Multi-mode Chromatography Research Articles

Multimode chromatography‐based techniques for high purity isolation of extracellular vesicles from human blood plasma

AbstractExtracellular vesicles (EVs) play a pivotal role in various biological pathways, such as immune responses and the progression of diseases, including cancer. However, it is challenging to isolate EVs at high purity from blood plasma and other biofluids due to their low abundance compared to more predominant biomolecular species such as lipoprotein particles and free protein complexes. Ultracentrifugation‐based EV isolation, the current gold standard technique, cannot overcome this challenge due to the similar biophysical characteristics of such species. We developed several novel approaches to enrich EVs from plasma while depleting contaminating molecular species using multimode chromatography‐based strategies. On average, we identified 716 ± 68 and 1054 ± 35 protein groups in EV isolates from 100 µL of plasma using multimode chromatography‐ and ultracentrifugation‐based techniques, respectively. The developed methods resulted in similar EV isolates purity, providing significant advantages in simplicity, throughput, scalability, and applicability for various downstream analytical and potential clinical applications.

A method for the quantitative analysis of polar anionic pesticides in milk/infant formula, cereals and fruit and vegetables using ion chromatography coupled to tandem mass spectrometry.

Polar pesticides such as anionic or ionisable compounds have always provided a challenge for analytical chemists. Methods of analysis have been developed using a range of techniques including normal phase chromatography, ion-pairing, derivatisation and HILIC or multi-mode chromatography. These work well with some of these compounds but, except for HILIC, all of them have their limitations and none of them cover the range required by legislation. Some of these compounds, glyphosate, chlorate and phosphonic acid, are found regularly in a range of food matrices, and therefore reliable methods of analysis are essential. This study describes an ion chromatography method with tandem mass spectrometry detection which not only covers the full range of compounds required by legislation but also can be expanded to include other anionic or ionisable pesticides and metabolites. These include glyphosate and its metabolites, glufosinate and its metabolites, ethephon and its metabolites as well as fosetyl aluminium, chlorate and perchlorate. The method is fully validated according to the performance criteria from the SANTE guidelines for the analysis of pesticides in food and feed over a wide range of matrices, including milk, infant formula, cereals and fruits and vegetables. Over 300 food samples have analysed as part of our routine monitoring program.

Ionic liquid and octadecylamine co-derived carbon dots for multi-mode high performance liquid chromatography

In this work, a novel kind of carbon dots (CDs) are synthesized by citric acid, 1-aminoethyl-3-methylimidazolium bromide ionic liquid and octadecylamine. Due to the variety of precursors, the synthesized CDs possess multiple functional groups including carboxyl, amino, hydroxyl and imidazole groups. One CDs functionalized silica gels (Si-CDs) chromatographic column can be applied in three separation modes including reversed-phase liquid chromatography, hydrophilic interaction liquid chromatography and ion exchange chromatography, suitable for separating compounds with different properties. Polycyclic aromatic hydrocarbons, alkylbenzenes, alkaloids, pesticides, antibiotics, sulfonamides, glycosides, inorganic anions, aromatic acids, nucleosides and bases as test compounds can be separated well on this Si-CDs column. For four adjacent analytes of triadimefon, tebuconazole, diniconazole and diclofop, the resolution reaches 3.77, 2.34 and 5.17 with an average column efficiency of 9500 plates/m. Retention mechanism contains hydrophobic, π-π stacking and electrostatic interactions according to linear solvation energy relationship. This Si-CDs column is used for real sample analysis of residual thiamphenicol and chloramphenicol in milk. With simplified sample pretreatment process, spiked recoveries between 89.3%-112% indicates the developed detection method based on this Si-CDs column is not only rapid but also reliable. Using CDs with multiple functional ligands as modifiers for multi-mode stationary phases avoids multi-step modification process of various small molecules on silica gels, which is helpful to the evolution of multi-mode chromatography and nanomaterials-based separation science.

"Production of Equine Anti-Daboia Siamensis Immunoglobulin F(ab’)2 Fragments with Ammonium Sulphate and Caprylic Acid Precipitation and by Multimode Chromatography"

"Production of Equine Anti-Daboia Siamensis Immunoglobulin F(ab’)2 Fragments with Ammonium Sulphate and Caprylic Acid Precipitation and by Multimode Chromatography"

Efficient production of immunologically active Shigella invasion plasmid antigens IpaB and IpaH using a cell-free expression system

