Surfactant-aided size exclusion chromatography

The flexibility and selectivity of size exclusion chromatography (SEC) for protein purification can be modified by adding non-ionic micelle-forming surfactants to the mobile phase. The micelles exclude proteins from a liquid phase similar to the exclusion effect of the polymer fibers of the size exclusion resin. This surfactant-aided size exclusion chromatography technology (SASEC) is demonstrated on the separation of two model proteins; bovine serum albumin (BSA) and myoglobin (Myo). The effect of the added surfactants on the distribution behavior of the proteins is predicted adequately by a size exclusion model presented in this paper.     

Measurements of protein-protein interactions by size exclusion chromatography

A method is presented for determining second virial coefficients (B(2)) of protein solutions from retention time measurements in size exclusion chromatography. We determine B(2) by analyzing the concentration dependence of the chromatographic partition coefficient. We show the ability of this method to track the evolution of B(2) from positive to negative values in lysozyme and bovine serum albumin solutions. Our size exclusion chromatography results agree quantitatively with data obtained by light scattering.     

Refolding of denatured ribonuclease observed by size exclusion chromatography

The unfolding and refolding of pancreatic ribonuclease have been observed by absorbance, fluorescence, and size exclusion chromatographic measurements in solutions of guanidinium chloride continuously maintained at pH 6.0 and 4 degrees C. The spectral measurements were fitted with a minimal number of kinetic phases while the chromatographic measurements were simulated from an explicit mechanism. All of the measurements are consistent with a minimal mechanism involving seven components. The folded components include the native protein and two transiently stable intermediates each having the same hydrodynamic volume. The intermediate having all native peptide isomers has an unfolding midpoint in 3.8 M denaturant while the intermediate having one nonnative peptide isomer has an unfolding midpoint in 1.3 M denaturant. The unfolded protein is distributed among four components having the same hydrodynamic volume but differing peptide isomers. At equilibrium, 10% of the denatured protein has all native isomers, 60% has one nonnative isomer, 5% has a different nonnative isomer, and 25% has both nonnative isomers. In low denaturant concentrations, the dominant component with one nonnative isomer can refold to transiently populate the compact intermediate with the same nonnative isomer.     

Liposome retention in size exclusion chromatography

Background  

Size exclusion chromatography is the method of choice for separating free from liposome-encapsulated molecules. However, if the column is not presaturated with lipids this type of chromatography causes a significant loss of lipid material. To date, the mechanism of lipid retention is poorly understood. It has been speculated that lipid binds to the column material or the entire liposome is entrapped inside the void.     

Monitoring xanthan quality during fermentation by size exclusion chromatography

Summary Xanthan concentration and molecular weight distribution are determined by size exclusion chromatography in the fermentation medium after dilution and cell removal by centrifugation. The analysis is rapid enough for process control. During a batch fermentation, the average molecular weight is found to be in the range of 7.2–9.3·10⁶ g/mole and to run through a maximum.     

Characterization and analysis of thermal denaturation of antibodies by size exclusion high-performance liquid chromatography with quadruple detection

Size exclusion chromatography (SEC) coupled with online light scattering, viscometry, refractometry, and UV-visible spectroscopy provides a very powerful tool for studying protein size, shape, and aggregation. This technique can be used to determine the molecular weight of the component peaks independent of the retention times in the SEC column and simultaneously measure the hydrodynamic radius and polydispersity of the protein. We applied this technology by coupling an Agilent Chemstation high-performance liquid chromatography system with a diode array UV-visible detector and a Viscotek 300 EZ Pro triple detector (combination of a light scattering detector, refractometer, and differential pressure viscometer) to characterize and compare the molecular properties of a number of monoclonal antibodies. Our studies reveal that different monoclonal immunoglobulin Gs (IgGs) and chimeric IgGs show slightly different retention times and therefore different molecular weights in gel filtration analysis. However, when they are analyzed by light scattering, refractometry, and viscometry, different IgGs have comparable molecular weight, molecular homogeneity (polydispersity), and size. Gel filtration coupled with UV or refractive index detection suggests that antibodies purified and formulated for preclinical and clinical development are more than 95% monomer with little or no detectable soluble aggregates. Light scattering measurements showed the presence of trace amounts of soluble aggregate in all the IgG preparations. The different IgG molecules showed different susceptibility to heat and pH. One of the murine antibodies was considerably less stable than the others at 55 degrees C. The application of this powerful technology for the characterization of monoclonal antibodies of therapeutic potential is discussed.     

