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 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.     

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.     

Membrane ion-exchange chromatography for process-scale antibody purification

Microemulsion electrokinetic chromatography (MEEKC) was evaluated as a screening tool for the indirect measurement of octanol–water partition coefficients (log P_o/w) of pesticide compounds. Over 80 pesticide compounds representing a variety of structural characteristics were studied, and good correlation of log P_o/w with the logarithm of the retention factor was found. The microemulsion system studied allowed the separation of compounds in the log P_o/w range of −1 to 7. In addition, a smaller set of simple organic molecules that vary in structural features was evaluated and compared to the pesticide log P_o/w calibration. The pesticide and simple organic molecule log P_o/w calibration lines were statistically similar. This suggests that a universal set of standard compounds may be employed for the log P_o/w calibration to provide measurements for a variety of compounds with good accuracy.     

Binary adsorption of globular proteins on ion-exchange media

Adsorption equilibrium of binary pairs of lysozyme (LYS), cytochrome c (CYC) and ribonuclease A (RNase) has been measured on different cation-exchange media at various solution conditions. Adsorption patterns largely follow the intrinsic protein–surface interactions, but can differ significantly for different pairs or even for one pair at different solution conditions. LYS/CYC adsorption shows similar behavior on all the adsorbents examined, with competitive adsorption dominated by LYS and the presence of LYS reducing the adsorption of CYC significantly. Simultaneous and sequential measurements for LYS/CYC show that the order of adsorption does not have a significant effect on the adsorption equilibrium. For LYS/RNase, LYS is consistently more strongly adsorbed. For CYC/RNase, both proteins can display significant adsorption, depending on the pH and salt concentration. A model based on colloidal energetics is developed to calculate the binary adsorption isotherms using parameter values obtained from single-component isotherms. The calculated adsorption is in good agreement with experimental results, with significantly better representation than for other commonly used binary isotherms.     

Comparison of agarose and dextran-grafted agarose strong ion exchangers for the separation of protein aggregates

The control of aggregate levels in recombinant protein based drugs is a primary concern during process development and manufacture. In recent years, a novel class of dextran-grafted ion exchange matrices has gained popularity for process scale protein purification due to increased mass transfer rates and higher dynamic binding capacity compared to conventional matrices. Using bovine serum albumin and a monoclonal antibody as model proteins, we studied Sepharose FF and Sepharose XL ion exchangers for the separation of protein aggregates. Experimental results comparing linear gradient elution, stepwise elution, and flow-through chromatography for aggregate separation are described. Differences in performance for the various ion exchangers are discussed and modeled. Strategies for the optimization of protein aggregate separation are provided.     

Preparative weak cation-exchange chromatography of monoclonal antibody variants I. Single-component adsorption

The retention behavior of a monoclonal antibody has been characterized on a weak cation exchanger, Fractogel EMD COO(-)(s). This new generation of resin materials comprise of a higher mechanical strength compared to softer gel-type matrices while maintaining elevated capacities, resulting in higher productivity and longer lifetimes. These parameters are extremely important when working with large bio-molecules such as proteins, and in particular monoclonal antibodies. In the first part of this work a parameter estimation strategy is presented to fully characterize the retention behavior of a single monoclonal antibody and determine suitable model parameters. Literature correlations were used for the estimation of mass transfer rates. The transport limiting parameter, pore diffusion, was regressed experimentally. Various methods for the adsorption isotherm determination have been applied, their combinations resulting in little experimental effort and accurate predictions of elution profiles. The process has been modelled with a complete pore diffusion model and the agreement between experimental and predicted profiles is good in general. However, a very marked sensitivity to changes in the effective pore diffusion coefficient has been observed. A correlation describing the effect of the separation conditions on the diffusion rate is therefore needed in order to have a fully predictive mathematical model.     

