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.     

Poly(glycidyl methacrylate)‐grafted hydrophobic charge‐induction agarose resins with 5‐aminobenzimidazole as a functional ligand

Hydrophobic charge‐induction chromatography is a new technology for antibody purification. To improve antibody adsorption capacity of hydrophobic charge‐induction resins, new poly(glycidyl methacrylate)‐grafted hydrophobic charge‐induction resins with 5‐aminobenzimidazole as a functional ligand were prepared. Adsorption isotherms, kinetics, and dynamic binding behaviors of the poly(glycidyl methacrylate)‐grafted resins prepared were investigated using human immunoglobulin G as a model protein, and the effects of ligand density were discussed. At the moderate ligand density of 330 μmol/g, the saturated adsorption capacity and equilibrium constant reached the maximum of 140 mg/g and 25 mL/mg, respectively, which were both much higher than that of non‐grafted resin with same ligand. In addition, effective pore diffusivity and dynamic binding capacity of human immunoglobulin G onto the poly(glycidyl methacrylate)‐grafted resins also reached the maximum at the moderate ligand density of 330 μmol/g. Dynamic binding capacity at 10% breakthrough was as high as 76.3 mg/g when the linear velocity was 300 cm/h. The results indicated that the suitable polymer grafting combined with the control of ligand density would be a powerful tool to improve protein adsorption of resins, and new poly(glycidyl methacrylate)‐grafted hydrophobic charge‐induction resins have a promising potential for antibody purification applications.     

Effects of ligand density and pore size on the adsorption of bovine IgG with DEAE ion-exchange resins

Immunoglobulin G is an important plasma protein with many applications in therapeutics and diagnostics, which can be purified effectively by ion exchange chromatography. The ligand densities and pore properties of ion-exchange resins have significant effects on the separation behaviors of protein, however, the understandings are quite limited. In this work, with bovine immunoglobulin as the model IgG, the adsorption isotherms and adsorption kinetics were investigated systematically with series of diethylaminoethyl ion-exchange resins with different ligand densities and pore sizes. The Langmuir equation and pore diffusion model were used to fit the experimental data. The influences of ligand density and pore size on the saturated adsorption capacity, the dissociation constant and the effective diffusivity were discussed. The adsorption capacities increased with the increase of ligand density and the decrease of pore size, and an integrative parameter was proposed to describe the combined effects of ligand density and pore size. It was also found that the effective pore diffusion coefficient of the adsorption kinetics was influenced by pore sizes of resins, but was relatively independent on the ligand densities of resins. For a given protein, the ligand density and pore size should be optimized for improving the protein adsorption.     

Role of the ligand density in cation exchange materials for the purification of proteins

The performance of functionalized materials, such as cation exchange resins, is dependent not only on the ligand type and ligand density, but also on the pore accessibility of the target molecule. In the case of large molecules such as antibodies this latter parameter becomes crucial, because the size of such molecules falls somewhere inside the pore size distribution of the resin. The influence of the ligand density and accessibility on the overall performance of the material is explored systematically. Five different materials, having the same chemistry as the strong cation exchange resin Fractogel EMD SO₃⁻ (M) , have been analyzed. These materials only differ in the ligand density. It is shown that the ligand density directly influences the porosity of the materials as well as the pore diffusivity and the dynamic binding capacity. For a given purification problem an optimal ligand density can be found. Based on the above results a new material is proposed, showing superior properties in terms of dynamic binding capacity. This is achieved by an optimization of the ligand density and by a decrease of the particle size of the stationary phase. The material properties are modeled with a general rate model. Further simulations were conducted to evaluate the performance of the new material in comparison with a conventional resin.     

