Comprehensive online multicolumn two-dimensional liquid chromatography-diode array detection-mass spectrometry workflow as a framework for chromatographic screening and analysis of new drug substances.

The analysis of complex mixtures of closely related species is quickly becoming a bottleneck in the development of new drug substances, reflecting the ever-increasing complexity of both fundamental biology and the therapeutics used to treat disease. Two-dimensional liquid chromatography (2D-LC) is emerging as a powerful tool to achieve substantial improvements in peak capacity and selectivity. However, 2D-LC suffers from several limitations, including the lack of automated multicolumn setups capable of combining multiple columns in both dimensions. Herein, we report an investigation into the development and implementation of a customized online comprehensive multicolumn 2D-LC-DAD-MS setup for screening and method development purposes, as well as analysis of multicomponent biopharmaceutical mixtures. In this study, excellent chromatographic performance in terms of selectivity, peak shape, and reproducibility were achieved by combining reversed-phase (RP), strong cation exchange (SCX), strong anion exchange (SAX), and size exclusion chromatography (SEC) using sub-2-µm columns in the first dimension in conjunction with several 3.0 mm × 50 mm RP columns packed with sub-3-µm fully porous particles in the second dimension. Multiple combinations of separation modes coupled to UV and MS detection are applied to the LC × LC analysis of a protein standard mixture, intended to be representative of protein drug substances. The results reported in this study demonstrate that our automated online multicolumn 2D-LC-DAD-MS workflow can be a powerful tool for comprehensive chromatographic column screening that enables the semi-automated development of 2D-LC methods, offering the ability to streamline full visualization of sample composition for an unknown complex mixture while maximizing chromatographic orthogonality. Graphical Abstract.

A Top-Down Proteomics Platform Coupling Serial Size Exclusion Chromatography and Fourier Transform Ion Cyclotron Resonance Mass Spectrometry.

Mass spectrometry (MS) based top-down proteomics provides rich information about proteoforms arising from combinatorial amino acid sequence variations and post-translational modifications (PTMs). Fourier transform ion cyclotron resonance (FT-ICR) MS affords ultrahigh resolving power and provides high-accuracy mass measurements, presenting a powerful tool for top-down MS characterization of proteoforms. However, the detection and characterization of large proteins from complex mixtures remain challenging due to the exponential decrease in S: N with increasing molecular weight (MW) and coeluting low-MW proteins; thus, size-based fractionation of complex protein mixtures prior to MS analysis is necessary. Here, we directly combine MS-compatible serial size exclusion chromatography (sSEC) fractionation with 12 T FT-ICR MS for targeted top-down characterization of proteins from complex mixtures extracted from human and swine heart tissue. Benefiting from the ultrahigh resolving power of FT-ICR, we isotopically resolved 31 distinct proteoforms (30-50 kDa) simultaneously in a single mass spectrum within a 100 m/ z window. Notably, within a 5 m/ z window, we obtained baseline isotopic resolution for 6 distinct large proteoforms (30-50 kDa). The ultrahigh resolving power of FT-ICR MS combined with sSEC fractionation enabled targeted top-down analysis of large proteoforms (>30 kDa) from the human heart proteome without extensive chromatographic separation or protein purification. Further separation of proteoforms inside the mass spectrometer (in-MS) allowed for isolation of individual proteoforms and targeted electron capture dissociation (ECD), yielding high sequence coverage. sSEC/FT-ICR ECD facilitated the identification and sequence characterization of important metabolic enzymes. This platform, which facilitates deep interrogation of proteoform primary structure, is highly tunable, allows for adjustment of MS and MS/MS parameters in real time, and can be utilized for a variety of complex protein mixtures.

Biochemical and biophysical characterization of a prokaryotic Mg2+ ion channel: Implications for cost-effective purification of membrane proteins.

