Investigation of protein binding affinity in multimodal chromatographic systems using a homologous protein library

A library of cold shock protein B mutant variants was employed to examine differences in protein binding behavior in ion exchange and multimodal chromatography. Single site mutations introduced at charged amino acids on the protein surface resulted in a homologous protein set with varying charge density and distribution. The retention times of the mutants varied significantly during linear gradient chromatography in both systems. The majority of the proteins were more strongly retained on the multimodal cation exchange resin as compared to the traditional cation exchanger. Further, the elution order of the mutants on the multimodal resin was different from that obtained with the ion exchanger. Quantitative structure–property relationship models generated using a support vector regression technique were shown to provide good predictions for the retention times of protein mutants on the multimodal resin. A coarse-grained ligand docking package was employed to examine the various interactions between the proteins and ligands in free solution. The multimodal ligand was shown to utilize multiple interaction types to achieve stronger retention on the protein surface. The use of this protein library in concert with the qualitative and quantitative analyses presented in this paper provides an improved understanding of protein behavior in multimodal chromatographic systems.     

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.     

Comparative performance of ion exchange and ion-paired reversed phase high performance liquid chromatography for the determination of nucleotides in biological samples.

We have compared anion exchange chromatography on APS-Hypersil (4.6 x 100 mm) eluted with a phosphate gradient with reversed phase chromatography on ODS-Hypersil (4.6 x 100 mm) in the presence of either tetrabutylammonium (TBA) or triethylammonium (TEA) ions with a methanol gradient. The systems have been compared both for ease of operation and for their resolving power with standard mixtures and acid extracts of both normal red cells (RBC) and ischaemic tissues. The two chromatographic modes exhibited similar separating efficiencies for standard mixtures of nucleotides but retention times were most stable using reversed phase liquid chromatography (RPLC) with TEA. Anion exchange columns slowly lost ion exchange capacity but selectivity was unchanged. RPLC in the presence of TBA gave reproducibile capacity factors only when operated isocratically due to irreversible changes to the silica surface. For RBCs the RPLC with TEA and anion exchange systems resolved 17 and 15 peaks, respectively, and for the ischaemic samples 22 and 14 peaks, respectively. However, nucleosides and bases were also resolved by the ODS column causing chromatographic crowding and uncertain peak identification.     

Desorption of plasmid DNA from anion exchangers: Salt concentration at elution is independent of plasmid size and load

Detailed studies on the sorption behavior of plasmids on anion exchangers are rare compared to proteins. In this study, we systematically compare the elution behavior of plasmid DNA on three common anion exchange resins using linear gradient and isocratic elution experiments. Two plasmids of different lengths, 8 and 20 kbp, were studied and their elution characteristics were compared to a green fluorescent protein. Using established methods for determining retention characteristics of biomolecules in ion exchange chromatography lead to remarkable results. In contrast to the green fluorescent protein, plasmid DNA consistently elutes at one characteristic salt concentration in linear gradient elution. This salt concentration was the same independent of plasmid size but differed slightly for different resins. The behavior is consistent also at preparative loadings of plasmid DNA. Thus, only a single linear gradient elution experiment is sufficient to design elution in a process scale capture step. At isocratic elution conditions, plasmid DNA elutes only above this characteristic concentration. Even at slightly lower concentrations most plasmids remain tightly bound. We hypothesize, that the desorption is accompanied by a conformational change leading to a reduced number of available negative charges for binding. This explanation is supported by structural analysis before and after elution.     

Optimization of l‐arginine purification from Corynebacterium crenatum fermentation broth

l ‐Arginine has many special physiological and biochemical functions, with wide applications in the food and pharmaceutical industry. Few studies on the purification of l ‐arginine from fermentation broth have been conducted; however, none of them were systematic enough for industrial scale‐up. Therefore, it is necessary to develop a highly efficient and systematic process for the purification of l ‐arginine from fermentation broth. In this study, we screened out a cation exchange resin, D155, having high exchange capacity, high selectivity, and easy elution capacity, and analyzed its adsorption isotherm, thermodynamics, and kinetics using different models. Further, the process parameters of fixed‐bed ion exchange adsorption and elution were optimized, and the penetration curve during the operation was modeled. Based on the fixed‐bed ion‐exchange parameters, a 30‐column continuous ion‐exchange system was designed, and the flow velocity in each zone was optimized. Finally, to obtain a high purity of l ‐arginine, the purification tests were conducted using anion exchange resin 711, and an l ‐arginine yield of 99.1% and purity of 98.5% was obtained. This effective and economical method also provides a promising strategy for separation of other amino acids from the fermentation broth, which is of great significance to the l ‐arginine fermentation industry.     

