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.     

Tailoring orthogonal proteomic routines to understand protein separation during ion exchange chromatography

Surface charge, molecular weight, and folding state are known to influence protein chromatographic behaviour onto ion exchangers. Experimentally, information related to such factors can be gathered via 2-DE methods. The application of 2-D PAGE under denaturing/reducing conditions was already shown to reveal separation trends within a large protein population from cell extracts. However, ion-exchange chromatography normally runs under native conditions. A tailored protocol consisting in a first separation based on IEF on Immobiline strips under native conditions followed by a second dimension SDS-PAGE run was adopted. The chromatographic versus electrophoretic separation behaviours of two model proteins, thaumatin (TAU) and BSA, were compared to better understand which proteomic routine would be better suited to anticipate IEX chromatographic separations. It was observed that the information contained in the pI value obtained with the adapted 2-DE protocol showed better correlation with the IEX chromatographic behaviour. On the other hand, chromatographic separations performed in the presence of urea as a denaturant have demonstrated the potential influence of hydrodynamic radius/conformation on protein separation. Moreover, the information provided by such 2-D system correlated well with the chromatographic behaviour of an additional set of pure proteins. An initial prediction of protein ion-exchange chromatographic behaviour could be possible utilizing an experimental approach based on 2-DE running under milder chemical conditions. This technique provides information that more closely resembles the separation behaviour observed with a complex biotechnological feedstock.     

Preparation of phenylboronic acid functionalized cation-exchange monolithic columns for protein separation and refolding

In this study, we described a simple and effective modification procedure to prepare poly (methacrylate-co-ethylene glycol dimethacrylate) monolithic columns functionalized with 3-aminophenylboronic acid. The column morphology, pore size and specific surface area of the fabricated monolith were characterized by scanning electron microscopy, X-ray photoelectron spectroscopy, thermogravimetric analysis, and mercury intrusion porosimeter, respectively. The frontal analysis was carried out for dynamic loading capacity of the model protein on the modified column. The chromatographic performance of the cation-exchange monolith was evaluated through separating a mixture of five proteins such as lysozyme, cytochrome c, ribonuclease A, trypsin and bovine serum albumin and one-step purification of lysozyme from egg whites, and the expected results were obtained. In addition, the functionalized column was used to refold ribonuclease A and cytochrome c, and this procedure was monitored by circular dichroism and fluorescence spectroscopy. Compared with the conventional dilution refolding method, the ion-exchange chromatography refolding method developed here is more effective for specific bioactivity recovery.     

Scope,limitations and mechanism of nucleotide separation by thin-layer chromatography with strong cation-exchange resin containing layers

Summary The scope and limitations of the application of the commercially available cation-exchange thin-layer chromatoplate (Fixion 50-X8) for the resolution of nucleotide mixtures is presented. A possible separation mechanism is proposed and discussed.Publication No. 11 in the series dealing with the separation of nucleic acid bases, nucleosides and nucleotides on thin-layers consisting of strong cation-exchange resins.     

Electrodialytic separation of potassium ions from sodium ions in the presence of crown ether using a cation-exchange membrane

The separation of potassium and sodium ions from their mixture was performed by electrodialyzing a mixed solution of potassium chloride and sodium chloride in the presence of 18-crown-6 using a commercial cation-exchange membrane. After 18-crown-6 had been impregnated in the membrane, the mixed solution containing 18-crown-6 was electrodialyzed as the desalting-side solution. The permeation of potassium ions through the membrane decreased remarkably and the electrical resistance of the membrane increased during electrodialysis with increasing concentration of 18-crown-6 in the solution. Because potassium ions form a more stable complex with 18-crown-6 than sodium ions and because the complex permeated through the membrane with difficulty, sodium ions are thought to selectively permeate through the membrane. The current efficiency in electrodialysis was greater than 97.0%. Received: 1 June 1999/Accepted in revised form: 13 August 1999     

Nature identification and morphology characterization of cation-exchange membrane fouling during conventional electrodialysis

The aim of this work was to study the effect of a concentrate solution pH value and of the composition in calcium, carbonate, and protein of a diluate solution to be treated by conventional electrodialysis on the fouling of cation-exchange membranes (CEM). It appeared that after demineralization of solutions containing CaCl(2) and CaCl(2)+Na(2)CO(3) using a concentrate solution maintained at a pH of 12, mineral fouling appeared on both sides of the CEM. The nature of the deposits was identified as calcium hydroxide and/or carbonate on both surfaces. The mineral fouling presented an aggregation-like crystal following a carnation-like pattern of aggregates of small rhombohedral crystals with CaCl(2) added alone, while CaCl(2)+Na(2)CO(3) yielded a smoother spherical crystal. Protein fouling was detected only on the CEM surface in contact with the diluate after demineralization of a solution containing CaCl(2)+Na(2)CO(3) using a concentrate pH value of 2.     

