Determination of diffusion coefficients of proteins in stationary phases by frontal chromatography

A strategy to determine effective diffusion coefficients of proteins in chromatographic gels is presented in this article. An experimental methodology based on frontal liquid chromatography was combined with a numerical methodology based on a mathematical model describing the chromatographic process including the extra-column dispersion, the dispersion due to the packed bed, the external mass transfer from the bulk phase to the stationary phase, and the diffusive transport within the stationary phase. The methodology has several advantages compared to previously reported methods to determine diffusion coefficients in that no other equipment than an HPLC is required, any class of stationary phases can be investigated as long as the experiments are performed under non-binding conditions, and no modification, e.g., moulding of slabs or membranes, to the stationary phase is required. To show the applicability of the methodology, the effective diffusion coefficients of lysozyme, bovine serum albumin, and immunoglobulin gamma in Sepharose CL-4B were determined and shown to be comparable with those determined with other methods.     

The Investigation of Protein Diffusion via H-Cell Microfluidics

In this study, we developed a microfluidics method, using a so-called H-cell microfluidics device, for the determination of protein diffusion coefficients at different concentrations, pHs, ionic strengths, and solvent viscosities. Protein transfer takes place in the H-cell channels between two laminarly flowing streams with each containing a different initial protein concentration. The protein diffusion coefficients are calculated based on the measured protein mass transfer, the channel dimensions, and the contact time between the two streams. The diffusion rates of lysozyme, cytochrome c, myoglobin, ovalbumin, bovine serum albumin, and etanercept were investigated. The accuracy of the presented methodology was demonstrated by comparing the measured diffusion coefficients with literature values measured under similar solvent conditions using other techniques. At low pH and ionic strength, the measured lysozyme diffusion coefficient increased with the protein concentration gradient, suggesting stronger and more frequent intermolecular interactions. At comparable concentration gradients, the measured lysozyme diffusion coefficient decreased drastically as a function of increasing ionic strength (from zero onwards) and increasing medium viscosity. Additionally, a particle tracing numerical simulation was performed to achieve a better understanding of the macromolecular displacement in the H-cell microchannels. It was found that particle transfer between the two channels tends to speed up at low ionic strength and high concentration gradient. This confirms the corresponding experimental observation of protein diffusion measured via the H-cell microfluidics.     

Effects of protein conformational changes on separation performance in electrostatic interaction chromatography: unfolded proteins and PEGylated proteins

As it is important to understand how protein conformational changes affect the separation performance in ion exchange chromatography (IEC), we investigated two model systems, unfolded proteins (lysozyme and bovine serum albumin) with urea and dithiothreitol, and PEGylated proteins (lysozyme attached with polyethyleneglycol molecular weight 5000). Linear gradient elution IEC experiments were carried out and the data were analysed by our model previously presented in order to obtain the binding site value B and the peak salt concentration I(R). Unfolded proteins (bovine serum albumin and lysozyme) with urea and dithiothreitol showed weaker retention and larger binding site values compared with the values for native proteins. Multiple PEGylated lysozyme peaks were separated, and eluted earlier than the native peak appeared. There is a good correlation between B and I(R) for PEGylated lysozymes.     

Generalized concentration dependence of globular protein self-diffusion coefficients in aqueous solutions.

The self-diffusion coefficients of globular proteins (myoglobin, bovine serum albumin, barstar, lysozyme) in aqueous solutions at different temperatures and pH values are obtained by pulsed-gradient spin-echo NMR, and their concentration dependence is analyzed. The generalized concentration dependence of globular protein self-diffusion coefficients is empirically established, and compared to the concentration dependence of diffusion coefficients of flexible polymers and rigid Brownian particles.     