Understanding and mitigating conductivity transitions in weak cation exchange chromatography

Large conductivity fluctuations were observed during a high pH wash step in a weak cation exchange chromatography process. These conductivity transitions resulted in a conductivity drop during pH increase and a conductivity rise during pH decrease. In some cases, the absolute conductivity change was greater than 6 mS/cm which was sufficient to affect target protein retention on the column. Further investigation revealed that wash buffer concentration, resin ligand density, and resin ligand pK have a profound effect on the magnitude of the conductivity transitions and the shape of corresponding pH traces. A potentiometric electrode selective for sodium ions was used to measure effluent counterion concentrations from two preparative resins during high pH washes, and the number of exchangeable counterions was compared to predictions made using ion exchange equilibrium theory. Results from this analysis show that conductivity transitions can be effectively mitigated without compromising process performance by optimizing the trade-off between wash buffer concentration and wash phase duration.     

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.     

Comparison of standard and new generation hydrophobic interaction chromatography resins in the monoclonal antibody purification process

Recent efforts to improve hydrophobic interaction chromatography (HIC) for use in monoclonal antibody (mAb) purification have focused on two approaches: optimization of resin pore size to facilitate mAb mass transport, and use of novel hydrophobic charge induction (HCIC) mixed mode ligands that allow capture of mAbs under low salt conditions. We evaluated standard HIC and new generation HIC and HIC-related chromatography resins for mAb purification process efficiency and product quality both as isolated chromatography steps and in purification process trains. We find that HIC resins with optimized pore size have significantly improved binding capacity which can increase HIC purification unit operation efficiency. The HCIC Mercapto-Ethyl-Pyridine (MEP) resin, which shows a different salt impact trend and impurity resolution pattern from standard HIC resin, can not only capture mAb from crude CHO fermentation supernatant but also substantially enhance mAb purification process flow efficiency when serving as a polishing role.     

Protein adsorption and transport in dextran-modified ion-exchange media. III. Effects of resin charge density and dextran content on adsorption and intraparticle uptake

Custom-synthesized variants of the commercial Capto S resin were used to examine the effects of resin charge density and dextran content on protein adsorption and intraparticle uptake. For the small protein lysozyme, resin charge density had the greatest effect on equilibrium capacity, consistent with calculations suggesting that lysozyme capacity should be limited by the available charge on the resin. Isocratic retention data and confocal microscopy imaging for this protein revealed a consistent ordering of the resins linking stronger protein-resin interactions with higher static capacities but slower intraparticle uptake rates over the range of properties studied. For the larger protein lactoferrin, it was found that increasing dextran content led to increased protein exclusion from the dextran layer, but that increasing resin charge density helped overcome the exclusion, presumably due to the increased electrostatic attraction between the resin and protein. Collectively examining the lysozyme and lactoferrin data along with information from previous studies suggests that a trade-off in maximizing dynamic capacities should exist between static capacities that increase to a finite extent with increased resin charge density and uptake rates that decrease with increased charge density. Column breakthrough data for lysozyme and lactoferrin appear to support the hypothesis, though it appears that whether a resin charge density is low or high must be considered in relation to the protein charge density. Using these trends, this work could be useful in guiding resin selection or design.     

Absorbents as packing materials in on-line coupling of reversed phase liquid chromatography and gas chromatography via a programmed temperature vaporizer

Dynamic binding capacity (DBC) measurements of cation-exchange resins were performed with two human monoclonal antibodies. DBC showed a pH dependent maximum, which was shifted to lower pH values with increasing buffer concentrations and increasing salting-out effect of the buffer anion according to the Hofmeister series. As this downshift correlates well with zeta potential values, a measurement of the latter allows the determination of the pH value for maximum DBC under a given set of conditions. Thus, the use of zeta potential values can accelerate the purification process development and helps to understand the protein adsorption mechanism.     

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.     