Although magnesium is the second most abundant cation present in the cell, the transport mechanism of Mg2+ across membranes is poorly understood. Importantly, the prokaryotic MgtE Mg2+ channel is related to mammalian SLC41A1 transporters and, therefore, biochemical and biophysical characterization of MgtE and its orthologs assumes significance. To date, the purification and structure determination of MgtE from Thermus thermophilus has been carried out using the widely used nonionic detergent, n-dodecyl-ß-d-maltopyranoside (DDM). However, DDM is an expensive detergent and alternative methods to produce high-quality proteins in stable and functional form will be practically advantageous to carry out structural studies in a cost-effective manner. In this work, we have utilized 'dual-detergent strategy' to successfully purify MgtE channel in a stable and functional form by employing relatively inexpensive detergents (Triton X-100 and Anzergent 3-14) for membrane solubilization and subsequently changed to DDM during purification. Our results show that Triton X-100 and Anzergent 3-14 extract MgtE well and the quality of purified protein is comparable to DDM-extracted MgtE. Interestingly, addition of high concentration of salt and glycerol during solubilization does not significantly affect the quantity and quality of MgtE. Importantly, limited proteolysis assay, circular dichroism spectroscopy and ensemble tryptophan fluorescence strongly support the use of Triton X-100, in particular, as an inexpensive, alternative detergent for the purification of MgtE without compromising the structural integrity of the channel and Mg2+-induced gating-related conformational dynamics. Overall, these results are relevant for the cost-effective purification of stable and functional membrane proteins in general, and magnesium channels, in particular.

A fully automated three-step protein purification procedure for up to five samples using the NGC chromatography system.

The drug discovery process in the biopharmaceutical industry usually starts with the generation of plasmids coding for certain proteins. Due to advances in cloning techniques the generation of thousands of different plasmids is not a limiting factor anymore. The next step is the expression and evaluation of the proteins. In recent years significant progress has been made in the miniaturization of protein expression and purification. These processes have been adapted to robotic platforms and hundreds of proteins can be expressed and purified in parallel. As a consequence of miniaturization, the protein purification is restricted to a one-step process. In addition the amount of purified protein is usually in the µg-range. This might be suitable if a sensitive initial screening assay is available. However, when larger amounts of proteins are required robotic platforms are no longer appropriate. In addition, a one-step purification procedure is often not sufficient to obtain pure protein preparations. To address this topic we have used the NGC chromatography system for automated purification of up to five samples using a three-step purification procedure. The first chromatographic step is the capture step followed by a desalting step. The final purification was done using size exclusion chromatography. This set-up reduces the overall-time needed for protein production, needs minimal operator invention, is easy to handle and thus increases the throughput.

Comprehensive two-dimensional liquid chromatography for the characterization of acrylate-modified hyaluronic acid.

Hyaluronic acid and its acrylate derivatives are important intermediates for various pharmaceutical, biomedical, and cosmetic applications due to their biocompatibility and viscoelasticity properties. However, these polymers are inherently difficult to characterize due to their significant heterogeneity regarding molar mass and chemical composition (degree of substitution, DS). The present study describes the development of a comprehensive online two-dimensional liquid chromatography (2D-LC) approach to characterize hyaluronic acid and its acrylate derivatives (DS ranging from 0.4 to 3.1) in terms of molar mass and degree of substitution. In the first dimension of the 2D-LC method, separation according to chemical composition/DS was achieved by using a stepwise solvent gradient and a reversed phase C8 column. Fractions from the first dimension were automatically transferred to the second dimension comprising size exclusion chromatographic separation of the fractions according to molar mass. It was found that the hyaluronic acid derivatives were broadly distributed with regard to both chemical composition and molar mass. Fractions with different degrees of substitution were identified, and their molar mass distributions were determined. The study proved that comprehensive 2D-LC is a powerful approach to reveal the complex nature of hyaluronic acid and its derivatives. Graphical abstract.

Isolation and Purification of a Neuroprotective Phlorotannin from the Marine Algae Ecklonia maxima by Size Exclusion and High-Speed Counter-Current Chromatography.

