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.     

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.     

Modeling of bispecific antibody elution in mixed‐mode cation‐exchange chromatography

The increasing demand for cost‐efficient manufacturing of biopharmaceuticals has been the main driving force for the development of novel chromatography resins, which resulted in the development of multimodal or mixed‐mode chromatographic resins. Most of them combine electrostatic and hydrophobic functionalities and are designed to deliver unique selectivity and increased binding capacities also at increased ionic strength. However, the mechanism of the protein–resin interaction in mixed‐mode chromatography is still not fully understood. The performance of protein separations in mixed‐mode chromatography is consequently difficult to predict. In this work, we present a model combining both salt and pH dependence to characterize and to predict protein retention in mixed‐mode chromatography. The model parameters are determined based on simple linear pH gradient elution experiments at different ionic strengths and they are directly transferable for the prediction of salt‐induced elution at fixed pH. Validity of the model is demonstrated for a bispecific antibody and its product‐related impurities.     

Colloidal stability of dextran-modified latex particles toward adsorption of concanavalin A

The colloidal stability of the dextran-modified poly(methyl methacrylate) (PMMA) latex particles toward adsorption of a carbohydrate-binding protein, concanavalin A (Con A), is primarily controlled by the charge neutralization mechanism. Formation of a crosslinked network structure via the specific affinity interactions between the dimeric Con A molecules and the dextran molecules anchored onto different latex particles may also have an impact on the coagulation kinetics. Judging from the data of coagulation kinetics, the colloidal stability of the latex particles toward added Con A in the decreasing order is: latex particles without dextran modification>latex particles with a dextran content of 2.15%>latex particles with a dex-tran content of 1.24% based on total polymer weight (PMMA+grafted dextran). The coagulation mechanisms involved in the adsorption of Con A onto the latex particles have been proposed to explain these experimental data. Charge neutralization of the negatively charged latex particles by adsorption of the positively charged Con A is the predominant destabilization mechanism. The ratio of the number of dextran active sites to that of Con A molecules plays an important role in the formation of the crosslinked network structure. The electrolytes in water cause a reduction in the electrostatic repulsion force among the interactive latex particles, but this ionic strength effect is not significant in comparison with charge neutralization. Received: 22 October 1997 Accepted: 24 December 1997     

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.     

Abnormality in using cyclic fatigue for ranking static fatigue induced slow crack growth behavior of polyethylene pipe grade resins

Slow crack growth behavior in polyethylene pipe grade resins were studied using both static fatigue (stress-rupture) and cyclic fatigue tests. This was done to better understand the applicability of cyclic fatigue in the prediction of slow crack growth ranking determined from the static fatigue test. In all polyethylene pipe grade resins tested at 80 °C, reduced crack growth failure times were exhibited when the cyclic fatigue test was employed. However, when applied to rank the resins through their slow crack failure times, the cyclic fatigue results did not always confirm those obtained from the static fatigue test. That is, in some cases, a resin with higher slow crack resistance ranking (longer failure times) than another resin in static fatigue exhibited lower ranking (shorter failure times) in the cyclic fatigue test. This abnormality of reversal in ranking is not a general observation but does occur. Based on the data obtained so far, when resins with smaller differences between static fatigue and cyclic fatigue slow crack growth failure times are compared with those resins having larger differences, the chances of correctly predicting the ranking obtained from static fatigue using cyclic fatigue tend to decrease. Hence, it is suggested that one needs to practice caution when using cyclic fatigue to predict the static fatigue ranking of resins for slow cracking resistance. Some insight into the cause of such abnormality is discussed with reference to creep-fatigue interactions.     

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.     

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.     

Effect of temperature and volume on the tensile and adhesive properties of photocurable resins

Knowing the mechanical properties of UV‐curable resins at cryogenic conditions is important to ongoing fusion‐energy research and to emerging aerospace applications. The tensile and interfacial shear strengths of two commercially available UV‐curable resins were measured at room‐temperature and cryogenic conditions for both bulk and reduced (subnanoliter) specimen volumes. The tensile properties of cured specimens are remarkably sensitive to both testing temperature and specimen size. For one type of resin, the cold (?150 °C) tensile strength of subnanoliter specimens is ～9× larger (179 ± 19 MPa) than bulk values at room temperature. The interfacial shear strength between SiC fibers and small volumes of resin volumes is comparable to the bulk, room‐temperature tensile strength, but it varies over a wide range at ?150 °C (15–53 MPa). All resins were fully cured, and an analysis of fractured surfaces revealed microstructural features. The enhanced strength in microscopic specimens may be related to inhomogeneous stress fields that develop during cure. © 2014 Wiley Periodicals, Inc. J. Polym. Sci., Part B: Polym. Phys. 2014 , 52, 936–945     