Surface-modified polystyrene beads as photografting imprinted polymer matrix for chromatographic separation of proteins

A new and facile fabricating method for lysozyme molecularly imprinted polymer beads (lysozyme-MIP beads) in aqueous media was presented. Mesoporous chloromethylated polystyrene beads (MCP beads) containing dithiocarbamate iniferter (initiator transfer agent terminator) were used as supports for the grafting of lysozyme imprinted copolymers with acrylamide and N,N′-methylenebisacrylamide through surface initiated living-radical polymerization (SIP). After the polymerization, a layer of lysozyme-MIP was formed on the MCP beads. The SIP allowed an efficient control of the grafting process and suppressed solution propagation. Therefore, the obtained lysozyme-MIP beads had a large quantity of well-distributed pores on the surface without any visible gel formation in solution and were more advantageous comparing with traditional MIPs which were prepared by traditionally initiated radical polymerization. The obtained composites were characterized by Fourier transform infrared spectroscopy, elemental analysis, nitrogen sorption analysis and scanning electron microscopy. Chromatographic behaviors of the column packed with lysozyme-MIP beads exhibited ability in separating lysozyme from competitive protein (bovine hemoglobin, bovine serum albumin, ovalbumin or cytochrome c) in aqueous mobile phase.     

Statistical fluctuation in zeta potential distribution of nanoliposomes measured by on‐chip microcapillary electrophoresis

The zeta potential of nanoliposomes with a diameter below 100 nm has been studied by the combined use of on‐chip microcapillary electrophoresis (μCE) and sensitive fluorescence imaging. Tracking the electrophoretic migration of individual nanoliposomes has enabled the accurate evaluation of the zeta potential distribution of nanoliposomes and the first observation of its abnormal broadening due to a statistical fluctuation phenomenon specific to the “nanoscale world.” The materials used for liposome preparation were phosphocholine as the neutral lipid, phosphatidylserine as the anionic lipid, and cholesterol. The size of the liposomes encapsulating calcein, a fluorescent dye used for imaging convenience, was tailored by extrusion through polycarbonate membrane filters of different pore sizes ranging from 50 to 1000 nm. The on‐chip μCE system comprised a μCE chip, a laser source, an inverted microscope, and an electron‐multiplying charge‐coupled device camera. The electrophoresis experiment using this system revealed that the relative standard deviation of the zeta potential distribution of nanoliposomes is inversely proportional to their diameter and apparently increases below 100 nm. This abnormal broadening of zeta potential distribution of nanoliposomes is explained by prominent discreteness effect of the number of anionic lipid molecules in nanoliposomes.     

High-speed separation of proteins by microchip electrophoresis using a polyethylene glycol-coated plastic chip with a sodium dodecyl sulfate-linear polyacrylamide solution

In this paper, we describe a method for size-based electrophoretic separation of sodium dodecyl sulfate (SDS)-protein complexes on a polymethyl methacrylate (PMMA) microchip, using a separation buffer solution containing SDS and linear polyacrylamide as a sieving matrix. We developed optimum conditions under which protein separations can be performed, using polyethylene glycol (PEG)-coated polymer microchips and electrokinetic sample injection. We studied the performance of protein separations on the PEG-coated PMMA microchip. The electrophoretic separation of proteins (21.5-116.0 kDa) was completed with separation lengths of 3 mm, achieved within 8 s on the PEG-coated microchip. This high-speed method may be applied to protein separations over a large range of molecular weight, making the PEG-coated microchip approach applicable to high-speed proteome analysis systems.     

Determination of biogenic amines in fresh and processed meat by suppressed ion chromatography-mass spectrometry using a cation-exchange column

A new method for simultaneous determination of underivatized biogenic amines based on the separation by cation-exchange chromatography and suppressed conductivity coupled with mass spectrometry detection has been developed. The method has been applied to the analysis of cadaverine, putrescine, histamine, agmatine, phenethylamine and spermidine in processed meat products. The amines were extracted from muscle tissue with methanesulfonic acid without any additional derivative step or sample clean-up. Biogenic amines were separated by the IonPac CS17 column, a cation-exchange column used with gradient elution, and detection was done by suppressed conductivity and mass spectrometry. Tyramine was simultaneously analysed by using a spectrophotometer (275 nm) before the suppressed conductivity detection. Linearity of response was obtained in the range 0.25-25 microg mL(-1). The detection limits ranged from 23 microg L(-1) for putrescine to 155 microg L(-1) for spermidine (suppressed conductivity) and from 9 microg L(-1) for agmatine to 34 microg L(-1) for spermidine (MS). Average recoveries from meat samples ranged from 85 to 97% and coefficients of variation ranged from 4.5 to 9.7%. The analysis of biogenic amines in fresh and processed meats (dry-cured, cooked and fermented products) can be used as a quality marker of raw material and for studying the relationship between their changes and the fermentation process involved in dry sausage ripening.     

