Adsorptive refolding of a highly disulfide-bonded inclusion body protein using anion-exchange chromatography

α-Fetoprotein (AFP) is a prospective biopharmaceutical candidate currently undergoing advanced-stage clinical trials for autoimmune indications. The high AFP expression yields in the form of inclusion bodies in Escherichia coli renders the inclusion body route potentially advantageous for process scale commercial manufacture, if high-throughput refolding can be achieved. This study reports the successful development of an ‘anion-exchange chromatography’-based refolding process for recombinant human AFP (rhAFP), which carries the challenges of contaminant spectrum and molecule complexity. rhAFP was readily refolded on-column at rhAFP concentrations unachievable with dilution refolding due to viscosity and solubility constraints. DEAE-FF functioned as a refolding enhancer to achieve rhAFP refolding yield of 28% and product purity of 95% in 3 h, at 1 mg/ml protein refolding concentration. Optimization of both refolding and chromatography column operation parameters (i.e. resin chemistry, column geometry, redox potential and feed conditioning) significantly improved rhAFP refolding efficiency. Compared to dilution refolding, on-column rhAFP refolding productivity was 9-fold higher, while that of off-column refolding was more than an order of magnitude higher. Successful demonstration that a simple anion-exchange column can, in a single step, readily refold and purify semi-crude rhAFP comprising 16 disulfide bonds, will certainly extend the application of column refolding to a myriad of complex industrial inclusion body proteins.     

Continuous matrix assisted refolding of alpha-lactalbumin by ion exchange chromatography with recycling of aggregates combined with ultradiafiltration

Continuous matrix assisted refolding (MAR) can be achieved on a solid support by using a continuous chromatographic system. Recycling the aggregate fraction, simultaneously formed during a refolding reaction, can further increase the refolding yield. Due to the nature of this reaction, aggregates are the main reason for a refolding yield below stoichiometric conversion. A preparative continuous annular chromatographic system (P-CAC) equipped with an ion exchange resin was used to continuously refold the model protein alpha-lactalbumin. For this purpose, this protein was denatured, reduced and adsorbed on the ion exchange resin. Elution was performed with or without redox reagents in the buffer system permitting fast formation of the native disulfide bonds. In the case redox reagents were present, the protein refolds then during its residence time on the matrix. However, aggregate formation is also increased and refolding yields are lower. Tightly bound aggregates were removed from the column by 2M guanidinium hydrochloride. In order to increase the system yield, this aggregate fraction was recycled after lowering the conductivity by ultradiafiltration and adjustment of the protein concentration by dilution. For on-column refolding, recycling of aggregates at a recycling rate of 0.17 increased the system yield from 25% to 30%. An algorithm was developed to show interdependencies of the single influencing parameters. The operability of the system was demonstrated but limitations due to instability of the P-CAC, especially inhomogeneous flow and peak wobbling, have to be considered.     

Size-exclusion chromatographic protein refolding: fundamentals, modeling and operation

Size-exclusion chromatography (SEC) has proven its capability to refold a variety of proteins using a range of gel filtration column materials, demonstrated in the growing body of experimental evidence. However, little effort has been allocated to the development of mechanistic models describing size-exclusion chromatographic refolding reactors (SECRR). Mechanistic models are important since they provide a link between process variables like denatured and reduced protein feed concentration (C_f,D&;R), flow rate, column length, etc., and performance indicators like refolding yield (Y_N), thereby opening the possibility for in silico design of SECRRs. A critical step, in the formulation of such models, is the selection of an adequate reaction mechanism, which provides the direct link between the separation and the refolding yield. Therefore, in this work we present a methodology using a SEC refolding reactor model, supported by a library of reaction mechanisms, to estimate a suitable reaction scheme using experimental SEC refolding data. SEC refolding data is used since it provides information about the mass distribution of monomers and aggregates after refolding, information not readily available from batch dilution refolding data alone. Additionally, this work presents (1) a systematic analysis of the reaction mechanisms considered using characteristic time analysis and Damköhler maps, revealing (a) the direct effect of a given reaction mechanism on the shape of the SEC refolding chromatogram (number of peaks and resolution) and (b) the effect that the competition between convection, refolding and aggregation is likely to have on the SEC refolding yield; (2) a comparison between the SECR reactor and the batch dilution refolding reactor based on mechanistic modeling, quantitatively showing the advantages of the former over the latter; and (3) the successful application of the modeling based strategy to study the SEC refolding data of an industrially relevant protein. In principle, the presented modeling strategy can be applied to any protein refolded using any gel filtration material, providing the proper mass balances and activity measurements are available.     