强阴离子交换色谱结合分子排阻色谱纯化血清中免疫球蛋白和白蛋白

动物血清中免疫球蛋白和白蛋白的等电点分别约为7.8和4.8,根据它们等电点的较大差别,利用Q SepharoseTM-XL强阴离子交换色谱结合分子排阻色谱同时分离纯化这2种蛋白。以0.02 mol/L pH 8.0的Tris-HCl缓冲液平衡离子交换色谱柱并将已稀释10倍的高免疫的兔血清上样,采用pH分段洗脱。在pH 6.0时以0.3 mL/min低流速洗脱得到高纯度的免疫球蛋白,继续在pH 4.0时洗脱,再辅以Sephadex G-75分子排阻色谱可获得纯度大于95%的白蛋白。对纯化后的蛋白进行活性检测,证明所纯化的免疫球蛋白和白蛋白都保持正常的生物活性。蛋白质含量测定说明免疫球蛋白的纯化回收率达到95%以上,而白蛋白的纯化回收率大于90%。该法简便快速,可同时从动物血清中纯化出保持生物活性的免疫球蛋白和白蛋白,纯化效率高。     

Biological Characterization of a Pituitary Monkey Lutropin Preparation after Chromatofocusing or after High Performance Anion Exchange Chromatography

Abstract

The Pharmacia Fast Protein Liquid Chromatography System equiped with a Mono P HR 5/10 chromatofocusing column was used to characterize the distribution of different bioactive forms of a cynomolgus (Macaca bascicularis) pituitary lutropin preparation. The results were compared with the profiles obtained after running the preparation on a Waters DEAE anion exchange column using eluants in the pH range from 6.0 to 8.0. The distribution of preserved biological activity were studied in respective eluted fractions from the different experiments of the applied preparation. Seventy-six percent of the bioactivity was recovered after chromatofocusing whereas preserved bioactivity after ion exchange chromatography ranged between 61–78% at pH 6.5 to 7.5. No bioactivity was restored after elution at pH 6.0 or 8.0.     

Column-switching system for selenium speciation by coupling reversed-phase and ion-exchange high-performance liquid chromatography with microwave-assisted digestion-hydride generation-atomic fluorescence spectrometry

Speciation of selenocysteine (SeCys), selenomethionine (SeMet), selenoethionine (SeET), selenite (Se(IV)) and selenate (Se(VI)) has been accomplished using high-performance liquid chromatography, with the aid of an anion exchange column and a reversed-phase column, both connected through a six-port switching valve. On-line microwave-assisted digestion and hydride generation steps were performed prior to the atomic fluorescence detection. The elution of the seleno amino acids was accomplished in the reversed-phased column using water as mobile phase. Selenite and selenate were separated in the anion exchange column, using gradient elution with an acetate buffer. The separation of the five selenium compounds took place in 15 min. The detection limits obtained ranged between 0.6 and 0.9 microg l(-1). Values of r>0.998 were obtained for linear fit graphs. A commercial available urine sample was analyzed, in which SeCys and Se(IV) were quantified.     

31ku激发子蛋白的液相色谱分离纯化

利用阴离子交换色谱和疏水相互作用色谱从烟草疫霉菌培养液中分离出一种新的31ku激发子蛋白，选择了阴离子交换色谱流动相的的最佳pH值6．0，建立了疏水相互作用色谱硫酸铵Tris缓冲液－水洗脱模式，简化了纯化步骤和减少了活性损失的危险。     

Retention of some glucosinolates in anion exchange chromatography. Part I: Qualitative study on a silica-trialkylammonium exchanger

Summary The retention and separation of glucosinolates, as organic anions, were studied on a silica-based strong anion exchanger under isocratic elution conditions. All glucosinolates carry the same functional ionic group (-OSO ₃ ^– ), however they do not have the same retention in anion exchange chromatography. The plots of capacity factors of organic anions versus the reciprocal of eluent ion concentration show good linearity. From the slope and y-intercept data the major retention mechanisms are interpreted as ion exchange and reversed-phase interactions. The effects of nature and concentration of the eluent ion and the influence of organic modifier addition to the aqueous buffered mobile phase are also investigated. Direct and indirect UV detection were used.Our results open the way for the development of new systems for intact glucosinolate analysis which are easier to use than the present ion-pairing chromatographic method.     