Comparative study of liquid uptake and permeation characteristics of sulfonated cation-exchange membranes in water and methanol

Liquid permeation and uptake measurements of pure water and methanol were carried out using three commercial cation-exchange membranes: Nafion-117 (perfluorinated polyethylene with pendant ether-linked side chains terminated with sulfonated groups), MK-40 (microparticles of polystyrene–divinylbenzene with sulfonic groups randomly dispersed in a polyethylene matrix) and CR61-CZL-412 (crosslinked sulfonated copolymer of styrene–divinylbenzene). Methanol uptake by the Nafion-117 membrane was higher than that of water, in contrast, for MK-40 and CR61-CZL-412 membranes the opposite behavior is observed. Differences in the water and methanol liquid uptakes by the membranes were discussed in terms of the chemical interaction between the liquids and the polymers, and also on the size of the liquid molecules. On the other hand, the methanol permeation flow values through the membranes were higher than those of water for all the studied membranes.     

The determination of reducing carbohydrates using a cation-exchange column and potentiometric detection with a metallic copper electrode

Summary A metallic copper electrode is employed as a potentiometric detector for reducing carbohydrates after their separation on a cation-exchange column. A post-column reactant solution comprising 1 mM copper (II) and 35 mM ammonia is mixed with the column effluent and the metallic copper electrode provides a steady baseline potential by responding in a Nernstian manner to the level of copper ions present. This in turn is affected by the presence of eluted reducing carbohydrates, leading to an indirect detection method for these species. The method is applied to the detection of maltose, lactose, xylose, glucose, sorbose, fructose and arabinose. The electrode response mechanism is discussed and detection limits in the low nanomole range are reported. The potentiometric detector is shown to be more sensitive than refractive index detection.     

Protein separation using a novel silica-based RPLC/IEC stationary phase modified with N-methylimidazolium ionic liquid

Ionic liquids (ILs) immobilized on silica as novel high performance liquid chromatography (HPLC) stationary phases have attracted considerable attention. However, it has not been applied to protein separation. In this paper, N-methylimidazolium IL-modified silica-based stationary phase (SilprMim) was prepared and investigated as a novel multi-interaction stationary phase charged positively for protein separation. The results indicate that all of the basic proteins tested cannot be absorbed on this novel stationary phase, whereas all of the acidic proteins tested can be retained, and the baseline separation of eight kinds of acidic protein standards can be achieved when performed in reversed phase/ ion-exchange chromatography (RPLC/IEC) mode. Compared with commonly used commercial octadecylated silica (ODS) column, the novel stationary phase can show selectivity and good resolution to acidic proteins, which has a promising application in the separation and analyses of acidic proteins from the complex samples in proteomics. In addition, the chromatographic behavior of proteins, the effect of the ligand structure and the retention mechanism on this stationary phase were also investigated.     

Inertness and stability of a maltose-silica for the separation of proteins by HPLC

Summary A stationary phase consisting of maltose on a silica support, designed for the separation of proteins, is described. When maltose is coupled to aminopropylsilica an effective shielding of the silica surface can be obtained. A size exclusion experiment is used to investigate pore structure and the behaviour of proteins. The stability of the surface layer is studied, as influenced by silica pretreatments and cross-linking reactions. Inertness is studied via the denaturation of trypsin in columns of different length at a number of temperatures. An example of a derivatization of the maltose layer is given which demonstrates its use as an interactive separation medium.     