Electrostatic model for protein adsorption in ion-exchange chromatography and application to monoclonal antibodies,lysozyme and chymotrypsinogen A

A model for the adsorption equilibrium of proteins in ion-exchange chromatography explicitly accounting for the effect of pH and salt concentration in the limit of highly diluted systems was developed. It is based on the use of DLVO theory to estimate the electrostatic interactions between the charged surface of the ion-exchanger and the proteins. The corresponding charge distributions were evaluated as a function of pH and salt concentration using a molecular approach. The model was verified for the adsorption equilibrium of lysozyme, chymotrypsinogen A and four industrial monoclonal antibodies on two strong cation-exchangers. The adsorption equilibrium constants of these proteins were determined experimentally at various pH values and salt concentrations and the model was fitted with a good agreement using three adjustable parameters for each protein in the whole range of experimental conditions. Despite the simplifications of the model regarding the geometry of the protein–ion-exchanger system, the physical meaning of the parameters was retained.     

A predictive approach to correlating protein adsorption isotherms on ion-exchange media

The equilibrium adsorption of three small basic proteins was measured on cation exchangers under various solution conditions and was used as the basis for developing a predictive approach for correlating adsorption behavior. A mechanistically based isotherm model is used to model the equilibrium adsorption so as to facilitate isotherm prediction using minimal experimental data. The model explicitly considers the contributions of protein-surface and protein-protein interactions, and decoupling them allows each to be correlated with different experimental measurements. Specifically, protein-surface interactions are related to chromatographic data in the form of the isocratic retention factor (k'), while protein-protein interactions are analyzed on the basis of high-coverage isotherm data on an arbitrary stationary phase. Analysis of experimental data within this framework reveals a high level of consistency. The model is also used to facilitate prediction of adsorption isotherms on other ion-exchange media using isotherms on one adsorbent.     

One-step purification of epigallocatechin gallate from crude green tea extracts by mixed-mode adsorption chromatography on highly cross-linked agarose media

(-)-Epigallocatechin gallate (EGCG) was purified in one step from a green tea polyphenol (GTP) crude extract by adsorption chromatography on a Superose 12 HR 10/30 column. The mobile phase used was a mixture of acetonitrile and water with an optimum mobile phase compositions regarding purity, recovery and separation time of 78/22 (v/v). Maximum practical sample loading was 100 mg GTP per run (corresponding to 4.2 mg/ml Superose). An EGCG purity of 99% with recoveries in the range 60-65% was achieved in one step directly from the crude GTP extract. Full column regeneration was obtained using solvents in the following order: 0.5 M NaOH, distilled water and 30% acetic acid.     

Cored anion-exchange chromatography media for antibody flow-through purification

Agarose-based anion-exchangers (e.g. quaternary amine, Q) have been widely used in monoclonal antibody flow-through purification to remove trace levels of impurities. Such media are often packed in a large column and the operation is usually robust but with limited throughput due to the compressibility of agarose and consequentially low bed permeability. In order to address this limitation, cored Q beads consisting of a rigid core and a thin agarose gel coating were developed and evaluated for protein flow-through chromatography. Using laboratory-scale columns it was found that, the cored beads indeed provide significantly enhanced rigidity and flow permeability relative to conventional homogeneous agarose resins. Depending on the structure and size of the cored beads, the permeability was 2-4-fold higher than that of a commonly used commercial agarose resin. Good virus and host cell protein clearance was achieved with the cored Q beads even at increased flow velocities. In addition, the impermeable core allows for more efficient use of buffers without loss of useful capacity in polishing applications. Process analyses based upon the experimental data demonstrated that the enhanced permeability achieved with the cored beads can significantly improve process throughput and economics.     

Studies of fish zona pellucida by high-performance ion-exchange chromatography on agarose columns and free zone electrophoresis

By either free zone electrophoresis or high-performance ion-exchange chromatography on DEAE agarose, zona pellucida from Baltic small herring (Clupea harengus L.) was separated into several fractions. These fractions had very similar protein compositions, since on polyacrylamide gel electrophoresis in sodium dodecyl sulphate they all gave the same pattern: chiefly one major and three minor bands corresponding to proteins with the following estimated molecular weights: 78 000, 96 000 (the major component), 115 000, and 125 000. It is likely that these proteins constitute the so-called supramolecular complexes of zona pellucida from Baltic small herring. Only one electrophoretic and one chromatographic fraction gave precipitin arcs on immunodiffusion with rabbit antiserum against zona pellucida from the fish Aristichthys nobilis (Richardson).     

