In situ ion exchange resin bags to estimate forest site quality

Cation and anion exchange resin bags were placed just under the humus layer at five adjacent forest sample sites with differing site quality classes in order to assess the available nutrient supply. For comparison, humus samples were collected from the same sites. Nutrients were extracted from humus samples by conventional extraction methods and by shaking together with ion exchange resin bags. Ca and Mg corresponded best to differences in site quality class, of all analysed ions in thein situ resin bag eluates. Thein situ resin bag adsorption of NH₄−N, Na and Mn also showed a positive correlation with site quality. The adsorption of PO₄−P was negatively correlated to site quality class. Inadequate amounts of exchange resin, or leaving resin bagsin situ for too long a time result in the replacement of already adsorbed ions by ions with higher ion exchange constants.     

Understanding the interaction of proteins to ion exchange chromatographic supports: A surface energetics approach

Ion exchange chromatography is one of the most widely used chromatographic technique for the separation and purification of important biological molecules. Due to its wide applicability in separation processes, a targeted approach is required to suggest the effective binding conditions during ion exchange chromatography. A surface energetics approach was used to study the interaction of proteins to different types of ion exchange chromatographic beads. The basic parameters used in this approach are derived from the contact angle, streaming potential, and zeta potential values. The interaction of few model proteins to different anionic and cationic exchanger, with different backbone chemistry, that is, agarose and methacrylate, was performed. Generally, under binding conditions, it was observed that proteins having negative surface charges showed strong to lose interaction (20 kT for Hannilase to 0.5 kT for IgG) with different anionic exchangers (having different positive surface charges). On the contrary, anionic exchangers showed almost no interaction (0–0.1 kT) with the positively charged proteins. An inverse behavior was observed for the interaction of proteins to cationic exchangers. The outcome from these theoretical calculations can predict the binding behavior of different proteins under real ion exchange chromatographic conditions. This will ultimately propose a better bioprocess design for protein separation.     

Fluidized bed ion exchange for improving purification of lactic acid from fermentation

A strong anionic exchange resin was used to recover lactic acid directly from fermentation in an upflow fluidized bed column, resulting in 0.18 g lactic acid/g resin bound with a subsequent elution of 94%. When the culture broth was heated and adjusted pH to 8.0, 0.4 g lactic acid was bound per g resin, with a subsequent elution of 90%. L(+) and D(–) lactic acid isomers distribution was analyzed in the elution product resulting in an increase of L(+) isomer concentration. The resin did not alter its binding capacity after 23 cycles.     

Cooperativity of covalent attachment and ion exchange on alcalase immobilization using glutaraldehyde chemistry: Enzyme stabilization and improved proteolytic activity

Alcalase was scarcely immobilized on monoaminoethyl-N-aminoethyl (MANAE)-agarose beads at different pH values (<20% at pH 7). The enzyme did not immobilize on MANAE-agarose activated with glutaraldehyde at high ionic strength, suggesting a low reactivity of the enzyme with the support functionalized in this manner. However, the immobilization is relatively rapid when using low ionic strength and glutaraldehyde activated support. Using these conditions, the enzyme was immobilized at pH 5, 7, and 9, and in all cases, the activity vs. Boc-Ala-ONp decreased to around 50%. However, the activity vs. casein greatly depends on the immobilization pH, while at pH 5 it is also 50%, at pH 7 it is around 200%, and at pH 9 it is around 140%. All immobilized enzymes were significantly stabilized compared to the free enzyme when inactivated at pH 5, 7, or 9. The highest stability was always observed when the enzyme was immobilized at pH 9, and the worst stability occurred when the enzyme was immobilized at pH 5, in agreement with the reactivity of the amino groups of the enzyme. Stabilization was lower for the three preparations when the inactivation was performed at pH 5. Thus, this is a practical example on how the cooperative effect of ion exchange and covalent immobilization may be used to immobilize an enzyme when only one independent cause of immobilization is unable to immobilize the enzyme, while adjusting the immobilization pH leads to very different properties of the final immobilized enzyme preparation. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2768, 2019.     