Purification of prostaglandin D synthase by ceramic- and size exclusion chromatography

Prostaglandin D synthase (L-PGDS) is a major glycosylated polypeptide in cerebrospinal fluid (CSF). The overexpression of L-PGDS in inflamed bovine mammary glands indicates its role as biomarker. No diagnostic tool for the quantitative detection of L-PGDS in cows has been reported. Immunometric ELISA tests might help to identify inflamed bovine tissue. The isolation of pure bovine L-PGDS, which is required for the generation of monoclonal antibodies, is an important prerequisite for a diagnostic ELISA test. Our goal was to identify a suitable technique to generate pure L-PGDS from bovine substrates. In the present study a two-step method for the purification of bovine CSF using ceramic hydroxyapatite chromatography followed by size exclusion chromatography is described. Subsequently, the identification of bovine L-PGDS was demonstrated by Western blot analysis and the high grade of the pure product was shown by 2-D PAGE. The yield of purified L-PGDS was 6.8 mg/l bovine CSF. L-PGDS from bovine CSF is shown to consist of multiple isoforms identical in molecular mass and pI values to those in previously described secretions of inflamed bovine mammary glands. In addition, the method was successfully applied to the purification of L-PGDS from human CSF.     

Distribution analysis of cellulose acetate phthalate by ion-exclusion-moderated size exclusion chromatography

Ion-exclusion is the electrostatic repulsive interaction between a charged polymer and charges of the same sign on the surface of a column packing. Controlled ion-exclusion allows compensation of hydrophobic adsorption in size exclusion chromatography of negatively charged cellulose acetate phthalate (CAP) polymers in acetone/water/LiCl (80/20) as a mobile phase. Properly selected low-ionic-strength conditions provide correct separation in size-exclusion mode also in binary solvent mixtures. Possible interfering effects related to light scattering at low-salt conditions are shown to be negligible if on-line concentration/light scattering detection is used. The absence of these interferences is easily checked by a comparison of experiments at two different low-salt concentrations. Molecular weight averages and distributions identical within the experimental error are obtained when both salt concentrations are properly selected.     

Aluminium speciation in soil solutions as studied by size exclusion chromatography

Size exclusion chromatography was used for the fractionation of the aqueous extracts taken from different soil horizons (LOf, Oh, Ah, 15 and 35 cm). The aluminium content in the fractions was determined by graphite furnace atomic absorption spectrometry. In the fractions obtained from the LOf, Oh and Ah horizons, a great part of the total aluminium was bound to organic molecules. Over 90% of the aluminium in mineral soil solutions (15 and 35 cm depth) was of low molecular weight or associated with those species.     

Assay for Rab geranylgeranyltransferase using size exclusion chromatography

A simple chromatographic assay for Rab geranylgeranyltransferase (Rab GGTase) has been developed. The method involves separation of the reaction mixture on a Sephadex G-25 superfine minicolumn. Addition of 2-propanol to the assay results in substantial (approximately 90%) decline of formation of noncovalent lipid-protein complexes, increasing reproducibility and reliability of the method. The activity of Rab prenyltransferase was measured in crude and partially purified enzyme preparations from wheat seedlings; measurements for several other plants and rat brain cytosol fractions are also presented. This method can be routinely applied to evaluate the activity of different protein prenyltransferases.     

Improved molecular weight analysis of streptococcal hyaluronic acid by size exclusion chromatography

Summary Four size exclusion chromatography (SEC) calibration techniques were tested for use in the molecular weight characterisation of Streptococcal Hyaluronic Acid (HA). An exponential equation, evaluated using the Hamielec method, was superior to the customary peak position method. It provided the most accurate estimates of the weight average molecular weight, Mw. The calibration was valid for HA in the range 800 – 2500 kDa, and permitted the calculation of both polydispersity and molecular weight distributions for HA from Streptococcal fermentations. This exponential calibration approach should have application in the characterisation of other large biopolymers, particularly where pore size of available SEC media is limiting.     

Hydraulic characteristics of aerobic granules using size exclusion chromatography

Elution of poly(ethylene glycol) of molecular weight 200-20,000 Da from a size exclusion chromatography column packed with phenol-fed aerobic granules of three different nominal sizes (types I-III) has been investigated. The pore sizes of the three types of granules were evaluated based on the mean hydraulic times of the elution curves that decreased directly proportional to the increased logarithm of the molecular mass of a standard tracer and increased as granule size decreased. The corresponding exclusion limits for types I-III granules were 139,000, 123,000, and 54,500 Da, respectively. A one-dimensional convection-dispersion model described the effective dispersion coefficients of the tracers through the granule column. The intra-granular permeabilities and convective and diffusional transit times through the granule interior were evaluated by a dual porosity model. For small molecules of molecular mass <5,000 Da, intra-granular convection dominated transport mechanisms at fast moving velocity. For comparatively larger molecules, diffusion barrier existed to limit nutrient supply to the granules. The size exclusion test provided intra granular transport characteristics using detailed analysis on the elution data.     