A novel protein refolding method integrating ion exchange chromatography with artificial molecular chaperone

Artificial molecular chaperone （AMC） and ion exchange chromatography （IEC） were integrated, thus a new refolding method, artificial molecular chaperone-ion exchange chromatography （AMC-IEC） was developed. Compared with AMC and IEC, the activity recovery of lysozyme obtained by AMC-IEC was much higher in the investigated range of initial protein concentrations, and the results show that AMC-IEC is very efficient for protein refolding at high concentrations. When the initial concentration of lysozyme is 180 mg/mL, its activity recovery obtained by AMC-IEC is still as high as 76.6%, while the activity recoveries obtained by AMC and IEC are 45.6% and 42.4%, respectively.     

Controlled oxidative protein refolding using an ion-exchange column

Column-based refolding of complex and highly disulfide-bonded proteins simplifies protein renaturation at both preparative and process scale by integrating and automating a number of operations commonly used in dilution refolding. Bovine serum albumin (BSA) was used as a model protein for refolding and oxido-shuffling on an ion-exchange column to give a refolding yield of 55% after 40 h incubation. Successful on-column refolding was conducted at protein concentrations of up to 10 mg/ml and refolded protein, purified from misfolded forms, was eluted directly from the column at a concentration of 3 mg/ml. This technique integrates the dithiothreitol removal, refolding, concentration and purification steps, achieving a high level of process simplification and automation, and a significant saving in reagent costs when scaled. Importantly, the current result suggests that it is possible to controllably refold disulfide-bonded proteins using common and inexpensive matrices, and that it is not always necessary to control protein-surface interactions using affinity tags and expensive chromatographic matrices. Moreover, it is possible to strictly control the oxidative refolding environment once denatured protein is bound to the ion-exchange column, thus allowing precisely controlled oxido-shuffling.     

Ion-exchange selectivity and chromatographic separation of trivalent metals on titanium antimonate

Summary The ion exchange selectivity of trivalent metal ions has been determined on titanium antimonate cation exchanger prepared by coprecipitation of antimony to titanium at different mole ratios. The selectivity sequence Al³⁺<Cr³⁺<Ga³⁺<In³⁺<Fe³⁺ was found for trivalent metal ions at an initial concentration of 10^–4 mol dm^–3 in nitric acid media. A high separation factor ^Ga/Al = Kd^Ga/Kd^Al, 4.8×10³, was observed for the Ga³⁺–Al³⁺ pair on titanium antimonate with an antimony to titanium ratio of 0.34. The effective separation of Ga³⁺ and In³⁺ from Al³⁺ was achieved using a 3 cm×0.5 cm i.d. column containing titanium antimonate with an antimony to titanium ratio of 0.34.     

Efficient refolding of a hydrophobic protein with multiple S-S bonds by on-resin immobilized metal affinity chromatography

The efficient refolding of recombinant proteins produced in the form of inclusion bodies (IBs) in Escherichia coli still is a complicated experimental problem especially for large hydrophobic highly disulfide-bonded proteins. The aim of this work was to develop highly efficient and simple refolding procedure for such a protein. The recombinant C-terminal fragment of human alpha-fetoprotein (rAFP-Cterm), which has molecular weight of 26 kDa and possesses 6 S-S bonds, was expressed in the form of IBs in E. coli. The C-terminal 7× His tag was introduced to facilitate protein purification and refolding. The refolding procedure of the immobilized protein by immobilized metal chelating chromatography (IMAC) was developed. Such hydrophobic highly disulfide-bonded proteins tend to irreversibly bind to traditionally used agarose-based matrices upon attempted refolding of the immobilized protein. Indeed, the yield of rAFP-Cterm upon its refolding by IMAC on agarose-based matrix was negligible with bulk of the protein irreversibly stacked to the resin. The key has occurred to be using IMAC based on silica matrix. This increased on-resin refolding yield of the target protein from almost 0 to 60% with purity 98%. Compared to dilution refolding of the same protein, the productivity of the developed procedure was two orders higher. There was no need for further purification or concentration of the renatured protein. The usage of silica-based matrix for the refolding of immobilized proteins by IMAC can improve and facilitate the experimental work for difficult-to-refold proteins.     