Hydrodynamics and mass transfer in two-phase aqueous extraction using spray columns

Spray columns can be used to isolate and purify proteins using the two-phase aqueous extraction technique based on polyethylene glycol (PEG) and dextran. The fractional dispersed phase (PEG) holdup and overall mass transfer coefficients were measured in a 9.7 mm i.d. spray column. We found that the dispersed phase holdup increased with increasing PEG phase velocity. The overall mass transfer coefficients for bovine serum albumin, normalized for the PEG holdup, were found to be independent of the PEG phase velocity. This result was expected, since true mass transfer coefficients do not vary with phase velocity.     

Transport of low molecular weight substances in protein crystals and films

Diffusion of synthetic dyes in lysozyme crystals and bovine serum albumin (BSA) amorphous films is shown to depend on the size of diffusate molecule far more strongly than in solutions. The diffusion coefficient increases from 10(-8) to 10(-13) cm2 sec-1 on about 1.5 divided by 2--fold increase in diffusate dimensions, the diffusion being completely arrested on reaching the limiting dimensions of about 14 A and 12 A for BSA films and lysozyme crystals, respectively. The diffusion in lysozyme crystals is anisotropic, with a diffusion maximum directed along their screw axes. The diffusion coefficient dependence on the charge of diffusate and its binding to proteins is discussed.     

Toward improving selectivity in affinity chromatography with PEGylated affinity ligands: The performance of PEGylated protein A

Chemical modification of macromolecular affinity chromatography ligands with polyethylene glycol chains or “PEGylation” can potentially improve selectivity by sterically suppressing non‐specific binding interactions without sacrificing binding capacity. For a commercial protein A affinity media and with yeast extract (YE) and fetal bovine serum (FBS) serving as mock contaminants, we found that the ligand accounted for more than 90% of the media‐associated non‐specific binding, demonstrating an opportunity for improvement. The IgG static binding affinity of protein A mono‐PEGylated with 5.0 and 20.7 kDa poly(ethylene glycol) chains was found to be preserved using a biomolecular interaction screening platform. Similar in situ PEGylations of the commercial protein A media were conducted and the modified media was functionally characterized with IgG solutions spiked with YE and FBS. Ligand PEGylation reduced the mass of media‐associated contaminants by a factor of two to three or more. Curiously, we also found an increase of up to 15% in the average recovery of IgG on elution after PEGylation. Combined, these effects produced an order of magnitude increase in the IgG selectivity on average when spiked with YE and a two‐ to three‐fold increase when spiked with FBS relative to the commercial media. Dynamic binding capacity and mass‐transfer resistance measurements revealed a reduction in dynamic capacity attributed to a decrease in IgG effective pore diffusivity and possibly slower IgG association kinetics for the PEGylated protein A ligands. Ligand PEGylation is a viable approach to improving selectivity in affinity chromatography with macromolecular ligands. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1364–1379, 2014     

Interaction mechanism of mono-PEGylated proteins in electrostatic interaction chromatography

The retention and binding mechanisms in electrostatic interaction-based chromatography (ion-exchange chromatography) of PEGylated proteins (covalent attachment of polyethylene glycol chains to protein) were investigated using our previously developed model. Lysozyme and bovine serum albumin were chosen as model proteins. The retention volume of PEGylated proteins shifted to lower elution volumes with increasing PEG molecular weight compared with the non-modified (native) protein retention volume. However, PEGylation did not affect the number of binding sites appreciably. The enzyme activity of PEGylated lysozyme measured with a standard insoluble substrate in suspension decreased considerably, whereas the activity with a soluble small-molecule substrate did not drop significantly. These findings indicate that when a protein is mono-PEG-ylated, the binding site is not affected and the elution volume reduces due to the steric hindrance between PEGylated protein and ion-exchange ligand.     

Purification and characterization of mouse interferon with novel affinity sorbents.