Phlorotannins are polyphenolic metabolites of marine brown algae that have been shown to possess health-beneficial biological activities. An efficient approach using a combination of high-speed counter-current chromatography (HSCCC) and size exclusion chromatography with a Sephadex LH-20 has been successfully developed for the isolation and purification of a neuroprotective phlorotannin, eckmaxol, from leaves of the marine brown algae, Ecklonia maxima. The phlorotannin of interest, eckmaxol, was isolated with purity >95% by HSCCC using an optimized solvent system composed of n-hexane-ethyl acetate-methanol-water (2:8:3:7, v/v/v/v) after Sephadex LH-20 size exclusion chromatography. This compound was successfully purified in the quantity of 5.2 mg from 0.3 kg of the E. maxima crude organic extract. The structure of eckmaxol was identified and assigned by NMR spectroscopic and mass spectrometric analyses. The purification method developed for eckmaxol will facilitate the further investigation and development of this neuroprotective agent as a drug lead or pharmacological probe. Furthermore, it is suggested that the combination of HSCCC and size exclusion chromatography could be more widely applied for the isolation and purification of phlorotannins from marine algae.

A brief practical review of size exclusion chromatography: Rules of thumb, limitations, and troubleshooting.

Through years of research, teaching, troubleshooting, and editing, I have developed a solid working understanding of many aspects of protein purification and protein biochemistry. I have also found that many researchers do not understand the basics, let alone the pitfalls, of the techniques they use. In the case of size exclusion chromatography (SEC), I summarize below some of the rules of thumb, suggestions, limitations and troubleshooting I have found most useful in helping my students and colleagues carry out successful SEC.

Submicron Protein Particle Characterization using Resistive Pulse Sensing and Conventional Light Scattering Based Approaches.

PURPOSE: Characterizing submicron protein particles (approximately 0.1-1µm) is challenging due to a limited number of suitable instruments capable of monitoring a relatively large continuum of particle size and concentration. In this work, we report for the first time the characterization of submicron protein particles using the high size resolution technique of resistive pulse sensing (RPS). METHODS: Resistive pulse sensing, dynamic light scattering and size-exclusion chromatography with in-line multi-angle light scattering (SEC-MALS) are performed on protein and placebo formulations, polystyrene size standards, placebo formulations spiked with silicone oil, and protein formulations stressed via freeze-thaw cycling, thermal incubation, and acid treatment. RESULTS: A method is developed for monitoring submicron protein particles using RPS. The suitable particle concentration range for RPS is found to be approximately 4 × 107-1 × 1011 particles/mL using polystyrene size standards. Particle size distributions by RPS are consistent with hydrodynamic diameter distributions from batch DLS and to radius of gyration profiles from SEC-MALS. RPS particle size distributions provide an estimate of particle counts and better size resolution compared to light scattering. CONCLUSION: RPS is applicable for characterizing submicron particles in protein formulations with a high degree of size polydispersity. Data on submicron particle distributions provide insights into particles formation under different stresses encountered during biologics drug development.

Medium Resolution 1 H-NMR at 62 MHz as a New Chemically Sensitive Online Detector for Size-Exclusion Chromatography (SEC-NMR).

A state-of-the-art, medium-resolution 1 H-NMR spectrometer (62 MHz) is used as a chemically sensitive online detector for size-exclusion chromatography of polymers such as polymethylmethacrylate (PMMA) and polystyrene (PS). The method uses protonated eluents and works at typical chromatographic conditions with trace amounts of analytes (<0.5 g L-1 after separation). Strong solvent suppression, e.g., by a factor of 500, is achieved by means of T1 -filtering and mathematical subtraction methods. Substantial improvements are made with respect to previous work in terms of the sensitivity (signal-to-noise ratio up to 130:1, PMMA OCH3 ) and selectivity (peak width, full width half maximum (FWHM) 4 Hz on-flow). Typical homopolymers and a blend are investigated to deformulate their composition along the dimensions of molecular weight and NMR chemical shift. These results validate this new hyphenated chromatography method, which can greatly facilitate analysis and is much more effective than previously published results.