Ion-exchange chromatography of glycolatocomplexes of rare earths

Summary The ion-exchange behavior of glycolatocomplexes of the rare earths (Sc, Y and lanthanides) with 1-octanesulfonate as the hydrophobic ion has been investigated in aqueous glycolate media. The system is capable of separating adjacent light intra rare earths, La-Ce-Pr-Nd-Y-Sm, and heavy rare earths, Ho-Er-Tm-Yb-Lu-Sc, from each other with good resolution by gradient elution at room temperature. Intermediate rare earths, Dy-Tb-Gd-Eu-Sm, are difficult to separate from each other. The position of Y in the eluate is different from that found with most other eluent systems used for rare earth separations, being between Nd and Sm.     

Modeling protein binding and elution over a chromatographic surface probed by surface plasmon resonance

Surface plasmon resonance (SPR) spectroscopy is used as a scaled-down, analytical, pseudo-chromatography tool for analyzing protein binding and elution over an ion-exchange surface under cyclic sorption conditions. A micrometric-scale adsorption surface was produced by immobilizing a typical ion exchange ligand – diethylaminoethyl (DEAE) – onto commercially available planar gold sensor chip surfaces pre-derivatized with a self-assembled monolayer of 11-mercaptoundecanoic acid with known density. An explicit mathematical formulation is provided for the deconvolution and interpretation of the SPR sensorgrams. An adsorption rate model is proposed to describe the SPR sensorgrams for bovine serum albumin, used here as model protein, when the DEAE surface is subjected to a cyclic series of binding and elution steps. Overall, we demonstrate that the adsorption rate model is capable of quantitatively describing BSA binding and elution for protein titers from dilute conditions up to overloaded conditions and a broad range of salt concentrations.     

Dispersive mixing and intraparticle partitioning of protein in size-exclusion chromatographic refolding

Refolding enables bioprocesses predicated on proteins expressed as inclusion bodies in Escherichia coli. Optimization of size-exclusion chromatography (SEC) refolding is a significant challenge because a wide range of factors, including the choice of gel media, the column dimensions and configuration, affect the final yield in a protein-specific manner. In this study, we investigated these factors by relating them to dispersive mixing and partitioning of refolding molecules within the SEC pore structure. Lysozyme was refolded using SEC resins giving different column dispersion and chromatography resolution. Despite a low separation resolution, the desalting SEC resin Sephadex G-25 resulted in a refolding yield that was 12-30% higher than those obtained with Superdex 75 and Superdex 200. This finding supported the notion that SEC refolding was enhanced by dispersive mixing, which was increased by a wide particle size distribution of the Sephadex G-25 used. Column dispersion was further improved by strategically placing an inlet gap before the packed resin beds, leading to a 20% increase in refolding yield. Refolding yield in Superdex 75 was 20% higher than that in Superdex 200 under conditions giving similar dispersive mixing. This yield enhancement is expected to be protein-specific since Superdex 75 was chosen to specifically maximize partitioning of lysozyme molecules within the resin particles, reducing the likelihood of aggregation during refolding. The highest refolding yield (65%) was achieved using a Sephadex G-25 column with a 15 mm inlet gap, suggesting that desalting systems optimized for dispersive mixing might be an economical and generic alternative for preparative SEC protein refolding.     

人尿中多胺的离子交换富集和气相色谱测定

以离子交换法对尿样中的多胺进行预分离富集，确定了最佳的离子交换条件，同时比较了三种不同酰化剂的酰化效果，确定了最佳酰化条件，并用毛细管气相色谱法测定了尿中腐胺，尸胺，精脒，精胺的含量，结果表明癌症病人尿中多胺含量高于正常人。     

Predicting protein retention time in ion-exchange chromatography based on three-dimensional protein characterization

The isoelectric point (pI), molecular weight (M_W) and aqueous two-phase partitioning coefficients of a set of model proteins were related to retention time in cation-exchange chromatography using partial least squares regression. A three-dimensional method which combined hydrophobic partitioning and two-dimensional electrophoresis was used to determine those three properties for a mixture of proteins. The regression models fit well (R² = 0.913 and 0.873 for two resin types) considering the limited property basis, and were able to predict results for a small test set of proteins. The models showed that greater size and charge increased retention time, while the net influence of hydrophobicity depended on the base matrix type. This establishes the potential for the intended application to complex mixtures of host cell proteins.     