液相色谱法分离纯化烟草疫霉菌激发子

利用阴离子交换色谱和疏水相互作用色谱从烟草疫霉菌培养液中分离了一种新的９０ＫＤ激发子蛋白,他阴离子交换色谱流动相的最佳ｐＨ值,建立了疏水相互作用色谱,硫酸铵与三羟甲基氨基甲烷（Ｔｒｉｓ）缓冲液／水洗脱模式,简化了纯化步骤和活性损失的危险。     

Separation properties of saccharides on a hydrophilic stationary phase having hydration layer formed zwitterionic copolymer

A novel water-holding adsorbent bonded with a zwitterionic polymer, diallylamine–maleic acid copolymer, was developed. With this adsorbent, hydrophilic solutes are partitioned by a hydration layer that forms on the zwitterions, as a main separating force. When the adsorbent was used to separate saccharides by normal-phase partition chromatography, the saccharides eluted in the order, mono-, di- and trisaccharide. The elution profile for mono- and di-saccharides was similar but not identical to that on anion exchange columns. This indicated that the adsorbent exhibited a complex retention behavior by the existence of both anion and cation exchange moieties in the functional polymer. Selecting Na⁺ as a counter-ion of the maleate moiety enhanced the retention of saccharide. When used in an high performance liquid chromatography (HPLC) system with gradient elution, the adsorbent enabled the simultaneous analysis of mono-, di- and oligosaccharides.     

Anion exchange liquid chromatography--inductively coupled plasma-mass spectrometry detection of the Co2+, Cu2+, Fe3+ and Ni2+ complexes of mugineic and deoxymugineic acid

Phytosiderophores, such as mugineic and deoxymugineic acid, are produced by graminaceous plant species in response to Fe deficiency conditions normally experienced in calcareous and alkaline non-calcareous soils. As these phytosiderophores have the ability to form thermodynamically stable complexes with other metal cations present in the growing medium, they have also been implicated in the transport and bioavailability of these metals in the environment. However, routine analytical methodology to detect the various metal complexes formed by these phytosiderophores is lacking. Therefore, as these complexes are negatively charged over a wide range of pH values, anion exchange liquid chromatography (AE LC) coupled to inductively coupled plasma-mass spectrometry (ICP-MS) was investigated as a means to separate and quantify these complexes. The metal-phytosiderophore complexes were prepared at pH 7 and separated by NaOH or NH4NO3 gradient elution on a Dionex AS11 anion exchange column. Of the metals tested only the Co2+ and Ni2+ complexes of mugineic and deoxymugineic acid were detected when using a 0-20mM NaOH gradient elution profile. However, the phytosiderophore complexes of Cu2+ and Fe3+ were also detected when using NH4NO3 as the mobile phase at pH 7. Base-assisted hydrolysis of the latter two complexes is proposed to explain their apparent 'instability' in the high pH NaOH mobile phase. The absolute detection limits of the developed methodologies for these metal complexes ranged from 0.1 to 2.8pmol. As phytosiderophore complexes with Cd2+ and Zn2+ were not detected, it was concluded that the dissociation kinetics of these metal-phytosiderophore complexes were too rapid for these complexes to be observed in the present chromatographic conditions.     

Reversed-phase liquid chromatography of proteins and peptides using multimodal copolymer-encapsulated silica

Multimodal copolymer-encapsulated particles for liquid chromatography were prepared by bonding 1-octadecene and unsaturated carboxylic acids on silica particles (5 microm diameter, 300 A pores) for liquid chromatography of proteins. These multimodal copolymer-encapsulated particles can provide both hydrophobic and hydrogen bonding interactions with polar compounds. The chromatographic performance of these multimodal copolymer-encapsulated particles for peptide and protein separations was evaluated under reversed-phase conditions. Compared with typical C8-bonded silica, polymer-encapsulated particles were more stable in acidic mobile phases and provided better recoveries, especially for large proteins (Mr>0.5 x 10(6)). Totally hydrophobic polymer-encapsulated particles were found to produce broad peaks for proteins, and significant improvements were observed by introducing hydrophilic groups (-COOH) onto the polymer-encapsulated surface to form a multimodal phase. For the reversed-phase liquid chromatography of peptides and proteins, improved selectivity and increased solute retention were found using the multimodal polymer-encapsulated particles. More peaks were resolved for the separation of complex peptide mixtures such as protein digests using the multimodal polymer-encapsulated particles as compared to totally hydrophobic polymer-encapsulated particles.     