Some ion-exchange resins for anion-chromatography

The quaternary ammonium salts, n-butyltrimethylammonium iodide, 1,1,3,3-tetramethylbutyltrimethylammonium iodide, n-octadecyltrimethylammonium iodide and tri-n-dodecylmethylammonium iodide were synthesized from commercially available amines and together with n-hexadecyltrimethylammonium bromide tested for retention by a series of macroreticular resins (XAD-2, XAD-4, XAD-7, XAD-8 and XAD-11) for use as "surface" ion-exchangers in the chromatography of anions. Exchange-capacity studies of the coated resins showed that the non-polar XAD-2 and XAD-4 resins had retention characteristics superior to those of the polar resins and that pore size in the resin was more important than surface area per unit weight of resin. Tri-n-dodecylmethylammonium salts in XAD-2 gave the highest exchange capacity, with best retention under elution conditions. Columns prepared from this anion-exchanger were used to separate and analyse simple mixtures of anions (chloride, nitrate and sulphate) each within the 1-30 ppm range, by single-column operation with indirect photometric detection and also by conductivity detection with background-ion suppression. Though of use for the determination of anions in simple mixtures, the resolution and performance were generally poorer than those displayed by a commercial (Dionex) column. This is at least partly attributable to the inferior column-packing properties of the granular XAD-resin.     

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.     

Effects of porous polystyrene resin parameters on Candida antarctica lipase B adsorption, distribution, and polyester synthesis activity

Polystyrene resins with varied particle sizes (35 to 350-600 microm) and pore diameters (300-1000 A) were employed to study the effects of immobilization resin particle size and pore diameter on Candida antarctica Lipase B (CALB) loading, distribution within resins, fraction of active sites, and catalytic properties for polyester synthesis. CALB adsorbed rapidly (saturation time 相似文献   

Evaluation of multi-modal high salt binding ion exchange materials

The performance and selectivity of novel cation and anion exchange multi-modal chromatographic materials were evaluated. Desorption profiles of 13 proteins possessing a range of properties (e.g. size, charge and hydrophobicity) were determined on the cation exchange materials. Batch experiments were carried out by loading individual proteins on each resin at low salt, and examining the desorption of the proteins during sequential washes with increasing salt concentrations. While all of the resins exhibited some binding of proteins at elevated salt concentrations, this effect was more pronounced on the resins with aromatic ligands as compared to the materials with aliphatic ligands. As expected, materials with higher ionic capacities exhibited higher binding at elevated salts. In addition, some proteins exhibited high binding at elevated salt concentrations to all of the resins. The combined effect of charge and other secondary interactions with these multi-modal chromatographic materials enables high salt binding of a range of proteins as well as unique selectivities for the recovery of certain classes of proteins. Since the anion exchange materials all exhibited high binding at elevated salt concentrations the work with these materials focused on a study of elution strategies to remove proteins from these aromatic based materials. After evaluating various elution protocols, a combined strategy of pH change and chaotropic salt were shown to minimize electrostatic and hydrophobic interactions and was found to be an effective elution strategy for this class of anion exchange materials using peanut lectin as a model protein.     

丙烯酸在强碱阴离子交换树脂中的聚合

研究了以水为介质，丙烯酸在强碱阴离子交换树脂中聚合时羧基分配系数随浸泡时间和反应时间的变化，以及树脂残阴交换量随反应时间的变化，结果是羧基分配系数在浸泡一小时后达到平衡，聚合反应开始后分配系数又迅速增大，三小时后达到最大值。残阴交换量随反应进行而减小，三小时后达到最小值。经分析认为丙烯酸的主要聚合场所是在树脂相，聚合过程中单体不断向树脂相扩散，基本不在液相聚合。     

Characterization of p-aminobenzamidine-based sorbent and its use for high-performance affinity chromatography of trypsin-like proteases

An affinity sorbent, hydrophilic polymer-based carrier of different pore size (Toyopearl) with immobilized p-aminobenzamidine (ABA), has been prepared. Its basic properties and some applications for protein purification were studied. ABA, which is a synthetic inhibitor for trypsin-like proteases, was covalently immobilized to Toyopearl by reductive amination. The ligand density and binding capacity for porcine trypsin varied depending on the pore size of Toyopearl. The maximum binding capacity of the immobilized p-aminobenzamidine Toyopearl (ABA-Toyopearl) for trypsin was more than 40 mg/ml gel. ABA-Toyopearl thus obtained was very stable below pH 8 and was successfully used for high-performance affinity chromatography of trypsin-like proteases such as trypsin, thrombin, tissue-type plasminogen activator or urokinase in a single step at 25 degrees C.     

