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.     

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.     

An SEC/MALS study of alternan degradation during size-exclusion chromatographic analysis

Ultrahigh-molar-mass (M) polymers such as DNA, cellulose, and polyolefins are routinely analyzed using size-exclusion chromatography (SEC) to obtain molar mass averages, distributions, and architectural information. It has long been contended that high-M polymers can degrade during SEC analysis; if true, the inaccurate molar mass information obtained can adversely affect decisions regarding processing and end-use properties of the macromolecules. However, most evidence to the effect of degradation has been circumstantial and open to alternative interpretation. For example, the shift in SEC elution volume as a function of increased chromatographic flow rate, observed using only a concentration-sensitive detector, may be the result of degradation or of elution via a nondegradatory slalom chromatography mechanism. Here, using both concentration-sensitive and multiangle static light-scattering detection, we provide unambiguous evidence that the polysaccharide alternan actually degrades during SEC analysis. The decrease in molar mass and size of alternan with increasing flow rate, measured using light scattering, allows ruling out an SC mode of elution and can only be interpreted as due to degradation. These findings demonstrate the extreme fragility of ultrahigh-M polymers and the care that must be taken for accurate characterization. Figure Scission of alternan chains in liquid chromatography.     

Size-exclusion chromatographic behavior of bovine serum albumin in uni-uni valent ions solutions

Summary Molecular interaction between uni-uni valent ions and bovine serum albumin was investigated by size-exclusion chromatography. Elution profiles are first presented for salt and protein solutions as samples with water as the mobile phase; then for water and protein as samples with salt solutions as the mobile phase. The results suggest the existence of a dynamic equilibrium between the salt ions and the protein ions as reactants and the ion-pair (salt-protein) complex as a product.     

Size-exclusion and high-performance liquid chromatography separation of peptides from peptic haemoglobin hydrolysate obtained by ultrafiltration

Summary Gel filtration (size-exclusion) and high-performance liquid chromatography have been used to separate peptic peptides from haemoglobin hydrolysate. Elution profiles on Sephadex G-25 displayed nine fractions with molecular weights lower than 6500 daltons. Each fraction was analysed for total amino acid content and showed less than 1% free amino acids. Reversed phase HPLC, using ammonium acetate buffer and acetonitrile as solvent, was applied to each fraction in order to obtain pure peptide peaks. The importance of acquiring a better knowledge of such an hydrolysate is discussed. Various potential applications of this type of hydrolysate, some of them already being undertaken, are envisaged.     

混合溶剂作淋洗剂的体积排除色谱(SEC)中溶剂化高分子峰和溶剂峰的关系

以混合溶剂作淋洗剂的体积排除色谱(SEC)中高分子样品一般会出现2个峰, 分别是溶剂化高分子峰和自由溶剂峰. 理论分析结果表明, 溶剂化高分子峰面积( A3eff )是“裸高分子”峰面积(A3)和被束缚溶剂峰面积(A1*)的加和, 被束缚溶剂峰面积(A1*)和自由溶剂峰面积(A2*)大小相等符号相反. 以聚苯乙烯(3)-氯仿(1)-甲醇(2)体系为研究对象, 分别对A3eff, A3及A2*进行了实验测定, 证实了理论推断的正确性, 表明由高分子峰和由溶剂峰来求算优先吸附系数是等价的.     

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.     

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.     

Direct gas chromatographic analysis of halogenated hydrocarbons at the part-per-trillion level

Large sample volume on-column injection (up to 250 μL) of n-pentane solutions of halogenated hydrocarbons has been employed for the direct measurement of both low-boiling and high-boiling compounds in what is essentially a single run. Two-bonded phase, fused-silica capillary columns are joined in series, through which the low-boiling compounds are first chromatographed and detected with an electron capture detector. High-boiling compounds are then trapped in a section joining the two columns, and subsequently chromatographed in the second column, using the same detector. This procedure permits analysis at the ppt level.     