Shigella spp. invade the colonic epithelium and cause bacillary dysentery in humans. Individuals living in areas that lack access to clean water and sanitation are the most affected. Even though infection can be treated with antibiotics, Shigella antimicrobial drug resistance complicates clinical management. Despite decades of effort, there are no licensed vaccines to prevent shigellosis. The highly conserved invasion plasmid antigens (Ipa), which are components of the Shigella type III secretion system, participate in bacterial epithelial cell invasion and have been pursued as vaccine targets. However, expression and purification of these proteins in conventional cell-based systems have been challenging due to solubility issues and extremely low recovery yields. These difficulties have impeded manufacturing and clinical advancement. In this study, we describe a new method to express Ipa proteins using the Xpress+TM cell-free protein synthesis (CFPS) platform. Both IpaB and the C-terminal domain of IpaH1.4 (IpaH-CTD) were efficiently produced with this technology at yields > 200 mg/L. Furthermore, the expression was linearly scaled in a bioreactor under controlled conditions, and proteins were successfully purified using multimode column chromatography to > 95% purity as determined by SDS-PAGE. Biophysical characterization of the cell-free synthetized IpaB and IpaH-CTD using SEC-MALS analysis showed well-defined oligomeric states of the proteins in solution. Functional analysis revealed similar immunoreactivity as compared to antigens purified from E. coli. These results demonstrate the efficiency of CFPS for Shigella protein production; the practicality and scalability of this method will facilitate production of antigens for Shigella vaccine development and immunological analysis.Key points• First report of Shigella IpaB and IpaH produced at high purity and yield using CFPS• CFPS-IpaB and IpaH perform similarly to E. coli–produced proteins in immunoassays• CFPS-IpaB and IpaH react with Shigella-specific human antibodies and are immunogenic in mice.Graphical abstract

Preparation and Evaluation of a Cholesterol Derivatized β-Cyclodextrin-bonded Phase for Achiral and Chiral HPLC.

A cholesterol mono-derivatized β-cyclodextrin was synthesized and bonded onto silica gel (SBA-15) to obtain a cholesterol mono-derivatized β-cyclodextrin-bonded stationary phase (CHCDP). The chemical structures of mono-derivatized β-cyclodextrin and CHCDP were characterized by infrared spectroscopy, mass spectrometry, elemental analysis and thermogravimetric analysis, correspondingly. Furthermore, the separation ability of CHCDP in terms of achiral compounds was systematically evaluated by separating benzene homologs, polycyclic aromatic hydrocarbons (PAHs) and some positional isomers. As a result, CHCDP completely separated five benzene homologs and nine PAHs within 30 min under the reversed-phase. In addition, the chiral chromatographic property of CHCDP was also evaluated by separating some racemic compounds including flavanones, triazoles, β-blockers, etc. The results showed that the CHCDP exhibited high enantioselectivities towards most of selected analytes. The enantioresolutions were in the range from 1.43 to 2.51 on CHCDP. Especially the resolutions of 2'-hydroxyflavanone, hexaconazole, Dns-serine and atenolol were as high as 1.94, 1.91, 2.15 and 1.57, respectively. Obviously, the CHCDP was a versatile stationary phase with chiral and achiral separation capabilities in multi-mode chromatography, which was related to the introduction of cholesterol to the port of cyclodextrin, enhancing the hydrophobic interaction of cyclodextrin with achiral compounds, while maintaining the inclusion complexation of it with chiral compounds as well.

Multimodal Chromatography for Purification of Biotherapeutics - A Review.

Process chromatography forms the core of purification of biotherapeutics. The unparalleled selectivity that it offers over other alternatives combined with the considerable robustness and scalability make it the unit operation of choice in downstream processing. It is typical to have three to five chromatography steps in a purification process for a biotherapeutic. Generally, these steps offer different modes of separation such as ion-exchange, reversed phase, size exclusion, and hydrophobic interaction. In the past decade, multimodal chromatography has emerged as an alternative to the traditional modes. It involves use of more than one mode of separation and typically combines ion-exchange and hydrophobic interactions to achieve selectivity and sensitivity. Over the last decade, numerous authors have demonstrated the significant potential that multimode chromatography offers as a protein purification tool. This review aims to present key recent developments that have occurred on this topic together with a perspective on future applications of multimodal chromatography.

Industrial Purification Strategies for Monoclonal Antibodies

The global monoclonal antibody market in 2016 was around USD 85.4 billion and is expected to reach USD 140 billion by end of 2024 i.e., approximately 1.5 fold increase in market rate within 10 years. The therapeutics purification process monoclonal antibody through chromatography technique is expected to grow by 5% from 2011 to 2017, with a prediction of reaching to USD 9 billion by end of 2017. The global market interest is shifting from monoclonal antibody to fragmented antibody; nearly 60 fragmented antibodies have gone into clinical trials as of 2010. In this review, recovery of monoclonal antibody by centrifugation, depth filtration followed by purification by chromatography techniques i.e., capture step by affinity chromatography, intermediate step by anionic chromatography in flow through mode and polishing step by cationic chromatography is reviewed. The production of antigen binding fragment is carried out by enzymatic method followed by affinity chromatography, multimode chromatography and cationic chromatography. Monoclonal antibodies have been developed by these methods are used for targeting range of diseases. Many of such monoclonal antibodies have been approved and many such is still in development pipeline. This review gives an overview on the structure of antibody, production of monoclonal antibody and the downstream purification of monoclonal antibody. The downstream purification covers the basic techniques followed for most of the mAb purification done commercially. The downstream purification steps discussed here include centrifugation, tangential flow filtration, depth filtration and chromatographic techniques.