Determination of selected neurotoxic insecticides in small amounts of animal tissue utilizing a newly constructed mini-extractor.

We developed a simple analytical method for the simultaneous determination of representatives of various groups of neurotoxic insecticides (carbaryl, chlorpyrifos, cypermethrin, and α-endosulfan and ß-endosulfan and their metabolite endosulfan sulfate) in limited amounts of animal tissues containing different amounts of lipids. Selected tissues (rodent fat, liver, and brain) were extracted in a special in-house-designed mini-extractor constructed on the basis of the Soxhlet and Twisselmann extractors. A dried tissue sample placed in a small cartridge was extracted, while the nascent extract was simultaneously filtered through a layer of sodium sulfate. The extraction was followed by combined clean-up, including gel permeation chromatography (in case of high lipid content), ultrasonication, and solid-phase extraction chromatography using C18 on silica and aluminum oxide. Gas chromatography coupled with high-resolution mass spectrometry was used for analyte separation, detection, and quantification. Average recoveries for individual insecticides ranged from 82 to 111%. Expanded measurement uncertainties were generally lower than 35%. The developed method was successfully applied to rat tissue samples obtained from an animal model dealing with insecticide exposure during brain development. This method may also be applied to the analytical treatment of small amounts of various types of animal and human tissue samples. A significant advantage achieved using this method is high sample throughput due to the simultaneous treatment of many samples. Graphical abstract Optimized workflow for the determination of selected insecticides in small amounts of animal tissue including newly developed mini-extractor.

Determination of pegfilgrastim aggregates by size-exclusion high-performance liquid chromatography on a methacrylate-based column.

A size-exclusion high-performance liquid chromatographic method using a methacrylate-based column was developed, validated and implemented for the determination of pegfilgrastim aggregates. The samples were directly injected into a TSKgel G4000PWXL column (7.5 mm × 300 mm, 10 µm, <500 A°) with a mobile phase of 100 mM phosphate, pH 2.5. Detection was made at 215 nm and analyses were run at a flow-rate of 0.6 ml/min at 10 °C. Vortex-mixing of samples produced oligomers, however, very high molecular weight aggregates were formed at high temperatures. The method exhibited linearity over the concentration range of 0.1-14 mg/ml for pegfilgrastim monomer and high molecular weight aggregates with a correlation coefficient of greater than 0.99. The method was specific and sensitive, with a lower quantification limit of 0.1 mg/ml and a detection limit of 0.02 mg/ml. Over 1200 samples were analyzed by the present method without significant change in the column performance.

Considerations for Sample Preparation Using Size-Exclusion Chromatography for Home and Synchrotron Sources.

The success of a SAXS experiment for structural investigations depends on two precise measurements, the sample and the buffer background. Buffer matching between the sample and background can be achieved using dialysis methods but in biological SAXS of monodisperse systems, sample preparation is routinely being performed with size exclusion chromatography (SEC). SEC is the most reliable method for SAXS sample preparation as the method not only purifies the sample for SAXS but also almost guarantees ideal buffer matching. Here, I will highlight the use of SEC for SAXS sample preparation and demonstrate using example proteins that SEC purification does not always provide for ideal samples. Scrutiny of the SEC elution peak using quasi-elastic and multi-angle light scattering techniques can reveal hidden features (heterogeneity) of the sample that should be considered during SAXS data analysis. In some cases, sample heterogeneity can be controlled using a small molecule additive and I outline a simple additive screening method for sample preparation.

A Successful Combination: Coupling SE-HPLC with SAXS.