Polyacrolein-isonicotinic acid hydrazone and polyacrylic acid-thiohydrazide resins-synthesis and sorption properties for precious and base metals

This investigation carries on previous studies on polyacrolein-styrene resins. Further attempts were made to convert the reactive aldehyde groups of the polymeric support to functional acrolein-isonicotinic acid hydrazone (P-NHZ) or acrylic acid-thiohydrazide derivatives (P-THZD). The conditions of synthesis and the basic sorption and desorption properties of the polymers synthesized are described. For gold and platinum group metals the resins show differences in loading capacities, rates of simultaneous sorption in static conditions and efficiencies in column tests. The elution by thiourea solution was utilized to separate Au, Pd and Pt from an excess of base metals. The P-NHZ resin seems to be more advantageous than the sulfur containing P-THZD resin.     

离子交换树脂的选择及柠檬酸溶液循环使用研究

用离子交换树脂法吸附柠檬酸溶液中的金属离子,苯乙烯系阳离子交换树脂的吸附性能较好,它对镍、铝离子的吸附容量均较大,且吸附前后柠檬酸溶液的浓度变化较小.静态条件下树脂对镍的吸附容量为16.83mg Ni/g干树脂,对铝为21.36mg Al/g干树脂;动态条件下树脂对镍的吸附容量为6.78mg Ni/g干树脂,对铝为31.8mgAl/g干树脂,吸附液流速为1m/h～3m/h.吸附后的柠檬酸溶液可循环使用.当用1mol/L硫酸解吸时,树脂对镍铝的解吸率可达90%以上.当硫酸中Ni2 为1.70mmol/L,Al3 为7.40mmol/L时,树脂的解吸率仍可达80%以上.     

Investigation into adsorption mechanisms of sulfonamides onto porous adsorbents

The presence of sulfonamide antibiotics in aquatic environments poses potential ecological risks and dangers to human health. In this study, porous resins as adsorbents for the removal of two sulfonamides, sulfadiazine and sulfadimidine, from aqueous solutions were evaluated. Activated carbon F-400 was included as a comparative adsorbent. Despite the different surface properties and pore structures of the three resins, similar patterns of pH-dependent adsorption were observed, implying the importance of sulfonamide molecular forms to the adsorption process on the resins. Sulfonamide adsorption to the three resins exhibited different ionic strengths and temperature dependence consistent with sulfonamide speciation and the corresponding adsorption mechanism. Adsorption of sulfadiazine to F-400 was relatively insensitive to pH and ionic strength as micropore-filling mainly contributed to adsorption. The adsorption mechanism of sulfadiazine to the hypercrosslinked resin MN-200 was similar to that of the macroporous resin XAD-4 at lower pH values, whereas it was almost identical to the aminated resin MN-150 at higher pH. This work provided an understanding of adsorption behavior and mechanism of sulfonamide antibiotics on different adsorbents and should result in more effective applications of porous resin for antibiotics removal from industrial wastewater.     

乙烯基酯树脂的微观结构及其力学性能研究

树脂的微观结构决定其力学性能 ,乙烯基酯树脂微观结构的一个重要特征就是凝胶粒子和两相结构的存在 .采用SEM、DSC研究了乙烯基酯树脂固化过程中凝胶粒子的形成过程及两相结构对固化放热的影响 ,结果表明 ,凝胶粒子在固化树脂中呈群状分布 ,每群凝胶粒子的最大尺寸为 2 0 μm左右 ,每群凝胶粒子由许多独立凝胶粒子组成 ;凝胶粒子的形成使树脂产生了两相结构 ,使树脂的恒温DSC残余放热呈双峰分布 ;树脂的固化条件影响其微观结构 .力学性能测试的结果表明树脂的固化条件影响其力学性能 ,低温固化树脂(80℃固化 )的后固化可以提高其拉伸强度和弯曲强度 ,对于高温固化树脂 (12 0℃固化 ) ,则出现相反的趋势 ,后固化降低了树脂的拉伸强度和弯曲强度 .     