Several novel selective sorbents for mouse interferon are described that exploit the hydrophobic property and glycoprotein nature of this molecule. Low-molecular-weight ligands (hydrocarbons) and high-molecular-weight ligands (bovine serum albumin) immobilized on agarose bind selectively mouse L-cell interferon. The high selectivity of binding is due primarily to a hydrophobic effect, although electrostatic forces are also apparently involved. Mouse L-cell interferon binds to immobilized serum albumin and can be completely recovered by raising the ionic strength of the eluant. The specific activity of interferon preparations can be increased 2,000-fold to a value of 3 x 10(8) reference units per mg of protein in a single step with full recovery of the antiviral activity. A selective adsorption, although to a lesser degree, can be also obtained on hydrocarbon-coated agarose (Affi-Gel 202), resulting in 300-fold purification on desorption. The existence of two major components of mouse interferon was revealed upon its chromatography on the following sorbents: (i) bovine serum albumin-agarose, (ii) omega-carboxypentyl-agarose; and (iii) Bandeiraea simplicifolia lectin-agarose. This report thus provides for the first time a means for efficient and clear-cut separation of interferon components, thus enabling their further characterization.     

Numerical inversion of the Perrin equations for rotational and translational diffusion constants by iterative techniques.

An iterative numerical technique is presented which allows the semiaxes for prolate and oblate ellipsoids to be determined from the Perrin equations for rotational and translational diffusion constants. The use of this inversion technique is illustrated by application to the proteins: lysozyme, bovine serum albumin, human transferrin, and bovine rhodopsin solubilized in digitonin.     

The measurement of insoluble proteins using a modified Bradford assay

A technique for determining the amount of thermally denatured, insoluble protein is described. The assay has been validated using four globular proteins, bovine serum albumin, beta-lactoglobulin, lysozyme, and ovalbumin. It consists of a resolubilization protocol, using 8 M urea and 5% 2-mercaptoethanol, linked to the Bradford dye binding assay. The resolubilization protocol was carried out at 100 degrees C to enable complete recovery of all insoluble proteins. Beta-Lactoglobulin resolubilization was completed after heating for 1 min, whereas samples of bovine serum albumin, lysozyme, and ovalbumin required heating for 1.5 min. The assay can measure protein concentrations as small as 10 micrograms, typically with standard deviations of 3%, thus comparing favorably with the standard Bradford assay. Other types of denaturation, such as chemical denaturation causing subsequent insolubility, may be studied with this technique providing that there is no interference with the Bradford assay.     

Binding capacity differences for antibodies and Fc-fusion proteins on protein A chromatographic materials

A range of studies were carried out to investigate the underlying reason for differences in dynamic binding capacities observed with various antibodies and Fc-fusion proteins during Protein A chromatography. Dynamic binding capacities were determined for these biomolecules using different protein A stationary phase materials. SEC was carried out to determine the relative sizes of the antibodies and fusion proteins. Pore diffusivities and static binding capacities were also determined on these Protein A resin materials. Trends in the dynamic binding capacities for these molecules did not correlate with differences in pore diffusion coefficients as might be expected for a mass transfer limited system. Instead, dynamic binding capacities were seen to follow the same trends as the static binding capacities and the apparent size of the molecules. Differences in static binding capacities were attributed to be due to differences in steric factor between the molecules. Solution binding stoichiometry studies were employed to estimate intra-Protein A steric effects while binding to the various domains within a Protein A ligand. In addition, steric hindrance was also found to exist between adjacent immobilized Protein A ligands on the chromatographic surface. The combination of intra and inter Protein A steric hindrances can explain differences in binding capacities observed between various antibody and Fc fusion proteins. The effect of Protein A ligand density on these supports was also examined and the results indicate that increasing Protein A ligand density leads to a situation of diminishing returns for binding capacity due to increased steric hindrance on the resin surface. The results presented in this paper show that steric hindrances can dominate over mass transfer effects in causing capacity variation between different molecules on the same stationary phase. This can lead to the development of more cost-efficient chromatographic stationary phases as well as provide information during the selection of Protein A media for preparative purification of monoclonal antibodies and Fc fusion proteins.     