Stability of tumor necrosis factor-alpha during ion exchange chromatography

Denaturation of target protein by various separation and purification steps contributes significant part to the total product loss in bioseparation. The conformational change and accompanying loss of activity of tumor necrosis factor- during ion exchange chromatography was reversible and was decreased by adding polyethylene glycol 200 at 2 to 5% (v/v) to the eluting solution.     

A cation exchange resin method for measuring long-term potassium release rates from soil

The aim of the present study was to determine long-term K release rates from soil by use of a modified resin method, where vigorous agitation and soil–resin separations were avoided to minimise the dissolution of the soil minerals. Resins saturated with Ca²⁺ or H⁺ were tested; Ca²⁺ because it is the most dominating cation on the soil exchange complex, and H⁺ because it is an important K⁺ counter-ion released from plant roots. The tested soil: resin systems included two rates of agitation; no agitation, and gentle agitation where the soil particles visually did not move relative to each other. The resin beds were kept physically separated from the soil particles by use of a specially designed extraction tube. Three soils of the same mineralogical and textural origin, but exposed to different K input levels during more than 80 years, were used. A pot experiment with ryegrass was carried out using the same three soils. Vigorous shaking of the resin–soil systems was not needed to obtain a sufficiently high and rapid K release to the resin; even without agitation long-term K release rates could be determined. The accumulated K release was in all combinations of agitation rate, resin saturation ion and soil K status proportional to the square root of extraction time after 1000 h of extraction, indicating diffusion controlled K release from the minerals. Resin extractable K was closely correlated with ryegrass K uptake, indicating similarity in the extraction process. In contrast, K extracted with ammonium acetate or nitric acid did not provide information about the ability of the soils to release K in the long term. Based on the criteria :(i) substantial K adsorption under slow K release conditions; and (ii) achievement of a stable long-term K release within a limited extraction period, the Ca-saturated resins with gentle agitation and H-saturated resins without agitation are concluded to be best suited for routine laboratory work.     

Ion exchange of amino acids at a natural organic ion exchanger: Thermodynamics and energetics

The thermodynamics and energetics of the ion exchange of four amino acids at a cellulosic ion exchanger have been studied. Experimental work included determination of ion exchange isotherms and the use of high-sensitivity titration microcalorimetry. A rigorous thermodynamic analysis of the data was developed allowing calculation of the standard free energy, the standard enthalpy, and standard entropy of exchange, and also the differential free energy, incremental enthalpy, and incremental entropy of exchange. The results show that the relative contributions of the enthalpy and entropy to the overall free energy differ markedly for the chosen amino acids. The reasons for these differences are analyzed and discussed. A knowledge of these fundamental thermodynamic properties indicates the solution conditions likely to give enhanced affinity of the ion exchanger for selected amino acids. The experimental techniques and analysis procedures developed are generally applicable to ion exchange separations of biomolecules. (c) 1995 John Wiley & Sons, Inc.     

离子交换色谱法对大肠杆菌表达的人重组骨形态发生蛋白-7的纯化

重组大肠杆菌高量表达重组人骨形态发生蛋白-7(rhBMP-7),每升培养液约得到湿菌体3g,其中目的蛋白约占菌体总蛋白量的40％。裂解离心,用低浓度变性剂洗涤初步纯化包涵体,上清中无目的蛋白损失;将包涵体溶解于高浓度变性剂溶液中,目的蛋白纯度提高到60％;然后在不同条件下用离子交换色谱法对变性状态下的蛋白质进行纯化,绝大部分杂蛋白被除去,目的蛋白纯度达95％以上;改变条件,可以减少rhBMP-7损失;用Western blot对目的蛋白进行特异性鉴定。     

L-谷氨酰胺在强酸性离子交换树脂上的稳定性

研究了谷氨酰胺在强酸性阳离子交换树脂「氢型」上离子交换时的化学稳定性,结果显示,在典型的及所述其他实验条件下,谷氨酰胺相当稳定。典型离交过程：１００ｍｌ去菌体谷氨酰胺发酵液含主要成份谷氨酰胺、谷氨酸和硫酸铵分别为３１２ｍｍｏｌ／Ｌ、６７．０ｍｍｏｌ／Ｌ和０．７５ｍｏｌ／Ｌ,离子交换柱床层体积２００ｍｌ,离交流速１．０ＢＶ／ｈ,０．５ｍｏｌ／Ｌ氨水洗脱流速０．５ＢＶ／ｈ。     