Multidimensional profiling of plasma lipoproteins by size exclusion chromatography followed by reverse-phase protein arrays

The composition of lipoproteins and the association of proteins with various particles are of much interest in the context of cardiovascular disease. Here, we describe a technique for the multidimensional analysis of lipoproteins and their associated apolipoproteins. Plasma is separated by size exclusion chromatography (SEC), and fractions are analyzed by reverse-phase arrays. SEC fractions are spotted on nitrocellulose slides and incubated with different antibodies against individual apolipoproteins or antibodies against various apolipoproteins. In this way, tens of analytes can be measured simultaneously in 100 μl of plasma from a single SEC separation. This methodology is particularly suited to simultaneous analysis of multiple proteins that may change their distribution to lipoproteins or alter their conformation, depending on factors that influence circulating lipoprotein size or composition. We observed changes in the distribution of exchangeable apolipoproteins following addition of recombinant apolipoproteins or interaction with exogenous compounds. While the cholesteryl ester transfer protein (CETP)-dependent formation of pre-β-HDL was inhibited by the CETP inhibitors torcetrapib and anacetrapib, it was not reduced by the CETP modulator dalcetrapib. This finding was elucidated using this technique.     

Determination of molecular weight of heparin by size exclusion chromatography with universal calibration

The molecular weight (MW) of heparin can be accurately determined by size exclusion chromatography using "universal calibration." A universal calibration curve was constructed for Superose 12 with standard pullulan samples and verified using characterized ficoll fractions. This calibration yielded the correct MW of heparin as determined by light scattering, when the ionic strength of the mobile phase was maintained over 1.0M. Sodium poly(styrenesulfonate) samples were not suitable standards because of adsorption at high salt concentration and repulsion from the packing material at low ionic strength. The extraordinarily high charge density of heparin leads to the need for high salt concentration to screen such repulsions.     

Characterization of gelatine and acid soluble collagen by size exclusion chromatography coupled with multi angle light scattering (SEC-MALS)

Acid soluble collagen (ASC) and a cattle hide gelatine were analyzed by size exclusion chromatography (SEC) coupled with multi angle light scattering (MALS). The SEC system was calibrated with ASC and its cyan bromide cleavage products. The accuracy of calibration was confirmed by MALS by measuring the mass-average molar masses (Mw). ASC acted as a mixture of two polymer standards of Mw = 90 and 180 kg/mol, respectively. The elution behavior of the gelatine in SEC-MALS was similar to that of ASC. Therefore, the determination of the molar mass distribution of this gelatine was possible either by SEC, using a calibration curve, or by MALS by direct measurement of Mw. According to the scaling law (1/2) = KMalpha, alpha = 0.78 was determined for the gelatine. This alpha could reflect a structure in solution, which is more similar to an ellipsoid than to a random coil.     

Analysis of oligomeric fermentation substrates and products by high pressure size exclusion chromatography

Summary Commercially available maltodextrins were subjected to high pressure size exclusion chromatography (HPSEC) on Toyo Soda G 2000 PW columns with water as the mobile phase. The elution profiles of these samples will allow researchers to select the correct maltodextrin for growth studies requiring specific dextrin oligomers. Characterization of the chromatography system with standards of known molecular weight allows estimation of the weight average molecular weight of polydisperse dextrins. The enzymatic hydrolysis of dextrin by bacterial-amylase and fungal glucoamylase was also monitored by HPSEC.     

Methodology for assaying recombinant interleukin-2 associated with liposomes by combined gel exclusion chromatography and fluorescence

A simple methodology based on fluorescence and gel exclusion chromatography (GEC) has been developed to assay recombinant Interleukin-2 (rIL-2) associated with vesicles. A Sephadex G75 column was used to separate the liposomes from non-entrapped rIL-2. The elution of the rIL-2 liposomes was monitored by coupling fluorescent and light scattering detection. The solubilisation of the vesicles with octylglucoside (OG) before the assay was necessary to avoid interference from light scattering. This methodology can be automated to yield an on-line system that can separate, solubilise and quantify rIL-2 in liposome samples. It can be extended to any protein associated with vesicles provided that the former can be detected by fluorescence.     

On-line separation of solute mixtures using reciprocating size exclusion chromatography

Solutes of different size in a mixture solution were separated on-line, using a semi-continuous reciprocating size exclusion chromatography. The band of fast-moving large molecules was isolated during the first half cycle, while the band of slow-moving small molecules was isolated during the second half cycle. After 7 cycles of frontal mode operation, 89% of the Blue Dextran in the feed was isolated as a pure solution. Vitamin B₁₂ of constant concentration was also isolated as a pure solution.     

Denaturation by urea and renaturation of 20 beta-hydroxysteroid dehydrogenase studied by high-performance size exclusion chromatography

The denaturation by urea and renaturation of 20 beta-hydroxysteroid dehydrogenase, a tetrameric enzyme consisting of four identical subunits, were followed by high-performance size exclusion chromatography to detect intermediates in the processes. During the denaturation process no intermediate form (structured monomers or dimers) between the tetramer and the denatured monomer was observed. During the renaturation process, carried out either with or without NADH, high molecular weight aggregates, native tetramers, and low molecular weight intermediates were evidenced and quantified. The contemporaneous measurement of recovery of activity unambiguously demonstrated that the tetrameric structure is essential for enzymatic activity.