Breakthrough performance of stacks of dye-cellulosic fabric in affinity chromatography of lysozyme

The breakthrough performance of stacks of dye-cellulosic fabric in affinity chromatography of lysozyme was investigated in batch and flow experiments. Breakthrough curves were significantly affected by flow rate and were not dependent on the feed solution concentration. System dispersion curves could not explain the flow-rate dependence. Breakthrough curves were analyzed by coupling the kinetic model for pore mass transfer as the only controlling resistance and a system dispersion model. From the analysis, pore film mass transfer resistance was found to be the leading rate-limiting factor when the residence time in the column is greater than 5 min. The model was used to predict the operating and design parameters needed to obtain sharp breakthrough curves. Selectivity studies using lysozyme and bovine serum albumin mixtures showed a high system selectivity for lysozyme.     

Affinity extraction of proteins with a reversed micellar system composed of Cibacron Blue-modified lecithin

Crude soybean lecithin was used as a novel surfactant to form reversed micelles in n-hexane. Cibacron Blue F-3GA (CB) was directly immobilized to the reversed micelles by a two-phase reaction. The reversed micellar system without CB showed low solubilizing capacity for low molecular weight proteins, lysozyme, and cytochrome c due to the weak electrostatic interactions. The introduction of CB significantly increased the solubilization of lysozyme because of its affinity binding to CB but showed no effect on the solubilization of cytochrome c since it did not bind to CB. Although bovine serum albumin had an affinity for CB, it was not extracted to the reversed micelles containing CB because its high molecular weight resulted in a significant steric hindrance effect. Thus the reversed micellar system had a high selectivity resulting from both biospecific and steric hindrance effects. The extraction yield of lysozyme decreased significantly with increasing ionic strength. Therefore, the back extraction of lysozyme was carried out using a stripping solution with an ionic strength of 0.865 mol/L. The overall recovery yield of lysozyme after back extraction could be increased to 87% by stripping for 2 h. The recovered lysozyme exhibited an activity equivalent to native lysozyme, and its secondary structure was also unchanged.     

Preferential hydration and solubility of proteins in aqueous solutions of polyethylene glycol

This paper is focused on the local composition around a protein molecule in aqueous mixtures containing polyethylene glycol (PEG) and the solubility of proteins in water + PEG mixed solvents. Experimental data from literature regarding the preferential binding parameter were used to calculate the excesses (or deficits) of water and PEG in the vicinity of β-lactoglobulin, bovine serum albumin, lysozyme, chymotrypsinogen and ribonuclease A. It was concluded that the protein molecule is preferentially hydrated in all cases (for all proteins and PEGs investigated). The excesses of water and deficits of PEG in the vicinity of a protein molecule could be explained by a steric exclusion mechanism, i.e. the large difference in the sizes of water and PEG molecules.

The solubility of different proteins in water + PEG mixed solvent was expressed in terms of the preferential binding parameter. The slope of the logarithm of protein (lysozyme, β-lactoglobulin and bovine serum albumin) solubility versus the PEG concentration could be predicted on the basis of experimental data regarding the preferential binding parameter. For all the cases considered (various proteins, various PEGs molecular weights and various pHs), our theory predicted that PEG acts as a salting-out agent, conclusion in full agreement with experimental observations. The predicted slopes were compared with experimental values and while in some cases good agreement was found, in other cases the agreement was less satisfactory. Because the established equation is a rigorous thermodynamic one, the disagreement might occur because the experimental results used for the solubility and/or the preferential binding parameter do not correspond to thermodynamic equilibrium.     

