of this mechanism, the breakthrough curve exhibits a tailing behavior, which appears to be associated with the slow binding sites. A detailed rate model taking into account intraparticle diffusion and binding kinetics is developed and is found capable of predicting both batch adsorption and breakthrough behavior over an ample range of experimental conditions. The corresponding effective diffusivity is independent of protein concentration in solution over the range 0.2–2 mg/cm³ and of protein binding as a result of the large pore size of the support matrix. Overall, the small particle size and low diffusional hindrance allow capture of IgG with short residence times while attaining substantial dynamic binding capacities.     

Solubility and binding properties of PEGylated lysozyme derivatives with increasing molecular weight on hydrophobic-interaction chromatographic resins

Dynamic binding capacities and resolution of PEGylated lysozyme derivatives with varying molecular weights of poly (ethylene) glycol (PEG) with 5 kDa, 10 kDa and 30 kDa for HIC resins and columns are presented. To find the optimal range for the operating conditions, solubility studies were performed by high-throughput analyses in a 96-well plate format, and optimal salt concentrations and pH values were determined. The solubility of PEG-proteins was strongly influenced by the length of the PEG moiety. Large differences in the solubilities of PEGylated lysozymes in two different salts, ammonium sulfate and sodium chloride were found. Solubility of PEGylated lysozyme derivatives in ammonium sulfate decreases with increased length of attached PEG chains. In sodium chloride all PEGylated lysozyme derivatives are fully soluble in a concentration range between 0.1 mg protein/ml and 10 mg protein/ml. The binding capacities for PEGylated lysozyme to HIC resins are dependent on the salt type and molecular weight of the PEG polymer. In both salt solutions, ammonium sulfate and sodium chloride, the highest binding capacity of the resin was found for 5 kDa PEGylated lysozyme. For both native lysozyme and 30 kDa mono-PEGylated lysozyme the binding capacities were lower. In separation experiments on a TSKgel Butyl-NPR hydrophobic-interaction column with ammonium sulfate as mobile phase, the elution order was: native lysozyme, 5 kDa mono-PEGylated lysozyme and oligo-PEGylated lysozyme. This elution order was found to be reversed when sodium chloride was used. Furthermore, the resolution of the three mono-PEGylated forms was not possible with this column and ammonium sulfate as mobile phase. In 4 M sodium chloride a resolution of all PEGylated lysozyme forms was achieved. A tentative explanation for these phenomena can be the increased solvation of the PEG polymers in sodium chloride which changes the usual attractive hydrophobic forces in ammonium sulfate to more repulsive hydration forces in this hydrotrophic salt.