Dynamic adsorption of diarrhetic shellfish poisoning (DSP) toxins in passive sampling relates to pore size distribution of aromatic adsorbent

Solid-phase adsorption toxin tracking (SPATT) technology was developed as an effective passive sampling method for dissolved diarrhetic shellfish poisoning (DSP) toxins in seawater. HP20 and SP700 resins have been reported as preferred adsorption substrates for lipophilic algal toxins and are recommended for use in SPATT testing. However, information on the mechanism of passive adsorption by these polymeric resins is still limited. Described herein is a study on the adsorption of OA and DTX1 toxins extracted from Prorocentrum lima algae by HP20 and SP700 resins. The pore size distribution of the adsorbents was characterized by a nitrogen adsorption method to determine the relationship between adsorption and resin porosity. The Freundlich equation constant showed that the difference in adsorption capacity for OA and DTX1 toxins was not determined by specific surface area, but by the pore size distribution in particular, with micropores playing an especially important role. Additionally, it was found that differences in affinity between OA and DTX1 for aromatic resins were as a result of polarity discrepancies due to DTX1 having an additional methyl moiety.     

Ion-exchange resins in non-aqueous solvents-I Sorption rates of p-nitroaniline and the effects of small amounts of water

The rates of sorption of p-nitroaniline onto three hydrogenform resins in methanol, ethanol, n-propanol, isopropanol, n-butanol, acetonitrile, benzene, acetic acid and dioxan are reported. Two of the resins are typical gel-type, microreticular, sulphonated resins and the third is a new, highly porous and rigid, macroreticular, sulphonated resin, Amberlyst 15. There appears to be a correlation between viscosity or dielectric constant and the time for maximum sorption or maximum distribution coefficient when the alcohols are used, but no correlation for all the solvents is apparent. The macroreticular resin still functions when dry, even in the presence of non-polar solvents, but the microreticular resin does not. Small amounts of water present in the solvent or resin aid the sorption of the amine onto both types of resin. The effect of mesh size and cross-linkage are examined.     

多孔离子交换树脂内Eu3+离子的分步扩散

用同位素交换法研究了Eu³⁺离子在D72和D751树脂内的扩散过程.应用分步孔道扩散方程将粒内有效扩散系数D_e分解为孔道扩散系数D_p和固相扩散系数D_g,表明该方程可用于描述多孔树脂内的动力学过程.实验表明,D_e、D_p、D_g均随反应温度的升高而增大.计算了实验条件下的Eu³⁺的自扩散活化能;D72树脂的D_p和D_g对温度的响应比D751树脂大,其D_e、D_p、D_g值亦均大于D751树脂;Eu³⁺在溶液中的自扩散系数D_s＞D_p,说明离子在树脂孔道内的自扩散不能完全等同于其在溶液中的自扩散.     

Fine Pore-Structure Engineering by Ligand Conformational Control of Naphthalene Diimide-Based Semiconducting Porous Coordination Polymers for Efficient Chemiresistive Gas Sensing

Exploring new porous coordination polymers (PCPs) that have tunable structure and conductivity is attractive but remains challenging. Herein, fine pore structure engineering by ligand conformation control of naphthalene diimide (NDI)-based semiconducting PCPs with π stacking-dependent conductivity tunability is achieved. The π stacking distances and ligand conformation in these isoreticular PCPs were modulated by employing metal centers with different coordination geometries. As a result, three conjugated PCPs (Co−pyNDI, Ni−pyNDI, and Zn−pyNDI) with varying pore structure and conductivity were obtained. Their crystal structures were determined by three-dimensional electron diffraction. The through-space charge transfer and tunable pore structure in these PCPs result in modulated selectivity and sensitivity in gas sensing. Zn−pyNDI can serve as a room-temperature operable chemiresistive sensor selective to acetone.