Purification of lysozyme by multistage affinity filtration

A multistage affinity filtration process was developed for the purification of proteins. An affinity adsorbent was prepared by immobilizing Cibacron Blue 3GA to TSK gel HW-65F. Adsorption equilibrium experiments showed that the blue TSK gel had a high affinity for lysozyme, while its binding to bovine serum albumin (BSA) was weaker. Using a three-stage affinity filtration system, lysozyme was purified from a model system (a mixture of lysozyme and BSA) and a natural source (chicken egg white). From the chicken egg white, the three-stage affinity filtration increased the recovery yield of lysozyme from 61 to 96%, compared with the one-stage process. A mathematical model taking into account the film and interior diffusions of protein and eluant was developed for the modeling and analysis of the experimental data. Both the experimental and modeling results indicate that the multistage affinity filtration technique can be employed for the selective recovery of proteins.     

A new approach to the depletion of albumin and immunoglobulin G from human serum

The use of proteomic analysis to find potential diagnostic biomarkers is limited by the presence of serum albumin (HSA) and immunoglobulin (IgG) at high concentrations in patients’ blood; these substances impede the detection of serum proteins with similar molecular weights. Recombinant HSA- and IgG-binding polypeptides are used as ligands in creating sorbents for complete removal of the proteins by affinity chromatography. The binding specificity of the sorbents for HSA and IgG is higher than that of the conventionally used antibodies. A composite sorbent enabling the depletion of HSA and IgG from serum by single-step affinity chromatography was obtained. The developed sorbents were used to prepare serum for proteomic analysis.     

Rate-limiting mass transfer in immunosorbents: characterisation of the adsorption of paraquat-protein conjugates to anti-paraquat Sepharose 4B

A series of paraquat-protein conjugates of different molecular size has been prepared by the coupling of paraquat hexanoate to the proteins lysozyme, ovalbumin, bovine serum albumin. The characteristics of the adsorption of these conjugates to an immunosorbent consisting of monoclonal anti-paraquat antibodies covalently immobilised to Sepharose 4B have been determined. Equilibrium adsorption isotherms were found to obey the Langmuir equation and indicated that 80% or more of the antibody binding sites were accessible to the conjugates. The rates of mass transfer of the conjugates to their adsorption sites on the immobilised antibodies was well described by a model in which mass transfer is controlled by transfer across the external film and diffusion within the porous adsorbent bead. The effective diffusivities of the conjugates within the immunosorbent were measured and has allowed the effect of the size of the adsorbing molecule on the rate of adsorption to be considered. The amount of paraquat that could be adsorbed and the rates of adsorption decreased as the size of the protein to which it is conjugated increased. The diffusivity of the conjugates within the pores of the adsorbent is reduced between two and five times compared to their diffusivities in free solution. The reduction is greater for the larger proteins and the variations of the effective diffusivities and the pore diffusivities with the molecular weight of the conjugate can be well described with simple correlations.     

Adsorption kinetics and equilibria of bovine serum albumin on rigid ion-exchange and hydrophobic interaction chromatography matrices in a stirred cell

Rigid adsorbents have advantages over soft gel media for downstream processing of proteins. The adsorption of bovine serum albumin (BSA) has been investigated on a rigid adsorbent based on a wide-pore, hydrophilically coated, silica-gel matrix. The effects of surface chemistry (weak anion exchanger and hydrophobic interaction chromatography) and particle size have been studied on the physical properties of the adsorbent and on the adsorption equilibria and adsorption kinetics. The rates of adsorption of BSA have been measured in a stirred cell and are found to be satisfactorily described by a two-step theoretical model, in which the mass transfer involves a pore diffusion resistance and an extra-particle film resistance. On the anion exchanger, the effective pore diffusivity decreases substantially with increasing protein concentration, approximately halving as the initial concentration rises from 0.7 to 2g/l. In the hydrophobic interaction chromatography medium, the pore diffusivity is less sensitive to protein concentration and is also reduced by a factor of about 4 by aggregation of the protein. Effective pore diffusivities with the "wide-pore" silica adsorbents in anion-exchange form are 36-94 times lower than the diffusivity in free solution and are comparable with the lower of the wide range of values published for soft gels.