The design of agarose beds for high-performance hydrophobic-interaction chromatography and ion-exchange chromatography which show increasing resolution with increasing flow rate

Agarose beads (agarose concentration: 15%; diameter: 15–70 μm) were shrunk, crosslinked and derivatized in organic solvents. As crosslinker and coupling agent γ-glycidoxypropyl tri-methoxysilane was used. Columns packed with nonporous beads of pentyl agarose and octyl agarose, prepared by this technique, were used for hydrophobic-interaction chromatography. Characteristic of these columns was that the resolution increased with an increase in flow rate (except for very low flow rates). This very attractive behavior, which violates the generally accepted theory of chromatography, was also exhibited by an ion-exchanger based on non-porous agarose beads.     

Incorporating water-release and lateral protein interactions in modeling equilibrium adsorption for ion-exchange chromatography

The equilibrium adsorption of two albumin proteins on a commercial ion exchanger has been studied using a colloidal model. The model accounts for electrostatic and van der Waals forces between proteins and the ion exchanger surface, the energy of interaction between adsorbed proteins, and the contribution of entropy from water-release accompanying protein adsorption. Protein-surface interactions were calculated using methods previously reported in the literature. Lateral interactions between adsorbed proteins were experimentally measured with microcalorimetry. Water-release was estimated by applying the preferential interaction approach to chromatographic retention data. The adsorption of ovalbumin and bovine serum albumin on an anion exchanger at solution pH>pI of protein was measured. The experimental isotherms have been modeled from the linear region to saturation, and the influence of three modulating alkali chlorides on capacity has been evaluated. The heat of adsorption is endothermic for all cases studied, despite the fact that the net charge on the protein is opposite that of the adsorbing surface. Strong repulsive forces between adsorbed proteins underlie the endothermic heat of adsorption, and these forces intensify with protein loading. It was found that the driving force for adsorption is the entropy increase due to the release of water from the protein and adsorbent surfaces. It is shown that the colloidal model predicts protein adsorption capacity in both the linear and non-linear isotherm regions, and can account for the effects of modulating salt.     

Purification of the isoflavonoid puerarin by adsorption chromatography on cross-linked 12% agarose

The isoflavonoid puerarin in extracts of the well-known traditional Chinese drug Radix puerariae (root of the plant Pueraria lobata) can be separated from other isoflavonoids by adsorption chromatography on the cross-linked 12% agarose gel Superose 12 equilibrated in distilled water. The adsorption is totally quenched by the addition of 50% acetic acid. The separation of the isoflavonoids is tentatively ascribed to interaction with the residues of the cross-linking reagents used in the manufacturing process of Superose 12. Thus, no useful separation can be achieved with non-cross-linked 12% agarose gel media. Symmetric elution profiles at high sample loadings (16 mg on a 24 ml column) suggest linear adsorption isotherms for the isoflavonoids.     

Optimization of monoclonal antibody purification by ion-exchange chromatography. Application of simple methods with linear gradient elution experimental data

Simple methods for the optimization of ion-exchange chromatography of proteins in our previous papers were applied to cation-exchange chromatography purification of monoclonal antibodies (Mab). We carried out linear gradient elution experiments, and obtained the data for the peak salt concentration and the peak width. From these data, the distribution coefficient as a function of salt concentration, and the height equivalent to a theoretical plate (HETP) as a function of mobile phase velocity were calculated. The optimized linear gradient elution conditions were determined based on the relationship between buffer consumption and separation time. The optimal stepwise elution conditions were determined based on the relationship between the distribution coefficient and the salt concentration.     

Properties and performance of novel high-resolution/high-permeability ion-exchange media for protein chromatography

There is continued interest in the development of stationary phases for protein chromatography that can provide high resolution at elevated flow rates of the mobile phase. When using porous particles, resolution and dynamic binding capacity decline rapidly as the flow rate is increased. Monolithic columns have been developed to overcome these limitations. However, there are difficulties in manufacturing homogeneous larger scale monoliths. In this paper we investigate the morphology and performance characteristics of columns based on new ion exchangers obtained by mechanically disrupting continuous beds of acrylamido-based polymeric media. Near colloidal suspensions of loose particles obtained with this procedure can be flow-packed in ordinary chromatography columns resulting in beds of unexpectedly high hydraulic permeability. Columns up to 2.2 cm in diameter were studied with both Q and S functionalized media. The hydraulic permeability and interparticle porosity of these columns were rather high. The permeabilities of the S and Q media were 1.5 x 10(-13) and 2.4 x 10(-13) m2, respectively, while the corresponding porosities were 60 and 70%. These porosity values are similar to those of monoliths, suggesting that these particles assemble under flow to give high-porosity bridged structures. The structure of these packed beds was further characterized by embedding small packed columns in resins and obtaining sections for microscopic observation. The sections reveal the presence of small aggregates of non-porous 1-3 microm particles, surrounded by flow channels several micrometers in size. The height equivalent to a theoretical plate under isocratic and gradient elution conditions and the dynamic binding capacity were determined for several proteins and were found to be virtually independent of flow.     