用D61大孔树脂从结晶母液中回收谷氨酸的研究

采用D61大孔氨型树脂,从等电点结晶母液中回收谷氨酸,上柱正交换,测出不同流率下的穿透曲线,得到交换区高度与交换速率的关系方程.同时采用3种试剂NH4Cl、(NH4)2SO4、NH3*H2O进行洗脱,其中NH3*H2O的洗脱效果较好,并测定了洗脱剂浓度和洗脱流率对洗脱效果的影响.     

离子交换法分离发酵液中鸟苷和肌苷

乌苷发酵液中乌苷与副产物肌苷的分离对乌苷工业有重要的意义。对乌苷和肌苷的分离条件进行研究,得到了最佳分离条件：采用Hd-8树脂,树脂床高度为4cm,前120mL采用0．05mol／L盐酸洗脱收集肌苷,120mL至240mL采用0．74mol／L pH5．0醋酸钠缓冲液洗脱收集乌苷,可以获得很好的效果。     

335弱碱性阴离子交换树脂对甘草酸的吸附过程

研究了335弱碱性阴离子交换树脂对甘草酸的吸附过程。拟合得到的吸附等温线方程为:c1/[q×(329-c1)]=0.035 8 1.872(c1/329),符合BET方程,计算得出335树脂的饱和吸附量是524.2 mg.g-1。通过吸附动力学曲线的研究,表明该树脂属于慢型吸附类型,得到树脂对甘草酸的吸附穿透曲线,穿透容量为42.00 mg.g-1,饱和容量近似为203.0 mg.g-1,交换柱的利用率小于0.206 9。用碱性洗脱液不易将树脂上吸附的甘草酸洗脱下来,利于甘草浸膏溶液中甘草酸和其它组分的分离。     

Impact of linker-drug on ion exchange chromatography separation of antibody-drug conjugates

ABSTRACT

Charge variants are important attributes of monoclonal antibodies, including antibody-drug conjugates (ADCs), because charge variants can potentially influence the stability and biological activity of these molecules. Ion exchange chromatography (IEX) is widely used for charge variants analysis of mAbs and offers the feasibility of fractionation for in-depth characterization. However, the conjugated linker-drug on ADCs could potentially affect the separation performance of IEX, considering IEX separation relies on surface charge distribution of analyte and involves the interaction between analyte surface and IEX stationary phase. Here, we investigated weak cation exchange chromatography (WCX) for its application in analyzing three ADCs (two broad distribution ADCs and an ADC with controlled conjugation sites) and the 2-drug/4-drug loaded species isolated from the two broad distribution ADCs using hydrophobic interaction chromatography. The major peaks in WCX profile were characterized via fraction collection followed by capillary electrophoresis-sodium dodecyl sulfate or peptide mapping. Results suggested that both the number of drug loads and conjugation sites could impact WCX separation of an ADC. The hypothesis was that the linker drugs could interfere with the ionic interaction between its surrounding amino acids on the mAb surface and column resin, which reduced the retention of ADCs on WCX column in this study. Our results further revealed that WCX brings good selectivity towards positional isomers, but limited resolution for different drug load, which causes the peak compositions of the two broad-distribution ADCs to be highly complex. We also compared results from WCX and imaged capillary isoelectric focusing (icIEF). Results showed that separation in icIEF was less influenced by conjugated linker drugs for the ADCs studied in this work, and better alignment was found between the two techniques for the ADC with controlled conjugate sites. Overall, this work provides insights into the complexity of WCX analysis of ADCs, which should be considered during method development and sample characterization.     

Gas phase hydrogen/deuterium exchange reactions of peptide ions in a quadrupole ion trap mass spectrometer

Hydrogen/deuterium exchange reactions of protonated and sodium cationized peptide molecules have been studied in the gas phase with a MALDI/quadrupole ion trap mass spectrometer. Unit-mass selected precursor ions were allowed to react with deuterated ammonia introduced into the trap cell by a pulsed valve. The reactant gas pressure, reaction time, and degree of the internal excitation of reactant ions were varied to explore the kinetics of the gas phase isotope exchange. Protonated peptide molecules exhibited a high degree of reactivity, some showing complete exchange of all labile hydrogen atoms. On the contrary, peptide molecules cationized with sodium exhibited only very limited reactivity, indicating a vast difference between the gas phase structures of the two ions. © 1997 Wiley-Liss Inc.     