Ion-exchange thin-layer chromatography of metal lons in organic solvent — Hydrochloric acid — Complexing agent media

Summary Thin-layers of strongly basic or strongly acidic ionexchange resins (Dowex 1 or Dowex 50), mixed with a plain cellulose, Avicel SF, have been used to investigate the chromatographic behaviour of 40 metal ions in acetic acid — hydrochloric acid — complexing agent media. The CIESE (combined ion-exchange-solvent extraction) effect, proposed by Korkisch, is noticed for scandium and thorium in the anion-exchange system involving trioctylphosphine oxide (TOPO) as the complexing agent, the system providing a basis on the specific separation of both elements. In the cation-exchange system involving TOPO, the metal ions are distributed chromatographically, so that the system allows multicomponent separations to be carried out. The system also reveals the CIESE effect for zirconium and hafnium.     

以四唑为固定相配体的新型离子交换固定相的制备及色谱性能

用硅胶与γ-缩水甘油基丙基三乙氧基硅烷和3-羟基丙腈反应, 再采用链接化学(Click chemistry)中腈与叠氮化钠进行的3+2环加成反应, 合成了以四唑基为配体的弱阳离子交换色谱固定相. 结果表明, 所制得的色谱柱(4.6 mm×50 mm, i.d.)对蛋白质具有良好的分离性能, 且质量回收率大于93%. 蛋白质在该固定相上的保留符合弱阳离子交换色谱机理, 但保留值随流动相pH的变化规律与蛋白质在以羧基为交换基团的固定相上的保留值的变化规律不同, 并对此现象进行了初步解释.     

Continuous chromatographic separation of glucose-fructose mixtures using anion-exchange resins

Summary A semi-continuous, counter-current chromatographic refiner (SCCR) was packed with an anion resin in the bisulphite form and used to separate mixtures of sugars from different feedstocks. Products with purities of more than 99% were achieved and solid concentrations of the fructose products of 12% w/v were possible. The deterioration of the anion resin was controlled by simple precautions and thus its useful life was substantially prolonged.     

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.     

聚(GMA-DVB-TAIC)型连续床的制备及其对蛋白质的色谱分离性能

甲基丙烯酸缩水甘油酯（GMA）为单体,二乙烯基苯（DVB）和三聚异氰尿酸三烯丙酯（TAIC）为交联剂,在致孔剂甲苯和正庚烷存在下,直接以Φ4．6×100mm色谱柱管为模具,通过原位聚合制备了聚（甲基丙烯酸缩水甘油酯-二乙烯基苯-三聚异氰尿酸三烯丙酯）（PGDT）型连续床．然后,利用二乙胺和骨架结构中的环氧基反应得到阴离子交换型连续床．对连续床的化学结构、孔结构及其对蛋白质的分离性能进行了研究．实验结果表明,连续床内部含有大量类似渠道的大孔,孔径为1～2μm．在流速高达3250cm／h时,背压仅为9．89Mpa．而且流速对色谱分离效率的影响小,高流速下仍能得到高分离效率,可以通过提高流速实现蛋白质的快速分离．     

Ion-exchange chromatography with an oxalic acid-α-hydroxyisobutyric acid eluent for the separation and quantitation of rare-earth elements in monazite and xenotime

Summary An oxalic acid-α-hydroxyisobutyric acid eluent has been used for the separation and determination of rare-earth elements by high-performance ion-exchange chromatography. Fifteen rare-earth elements were separated within less than 25 min on a 150×4.6 mm i.d. column packed with 5-μm sulfonic acid-bonded silica particles by elution with a combined gradient of 0.60–9.0 mM oxalic acid and 19.0–5.0 mM α-hydroxyisobutyric acid at pH 4.6. Detection and quantitation of the separated rare-earth elements was accomplished by visible-absorbance measurements at 600 nm after postcolumn reaction with arsenazo I. The gradient of the two complexing agents was optimized to enable the separation of yttrium(III) without interference from other elements, especially dysprosium(III) and terbium(III). Mass detection limits of the elements were in the range of 2–4 ng. Finally, the chromatographic system was applied to the quantitative analysis of rare-earth elements in monazite and xenotime.     

The separation of synthetic mixtures of glucose and fructose and also inverted sucrose feedstocks using countercurrent chromatographic techniques

Summary A pilot scale semi-continuous countercurrent chromatographic Refiner (SCCR4) unit packed with a Zerolit 225 resin in the Ca²⁺ form has been used to separate synthetic mixtures of glucose and fructose and also inverted sucrose feedstocks into glucose-rich and fructose-rich products.For the same process conditions less pure fructose-rich products were found when using inverted sucrose feedstocks than with synthetic mixtures of glucose and fructose. However, anion-exchange resins Duolite A113 in the (HSO ₃ ^– ) form did not produce any variation in product purities with change of feed source.