Exploration of overloaded cation exchange chromatography for monoclonal antibody purification

Cation exchange chromatography using conventional resins, having either diffusive or perfusive flow paths, operated in bind-elute mode has been commonly employed in monoclonal antibody (MAb) purification processes. In this study, the performance of diffusive and perfusive cation exchange resins (SP-Sepharose FF (SPSFF) and Poros 50HS) and a convective cation exchange membrane (Mustang S) and monolith (SO(3) Monolith) were compared. All matrices were utilized in an isocratic state under typical binding conditions with an antibody load of up to 1000 g/L of chromatographic matrix. The dynamic binding capacity of the cation exchange resins is typically below 100 g/L resin, so they were loaded beyond the point of anticipated MAb break through. All of the matrices performed similarly in that they effectively retained host cell protein and DNA during the loading and wash steps, while antibody flowed through each matrix after its dynamic binding capacity was reached. The matrices differed, though, in that conventional diffusive and perfusive chromatographic resins (SPSFF and Poros 50HS) demonstrated a higher binding capacity for high molecular weight species (HMW) than convective flow matrices (membrane and monolith); Poros 50HS displayed the highest HMW binding capacity. Further exploration of the conventional chromatographic resins in an isocratic overloaded mode demonstrated that the impurity binding capacity was well maintained on Poros 50HS, but not on SPSFF, when the operating flow rate was as high as 36 column volumes per hour. Host cell protein and HMW removal by Poros 50HS was affected by altering the loading conductivity. A higher percentage of host cell protein removal was achieved at a low conductivity of 3 mS/cm. HMW binding capacity was optimized at 5 mS/cm. Our data from runs on Poros 50HS resin also showed that leached protein A and cell culture additive such as gentamicin were able to be removed under the isocratic overloaded condition. Lastly, a MAb purification process employing protein A affinity chromatography, isocratic overloaded cation exchange chromatography using Poros 50HS and anion exchange chromatography using QSFF in flow through mode was compared with the MAb's commercial manufacturing process, which consisted of protein A affinity chromatography, cation exchange chromatography using SPSFF in bind-elute mode and anion exchange chromatography using QSFF in flow through mode. Comparable step yield and impurity clearance were obtained by the two processes.     

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.     

Investigation of the adsorption behavior of glycine peptides on 12% cross-linked agarose gel media

The highly cross-linked 12% agarose gel Superose 12 10/300 GL causes retardation of glycine peptides when mobile phases containing varying concentrations of acetonitrile in water are used. An investigation has been made into the retention mechanism behind this retardation using the glycine dipeptide (GG) and tripeptide (GGG) as models. The dependence of retention times of analytical-size peaks under different experimental conditions was interpreted such that the adsorption most probably was caused by the formation of hydrogen bonds but that electrostatic interactions cannot be ruled out. Thereafter, a nonlinear adsorption study was undertaken at different acetonitrile content in the eluent, using the elution by characteristic points (ECPs) method on strongly overloaded GG and GGG peaks. With a new evaluation tool, the adsorption energy distribution (AED) could be calculated prior to the model selection. These calculations revealed that when the acetonitrile content in the eluent was varied from 0% to 20% the interactions turned from (i) being homogenous (GG) or mildly heterogeneous (GGG), (ii) via a more or less stronger degree of heterogeneity around one site to (iii) finally a typical bimodal energy interaction comprising of two sites (GG at 20% and GGG at 10% and 20%). The Langmuir, Tóth and bi-Langmuir models described these interesting adsorption trends excellently. Thus, the retardation observed for these glycine peptides is interpreted as being of mixed-mode character composed of electrostatic bonds and hydrogen bonds.