A monodispersed and ideal solution is a central (unique?) requirement of SAXS to allow one to extract structural information from the recorded pattern. On-line Size Exclusion Chromatography (SEC) marked a major breakthrough, separating particles present in solution according to their size. Identical frames under an elution peak can be averaged and further processed free from contamination. However, this is not always straightforward, separation is often incomplete and software have been developed to deconvolve the contributions from the different species (molecules or oligomeric forms) within the sample. In this chapter, we present the general workflow of a SEC-SAXS experiment. We present recent instrumental and data analysis improvements that have improved the quality of recorded data, extended its potential and turn it into a mainstream approach. We describe into some details two specific applications of SEC-SAXS that provide more than just separating associated forms from the particle of interest.

Methods of isolating extracellular vesicles impact down-stream analyses of their cargoes.

Viable tumor cells actively release vesicles into the peripheral circulation and other biologic fluids, which exhibit proteins and RNAs characteristic of that cell. Our group demonstrated the presence of these extracellular vesicles of tumor origin within the peripheral circulation of cancer patients and proposed their utility for diagnosing the presence of tumors and monitoring their response to therapy in the 1970s. However, it has only been in the past 10 years that these vesicles have garnered interest based on the recognition that they serve as essential vehicles for intercellular communication, are key determinants of the immunosuppressive microenvironment observed in cancer and provide stability to tumor-derived components that can serve as diagnostic biomarkers. To date, the clinical utility of extracellular vesicles has been hampered by issues with nomenclature and methods of isolation. The term "exosomes" was introduced in 1981 to denote any nanometer-sized vesicles released outside the cell and to differentiate them from intracellular vesicles. Based on this original definition, we use "exosomes" as synonymous with "extracellular vesicles." While our original studies used ultracentrifugation to isolate these vesicles, we immediately became aware of the significant impact of the isolation method on the number, type, content and integrity of the vesicles isolated. In this review, we discuss and compare the most commonly utilized methods for purifying exosomes for post-isolation analyses. The exosomes derived from these approaches have been assessed for quantity and quality of specific RNA populations and specific marker proteins. These results suggest that, while each method purifies exosomal material, there are pros and cons of each and there are critical issues linked with centrifugation-based methods, including co-isolation of non-exosomal materials, damage to the vesicle's membrane structure and non-standardized parameters leading to qualitative and quantitative variability. The down-stream analyses of these resulting varying exosomes can yield misleading results and conclusions.

Pre-equilibration kinetic size-exclusion chromatography with mass spectrometry detection (peKSEC-MS) for label-free solution-based kinetic analysis of protein-small molecule interactions.

Here we introduce pre-equilibration kinetic size-exclusion chromatography with mass-spectrometry detection (peKSEC-MS), which is a label-free solution-based kinetic approach for characterizing non-covalent protein-small molecule interactions. In this method, a protein and a small molecule are mixed outside the column and incubated to approach equilibrium. The equilibrium mixture is then introduced into the SEC column to initiate the dissociation process by separating small molecules from the complex inside the column. A numerical model of a 1-dimensional separation was constructed to simulate mass chromatograms of the small molecule for varying rate constants of binding.

Isolation and purification of monosialotetrahexosylgangliosides from pig brain by extraction and liquid chromatography.

Monosialotetrahexosylganglioside (GM1), one of glycosphingolipids containing sialic acid, plays particularly important role in fighting against paralysis, dementia and other diseases caused by brain and nerve damage. In this work, a simple and highly efficient method with high yield was developed for isolation and purification of GM1 from pig brain. The method consisted of an extraction by chloroform-methanol-water and a two-step chromatographic separation by DEAE-Sepharose Fast Flow anion-exchange medium and Sephacryl S-100 HR size-exclusion medium. The purified GM1 was proved to be homogeneous and had a purity of >98.0% by high-performance anion-exchange and size-exclusion chromatography. The molecular weight was 30.0 kDa by high-performance size-exclusion chromatography and 1546.9 Da by electrospray ionization mass spectrometry. The chromogenic reaction by resorcinol-hydrochloric acid solution indicated that the purified GM1 showed a specific chromogenic reaction of sialic acid. Through this isolation and purification program, ~1.0 mg of pure GM1 could be captured from 500 g wet pig brain tissue and the yield of GM1 was around 0.022%, which was higher than the yields by other methods. The method may provide an alternative for isolation and purification of GM1 in other biological tissues.