Open-tubular nanoelectrochromatography (OT-NEC): gel-free separation of single stranded DNAs (ssDNAs) in thermoplastic nanochannels

Electrophoresis or electrochromatography carried out in nanometer columns (width and depth) offers some attractive benefits compared to microscale columns. These advantages include unique separation mechanisms that are scale dependent, fast separation times, and simpler workflow due to the lack of a need for column packing and/or wall coatings to create a stationary phase. We report the use of thermoplastics, in this case PMMA, as the substrate for separating single-stranded DNAs (ssDNAs). Electrophoresis nanochannels were created in PMMA using nanoimprint lithography (NIL), which can produce devices at lower cost and in a higher production mode compared to the fabrication techniques required for glass devices. The nanochannel column in PMMA was successful in separating ssDNAs in free solution that was not possible using microchip electrophoresis in PMMA. The separation could be performed in <1 s with resolution >1.5 when carried out using at an electric field strength of 280 V/cm and an effective column length of 60 μm (100 nm × 100 nm, depth and width). The ssDNAs transport through the PMMA column was driven electrokinetically under the influence of an EOF. The results indicated that the separation was dominated by chromatographic effects using an open tubular nano-electrochromatography (OT-NEC) mode of separation. Interesting to these separations was that no column packing was required nor a wall coating to create the stationary phase; the separation was affected using the native polymer that was UV/O₃ activated and an aqueous buffer mobile phase.     

Fast separation of hen egg white protein with a phosphorylcholine type zwitterionic ion chromatography stationary phase

In this work,a kind of preparation method of zwitterionic ion chromatography(ZIC) stationary phase modified with phosphorylcholine(PC) was obtained by hydrolyzing after bonding phosphorylcholine dichloride to diol-silica to better explore the characteristics of the PC groups as ZIC stationary phase ligand in simultaneous separation of acidic proteins and basic proteins. The results showed that tw’o kinds of acidic proteins and three kinds of basic proteins can be separated completely,meanwhile,hen egg white was separated and purified and three kinds of egg white components ovalbumin,G2 ovoglobulin and ovotransfemin proteins were separated completely by one single step on PC-ZIC column,the purity of all proteins reached above 95%.PC-ZIC stationary phase was successfully improved with better separation capacity and selectivity than previously reported in this paper.     

Use of zwitterionic buffer additives to improve the separation of proteins in capillary zone electrophoresis

In CZE, the adsorption of the proteins on the capillary wall is a common problem. This paper describes the simple method of utilizing zwitterionic buffer additives to improve the separation of proteins in untreated fused silica capillaries at neutral pH. Three kinds of zwitterion are evaluated in the separation of acidic, neutral, and basic proteins, including their effect on protein efficiency, mobility, separation, and resolution; the difference between the effects of the different additives are also highlighted. The method has proved to be a possible means of reducing protein adsorption, especially for basic proteins.     

共价有机骨架材料设计及其在分离领域的应用

共价有机骨架(COFs)材料是由有机小分子单体通过共价键连接形成的结晶多孔聚合物。与传统的线性聚合物不同的是,COFs可以在二维和三维空间上对其骨架结构进行控制,从而合成具有高度有序的刚性多孔结构,并且能够调节骨架的化学和物理性质。这种由COF形成的纳米级孔道和空间为分子存储、释放和分离提供了理想的环境。因此它在能量储存、分离、催化等领域有着广泛的应用前景。本文综述了近年来COFs材料的研究进展,主要包括材料的合成策略及其在分离领域的应用,并对COFs材料未来的发展方向进行了展望。     

A multi-residue cation-exchange clean up procedure for basic drugs in produce of animal origin

There is considerable interest in maximising the amount of information obtained from animal product analyses, when screening for the presence of veterinary drug residues. One of the barriers to effective multi-residue analysis to date has been a lack of effective clean up procedures to isolate a wide range of residues from the potential interferents, which may be present in both simple and complex (including processed) foods. A cation-exchange clean up has, therefore, been developed for use with acetonitrile extracts of foods, when analysing for several basic drug groups (sulfonamides, benzimidazoles, levamisole, nitroimidazoles, tranquillisers and fluroquinolones). The clean up procedure has also been shown to be effective using a modified extraction solvent for malachite green and leucomalachite green in fish.Several of the key parameters that influence analyte recovery have been investigated and in an optimised procedure, tissue/biofluid samples containing sulfonamides, benzimidazoles, levamisole, nitroimidazoles, tranquillisers and fluoroquinolones are first extracted with acetonitrile. The extract is then dried with sodium sulfate and acidified with glacial acetic acid before loading onto a Bond Elut, strong cation-exchange (SCX) solid phase extraction (SPE) cartridge. Extracts from fish containing malachite green and leucomalachite green can be cleaned up using the same SCX SPE procedure following extraction with citrate buffer/acetonitrile. Typical recoveries of drugs from low level fortified tissues using the optimised procedure lie in the range 53-104% with the exception of carazolol from pig kidney (31%), malachite green from trout (42-51%) and ciprofloxacin from chicken muscle (44%) and from egg (21%).