Membrane chromatography: Protein purification from E. coli lysate using newly designed and commercial anion-exchange stationary phases

This contribution describes the purification of anthrax protective antigen (PA) protein from Escherichia coli lysate using bind-and-elute chromatography with newly designed weak anion-exchange membranes. Protein separation performance of the new AEX membrane adsorber was compared with the commercial Sartobind^® D membrane adsorber and HiTrap™ DEAE FF resin column under preparative scale conditions. Dynamic protein binding capacities of all three stationary phases were determined using breakthrough curve analysis. The AEX membrane showed higher binding capacities than the Sartobind^® D membrane at equivalent volumetric throughput and higher capacities than the HiTrap™ DEAE FF resin column at 15 times higher volumetric throughput. Anion-exchange chromatography was performed using all three stationary phases to purify PA protein. Quantitative SDS-PAGE analysis of effluent fractions showed that the purity of PA protein was higher for membrane adsorbers than the HiTrap™ DEAE FF resin column and was the same for the new AEX membrane and Sartobind^® D membrane adsorbers. The effects of E. coli lysate load volume and volumetric flow rate on PA protein separation resolution using the membrane adsorbers were minor, and the peak elution profile remained un-changed even under conditions where >75% of the total protein dynamic binding capacity of the membranes had been utilized. PA protein peak resolution was higher using pH-gradient elution than with ionic strength gradient elution. Overall, the results clearly demonstrate that membrane chromatography is a high-capacity, high-throughput, high-resolution separation technique, and that resolution in membrane chromatography can be higher than resin column chromatography under preparative conditions and at much higher volumetric throughput.     

新型吸附树脂对水中苯的吸附行为

研究表明,苯对人的毒性作用表现在中枢神经系统,65g／m^3的苯可引起广泛的出血导致死亡。反复暴露的低浓度苯主要对血液及造血组织产生毒性作用。根据苯吸入致白血病的流行病学研究数据,采用定量外推法计算出饮用水苯质量浓度为0．01mg/L时终身患癌的超额危险度为10^-5,因此饮用水中的苯的限值为0．01mg／L。因此,有效去除饮水中的苯具有重要的意义。     

Adsorption equilibrium of fructosyltransferase on a weak anion-exchange resin

The adsorption equilibrium of a glycoprotein, fructosyltransferase from Aureobasidium pullulans, on an anion-exchange resin, Sepabeads FP-DA activated with 0.1M NaOH, was investigated. The adsorption isotherms were determined at 20 degrees C in a phosphate-citrate buffer with pH 6.0 using the static method. Sodium chloride was used to adjust the ionic strength in the range from 0.0215 to 0.1215 mol dm(-3) which provided conditions varying from a weak effect of salt concentration on protein binding to its strong suppression. The equilibrium data were very well fitted by means of the steric mass-action model when the ion-exchange capacity of 290 mmol dm(-3) was obtained from independent frontal column experiments. The model fit provided the protein characteristic charge equal to 1.9, equilibrium constant 0.326, and steric factor 1.095 x 10(5).     

胺基修饰的高度交联聚苯乙烯树脂对间苯二酚吸附行为研究

研究了AH系列胺基修饰的超高交联树脂对水溶液中间苯二酚的静态吸附行为特征,结果表明,它们对间苯二酚的吸附容量明显高于母体交联树脂NDA-100和大孔弱碱性阴离子交换树脂D301.AH系列树脂与吸附质分子之间不仅有范德华作用力,还存在着氢键等作用力.该类树脂对间苯二酚的吸附为自发的放热过程,属于以物理作用为主兼有弱化学作用的吸附过程.吸附速率符合准一级动力学方程,表观吸附速率常数随树脂胺基含量的升高而降低.     

大孔树脂对茄尼醇吸附行为的研究

从6种大孔树脂中筛选出用于茄尼醇分离较好的树脂NKA,并进一步研究了其对茄尼醇吸附行为,结果表明,吸附等温线服从Langmuir方程和Freundlich方程,且吸附过程表现为优惠吸附.在温度为283～313K,吸附量为15～35mg/g的条件下,吸附焓变为-16.20～16.57kJ/mol,自由能变为.3.142～3.459kJ/mol,吸附熵变为-47.43～41.17J/mol.K.NKA树脂对茄尼醇吸附速率较快,吸附过程符合一级吸附动力学方程,吸附过程主要受液膜扩散控制.     

Relation of structure to performance characteristics of monolithic and perfusive stationary phases

Commercially available polymer-based monolithic and perfusive stationary phases were evaluated for their applicability in chromatography of biologics. Information on bed geometry, including that from electron microscopy (EM), was used to interpret and predict accessible volumes, binding capacities, and pressure drops. For preparative purification of biologics up to at least 7 nm in diameter, monoliths and perfusive resins are inferior to conventional stationary phases due to their low binding capacities (20–30 g/L for BSA). For larger biologics, up to several hundred nanometers in diameter, calculations from EM images predict a potential increase in binding capacity to nearly 100 g/L. The accessible volume for adenovirus calculated from the EM images matched the experimental value. While the pores of perfusive resins are essentially inaccessible to adenovirus under binding conditions, under non-adsorbing conditions the accessible intrabead porosity is almost as large as the interbead porosity. Modeling of breakthrough curves showed that the experimentally observed slow approach to full saturation can be explained by the distribution of pore sizes.