Size-exclusion chromatography-from high-performance to ultra-performance.

Size-exclusion chromatography (SEC) enables measurement of the average molecular weights and molecular-weight distributions of polymers. Because these characteristics may, in turn, be correlated with important performance characteristics of plastics, SEC is an essential analytical technique for characterization of macromolecules. Although SEC is one of the oldest instrumental chromatographic techniques, it is still under continuous development, as a result of the great demand for increased resolution and faster analysis in SEC. Ultra-high-pressure size-exclusion chromatography (UHPSEC) was recently introduced to satisfy the growing demands of analytical chemists. Using instrumentation capable of generating very high pressures and columns packed with small particles, this technique enables greater separation efficiency and faster analysis than are achieved with conventional SEC. UHPSEC is especially advantageous for high-resolution analysis of oligomers, for very rapid polymer separations, and as a second dimension in comprehensive two-dimensional liquid chromatography of polymers. In this paper we discuss the benefits of UHPSEC for separation of macromolecules, with examples from the literature.

The application of hybrid pixel detectors for in-house SAXS instrumentation with a view to combined chromatographic operation.

This study analyses the potential for laboratory-based size-exclusion chromatography (SEC) integrated small-angle X-ray scattering (SAXS) instrumentation to characterize protein complexes. Using a high-brilliance home source in conjunction with a hybrid pixel X-ray detector, the efficacy of SAXS data collection at pertinent protein concentrations and exposure times has been assessed. Scattering data from SOD1 and from the complex of SOD1 with its copper chaperone, using 10 min exposures, provided data quality in the range 0.03 < q < 0.25 Å(-1) that was sufficient to accurately assign radius of gyration, maximum dimension and molecular mass. These data demonstrate that a home source with integrated SEC-SAXS technology is feasible and would enable structural biologists studying systems containing transient protein complexes, or proteins prone to aggregation, to make advanced preparations in-house for more effective use of limited synchrotron beam time.

Sample clean-up by sol-gel immunoaffinity chromatography for the determination of bisphenol A in food and urine.

Bisphenol A (2,2-bis(4-hydroxyphenyl)propane, BPA) is an industrial chemical mainly used as a monomer in the synthesis of polycarbonates and epoxy resins. BPA has been shown to elicit estrogenic effects via binding to the nuclear estrogen receptors α and ß. Food is considered as the major source of BPA exposure for the general human population. When incorporated into the body, BPA is metabolised in the liver, mainly to BPA glucuronide, and excreted via the urine. The present paper presents analytical methods for the determination of BPA concentrations in foodstuffs and the determination of free and total (free plus conjugated) BPA in urine samples. The paper provides protocols for the preparation and operation of sol-gel immunoaffinity columns and their application to remove interfering matrix compounds and to enrich BPA. In addition, the paper points out major sources of systematic errors in BPA analysis and describes how they can be avoided.

Tris(hydroxymethyl)aminomethane-functionalized agarose particles: parameters affecting the binding of bovine serum albumin.

A new protein adsorbent is introduced based on the coupling of the common buffer ion, tris(hydroxymethyl)aminomethane, to the agarose gel Sepharose HP from GE Healthcare Bio-Sciences AB, Uppsala, Sweden. The article describes the synthesis of the new adsorbent and the use of BSA as a model in a binding study. By optimization of the coupling procedure, a maximum ligand density of 63.5 µmol/mL gel could be obtained. Adsorption equilibria were investigated in the pH range 5.0-8.0 and at salt concentrations of 0-0.4 mol/L. Binding of BSA under different conditions indicated that both electrostatic interaction and hydrogen bonding were involved in the adsorption process where the former played a major role.