离子交换与离子排斥色谱柱串联体系中阳离子的多峰现象及其机理

研究了串联柱体系中阳离子的“多峰现象”。在阳离子交换柱后面接上阴离子分析用的离子排斥柱构成一个串联柱体系,当以酒石酸（ＴＡ）和吡啶二羧酸（ＰＤＣ）的混合溶液作淋洗液时,每一种阳离子同时出现３个色谱峰。这是因为从阳离子交换柱流出的阳离子与有络合作用的两种淋洗剂阴离子形成络合物,使流动相中淋洗剂阴离子浓度减少以及两种淋洗剂阴离子在离子排斥柱中被保留且保留值不同。     

Preparation of a weak anion exchange/hydrophobic interaction dual‐function mixed‐mode chromatography stationary phase for protein separation using click chemistry

In this study, 3‐diethylamino‐1‐propyne was covalently bonded to the azide‐silica by a click reaction to obtain a novel dual‐function mixed‐mode chromatography stationary phase for protein separation with a ligand containing tertiary amine and two ethyl groups capable of electrostatic and hydrophobic interaction functionalities, which can display hydrophobic interaction chromatography character in a high‐salt‐concentration mobile phase and weak anion exchange character in a low‐salt‐concentration mobile phase employed for protein separation. As a result, it can be employed to separate proteins with weak anion exchange and hydrophobic interaction modes, respectively. The resolution and selectivity of the stationary phase were evaluated in both hydrophobic interaction and ion exchange modes with standard proteins, respectively, which can be comparable to that of conventional weak anion exchange and hydrophobic interaction chromatography columns. Therefore, the synthesized weak anion exchange/hydrophobic interaction dual‐function mixed‐mode chromatography column can be used to replace two corresponding conventional weak anion exchange and hydrophobic interaction chromatography columns to separate proteins. Based on this mixed‐mode chromatography stationary phase, a new off‐line two‐dimensional liquid chromatography technology using only a single dual‐function mixed‐mode chromatography column was developed. Nine kinds of tested proteins can be separated completely using the developed method within 2.0 h.     

Purification of oligouronides using hollow-fiber membrane functionalised with L-histidine

The pseudobioaffinity chromatography with L-histidines immobilized onto poly(ethylene-vinyl alcohol) hollow-fiber membranes was studied for a selective purification of anionic oligosaccharides. Oligoglucuronans with different degrees of polymerization and acetylation, prepared by an enzymatic degradation of bacterial polyglucuronic acid, were used as models. The adsorption and elution parameters were studied to optimise the selective adsorption. A better understanding of the physico-chemical phenomena governing this selectivity was attempted and a few hypotheses on the mechanism of selectivity are proposed.     

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.     

Simultaneous determination of 13 carbohydrates using high‐performance anion‐exchange chromatography coupled with pulsed amperometric detection and mass spectrometry

A simple, accurate, and highly sensitive method was developed for the determination of 13 carbohydrates in polysaccharide of Spirulina platensis based on high‐performance anion‐exchange chromatography coupled with pulsed amperometric detection and mass spectrometry. Samples were extracted with deionized water using ultrasonic‐assisted extraction, and the ultrasound‐assisted extraction conditions were optimized by Box–Behnken design. Then the extracted polysaccharide was hydrolyzed by adding 1 mol/L trifluoroacetic acid before determination by high‐performance anion‐exchange chromatography coupled with pulsed amperometric detection and confirmed by high‐performance anion‐exchange chromatography coupled with mass spectrometry. The high‐performance anion‐exchange chromatography coupled with pulsed amperometric detection method was performed on a CarboPac PA20 column by gradient elution using deionized water, 0.1 mol/L sodium hydroxide solution, and 0.4 mol/L sodium acetate solution. Excellent linearity was observed in the range of 0.05–10 mg/L. The average recoveries ranged from 80.7 to 121.7%. The limits of detection and limits of quantification for 13 carbohydrates were 0.02–0.10 and 0.2–1.2  μg/kg, respectively. The developed method has been successfully applied to ambient samples, and the results indicated that high‐performance anion‐exchange chromatography coupled with pulsed amperometric detection and mass spectrometry could provide a rapid and accurate method for the simultaneous determination of carbohydrates.     

Effects of ion association of lipophilic quaternary ammonium salts in ion-exchange and potentiometric selectivity

It has been found experimentally and substantiated theoretically that the anion-exchange selectivity in water—lipophilic quaternary ammonium salts (QAS) toluene solutions systems as well as the potentiometric selectivity of plasticized PVC membranes containing QAS, are strongly influenced by ion association. In particular, it has been demonstrated that varying the steric accessibility of QAS exchange center is a powerful tool for the selectivity control. The experimental values of the selectivity change caused by variations in the QAS exchange center steric accessibility were about 3 orders of magnitude when the single-charged ions were exchanged for the single-charged ones and more than seven orders when double-charged ions were exchanged for the single-charged. The above effects have also been observed for the potentiometric selectivity of QAS-based PVC membranes and, to some extent, for the potentiometric selectivity of the neutral anion carrier-based membranes doped by QAS to provide anion permselectivity. This fact is of immediate practical interest for the development of ISE with improved selectivity. The obtained results allow to revise the generally accepted idea of QAS as “nonselective” ion-exchangers and to suggest specific ways for controlling the ion-exchange and potentiometric selectivity using the ion association as a tool.