Size-exclusion chromatography of perfluorosulfonated ionomers

A size-exclusion chromatography (SEC) method in N,N-dimethylformamide containing 0.1 M LiNO₃ is shown to be suitable for the determination of molar mass distributions of three classes of perfluorosulfonated ionomers, including Nafion^®. Autoclaving sample preparation is optimized to prepare molecular solutions free of aggregates, and a solvent exchange method concentrates the autoclaved samples to enable the use of molar-mass-sensitive detection. Calibration curves obtained from light scattering and viscometry detection suggest minor variation in the specific refractive index increment across the molecular size distributions, which introduces inaccuracies in the calculation of local absolute molar masses and intrinsic viscosities. Conformation plots that combine apparent molar masses from light scattering detection with apparent intrinsic viscosities from viscometry detection partially compensate for the variations in refractive index increment. The conformation plots are consistent with compact polymer conformations, and they provide Mark–Houwink–Sakurada constants that can be used to calculate molar mass distributions without molar-mass-sensitive detection. Unperturbed dimensions and characteristic ratios calculated from viscosity–molar mass relationships indicate unusually free rotation of the perfluoroalkane backbones and may suggest limitations to applying two-parameter excluded volume theories for these ionomers.     

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.     

The selective abstraction of aliphatic aldehydes by gas chromatographic stationary phases containing N,N′-disubstituted-p-phenylenediamines

Summary The selective on-column abstraction of aliphatic aldehydes by gas chromatographic stationary phases containing N-iso-propyl N′-phenyl-p-phenylenediamine is described.     

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.     

Gas chromatographic determination of serum bile acids — Application of a novel extraction procedure

Summary The profile of serum bile acids is a result of their liver metabolism and enterohepatic circulation.In the present work size exclusion chromatography is used for extraction of serum bile acids to optimize the methodology for analyzing serum bile acids by high resolution gas chromatography.Compared to other extraction methods like adsorption-[1–3] or reversed phase chromatography [4,5], this novel technique yielded a satisfactory recovery (75–104%) with high reproducibility. Therefore a reliable determination of serum bile acids is possible.     

Automated high-performance liquid chromatographic determination of amphetamine in biological fluids using column-switching and on-column derivatization

Summary A rapid and simple liquid-chromatographic method has been developed for on-line quantification of amphetamine in biological fluids. Untreated samples (20 μL) are injected directly into the chromatographic system and purified on a 20 mm×2.1 mm i.d. pre-column packed with 30 μm Hypersil C₁₈ stationary phase. After clean-up the analyte is transferred to the analytical column (125 mm×4 mm i.d., 5 μm LiChrospher 100 RP₁₈) for derivatization and separation using a mixture of acetonitrile and the derivatization reagent (o-phthaldialdehyde andN-acetyl-L-cysteine) as the mobile phase. The experimental conditions for on-line derivatization and resolution of the amphetamine have been optimized, and the results have been compared with those obtained by derivatizing the analyte in pre-column mode. The method described has been applied to the determination of amphetamine in plasma and urine. Good linearity and reproducibility were obtained in the 0.1–10.0 μg mL⁻¹ concentration range, and limits of detection were 25 ng mL⁻¹ and 10 ng mL⁻¹ with UV and fluorescence detection, respectively. The procedure described is very simple and rapid, because no off-line manipulation of the sample is required; the total analysis time is approximately 8 min.     

用蛋白电泳和高效凝胶排阻色谱法分析还原脲变性蛋白溶菌酶稀释复性过程的集聚体

本文利用蛋白电泳和高效凝胶排阻层析法分析了还原脲变性蛋白溶菌酶稀释复性过程中的集聚体。当用复性液稀释复性还原脲变性蛋白溶菌酶时,会迅速产生可观量的沉淀。沉淀和上清液的不连续十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和高效凝胶排阻层析分析结果表明,还原脲变性蛋白溶菌酶在稀释复性过程中除了能够复性成天然态蛋白溶菌酶分子外,还会形成可溶的蛋白溶菌酶分子二聚体和三聚体,二聚体和三聚体主要是靠分子间二硫键的错配连接而成的;可溶的蛋白溶菌酶分子二聚体之间通过非共价键相互作用而形成集聚体沉淀,而可溶的三聚体溶菌酶分子则仍处于复性液上清液中。     

Influence of injection systems on the gas chromatographic analysis of oxygenated dibenzothiophenes

Analytical methods are described for identification and monitoring of the oxygenated metabolites of dibenzothiophene. Since such compounds are thermolabile, GC analysis is seen to be hardly feasible with conventional injectors such as all-glass moving needle or splitless injectors. Only on-column injection gives no degradation products. In addition, reversed-phase HPLC is particularly suitable for the analysis of the sulfone or sulfoxides of dibenzothiophenes.