Strategic assay deployment as a method for countering analytical bottlenecks in high throughput process development: Case studies in ion exchange chromatography

High throughput approaches to facilitate the development of chromatographic separations have now been adopted widely in the biopharmaceutical industry, but issues of how to reduce the associated analytical burden remain. For example, acquiring experimental data by high level factorial designs in 96 well plates can place a considerable strain upon assay capabilities, generating a bottleneck that limits significantly the speed of process characterization. This article proposes an approach designed to counter this challenge; Strategic Assay Deployment (SAD). In SAD, a set of available analytical methods is investigated to determine which set of techniques is the most appropriate to use and how best to deploy these to reduce the consumption of analytical resources while still enabling accurate and complete process characterization. The approach is demonstrated by investigating how salt concentration and pH affect the binding of green fluorescent protein from Escherichia coli homogenate to an anion exchange resin presented in a 96‐well filter plate format. Compared with the deployment of routinely used analytical methods alone, the application of SAD reduced both the total assay time and total assay material consumption by at least 40% and 5%, respectively. SAD has significant utility in accelerating bioprocess development activities. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Modeling of direct recovery of lactic acid from whole broths by ion exchange adsorption

Lactic acid fermentation process with L. casei CRL 686 was performed. The static adsorption isotherm over a strong anionic exchange resin, Amberlite^TM IRA-400 was measured, and the static binding capacity parameters were quantified. Early recovery of lactic acid from this lactate producer from unclarified culture broth was performed in a liquid solid fluidized bed, with the resin as the solid adsorbent, and the dynamic adsorption capacity was calculated. Good agreement was found between static and dynamic binding capacity values. The fluidized bed height was twice the settled bed height and the overall process was controlled by the liquid solid mass transfer. This operation was also simulated by continuously well stirred tanks arranged in series and superficial solid deactivation as in a gas solid catalytic reactor. The deactivation process takes into account liquid channeling and agglomerations of solid induced by the viscosity of the broth and also by the cells during the adsorption. These patterns were also verified by experimental observations, and are in agreement with the results found in the literature. The breakthrough data together with others from previous works were satisfactorily fitted until the 90% dimensionless concentration was reached for both culture broths. The model could be used in future studies on predictions about the liquid solid fluidized bed behavior and other different operating conditions.     

Assessment of the phytotoxicity of chromium in soils using the selective ion exchange resin extraction method

Chromium in soils is present in the form of Cr(VI) oxyanions or Cr(III) cations. The toxicity and mobility of Cr(VI) are higher than those of Cr(III), thus it is essential that the availability of Cr(VI) in soils be accurately estimated in order to assess the phytotoxicity of Cr and its resultant health hazards to animals and humans. In this study, the Cu-saturated selective ion exchange resin (DOWEX M4195) was used as an infinite sink to test the feasibility of using the resin for extracting available Cr(VI) from soil. In the experiments, the results show that the resin had a high affinity for Cr(VI) and that Cr(VI) adsorbed by resins could be desorbed by using 10% NaCl (pH 4). In addition, the adsorption and desorption of Cr(VI) were not affected by pH levels, the forms of Cr(VI) or the presence of major anions in the soil solution. The above results indicate that the Cu-saturated resin can selectively adsorb Cr(VI) from solution. In the soil extraction experiments, three Cr(VI)-spiked soils were processed using the Cu-saturated resin extraction method. The results show that amounts of soil Cr extractable by the resin had a significant negative correlation to the height of wheat seedlings grown in the Neubauer test. Comparing this to the commonly used extractant, 0.1 M HCl, the amount of soil Cr, extractable by the resin, had a higher correlation to plant height. The results suggest that the selective ion exchange resin method developed in this study is useful in evaluating the quantities of plant-available Cr(VI) in soil and can, therefore, assess the phytotoxicity of Cr in soil.