Metformin Improves Diabetic Bone Health by Re-Balancing Catabolism and Nitrogen Disposal.

ObjectiveMetformin, a leading drug used to treat diabetic patients, is reported to benefit bone homeostasis under hyperglycemia in animal models. However, both the molecular targets and the biological pathways affected by metformin in bone are not well identified or characterized. The objective of this study is to investigate the bioengergeric pathways affected by metformin in bone marrow cells of mice.Materials and MethodsMetabolite levels were examined in bone marrow samples extracted from metformin or PBS -treated healthy (Wild type) and hyperglycemic (diabetic) mice using liquid chromatography-mass spectrometry (LC-MS)-based metabolomics. We applied an untargeted high performance LC-MS approach which combined multimode chromatography (ion exchange, reversed phase and hydrophilic interaction (HILIC)) and Orbitrap-based ultra-high accuracy mass spectrometry to achieve a wide coverage. A multivariate clustering was applied to reveal the global trends and major metabolite players.ResultsA total of 346 unique metabolites were identified, and they are grouped into distinctive clusters that reflected general and diabetes-specific responses to metformin. As evidenced by changes in the TCA and urea cycles, increased catabolism and nitrogen waste that are commonly associated with diabetes were rebalanced upon treatment with metformin. In particular, we found glutamate and succinate whose levels were drastically elevated in diabetic animals were brought back to normal levels by metformin. These two metabolites were further validated as the major targets of metformin in bone marrow stromal cells.ConclusionOverall using limited sample size, our study revealed the metabolic pathways modulated by metformin in bones which have broad implication in our understanding of bone remodeling under hyperglycemia and in finding therapeutic interventions in mammals.

Multi-mode chromatographic evaluation of a new lysine-silica stationary phase for high-performance liquid chromatography

A new stationary phase based on silica bonded with N-terminally protected lysine was synthesized using graft-to methodology and subsequently characterized by elemental analysis, thermogravimetric analysis, and diffuse reflectance infrared Fourier transform spectroscopy. The grafted phase was then applied to multi-mode chromatography, including effective separation of hydrophobic polyaromatic compounds in reversed-phase and polar biomolecules in hydrophilic interaction liquid chromatography.

Unfolding of a model protein on ion exchange and mixed mode chromatography surfaces

Recent studies with proteins indicate that conformational changes and aggregation can occur during ion exchange chromatography (IEC). Such behavior is not usually expected, but could lead to decreased yield and product degradation from both IEC and multi mode chromatography (MMC) that has ligands of both hydrophobic and charged functionalities. In this study, we used hydrogen exchange mass spectrometry to investigate unfolding of the model protein BSA on IEC and MMC surfaces under different solution conditions at 25°C. Increased solvent exposure, indicating greater unfolding relative to that in solution, was found for protein adsorbed on cationic IEC and MMC surfaces in the pH range of 3.0 to 4.5, where BSA has decreased stability in solution. There was no effect of anionic surfaces at pH values in the range from 6.0 to 9.0. Differences of solvent exposure of whole molecules when adsorbed and in solution suggest that adsorbed BSA unfolds at lower pH values and may show aggregation, depending upon pH and the surface type. Measurements on digested peptides showed that classifications of stability can be made for various regions; these are generally retained as pH is changed. When salt was added to MMC systems, where electrostatic interactions would be minimized, less solvent exposure was seen, implying that it is the cationic moieties, rather than the hydrophobic ligands, which cause greater surface unfolding at low salt concentrations. These results suggest that proteins of lower stability may exhibit unfolding and aggregation during IEC and MMC separations, as they can with hydrophobic interaction chromatography.

Simple and Sensitive Method for Measurement of Metoclopramide in Cattle Plasma by LC-MS/MS Using a Multimode Chromatography

A quantitative assay method using LC/ESI-MS/MS for simultaneous determination of MCP in cattle plasma was developed and validated. Chromatographic separation was carried out using a multimode column (2 × 150 mm, 3 μm) with gradient elution (0.05% formic acid/methanol with 0.05% formic acid). MCP and levosulpiride (internal standard) were analyzed in the precursor/product ion pair of m/z 300.1/226.9 and 342.0/112.0, respectively. Linear calibration curves were obtained in the range of 2.5-500 ng/ml (R(2)>0.999) with a lower limit of quantification of 0.05 ng/ml. Mean recoveries were 96-103%, and the coefficient of variation was less than 6.5%. Plasma MCP concentrations after intravenous administration at 0.4 mg/kg to 12 cattle were determined by the LC-MS/MS method.

Synthesis and characterization of phenylpropanolamine bonded silica for multimode liquid chromatography of small molecules

A multimodal silica support carrying both anionexchange and hydrophobic functions was prepared by bonding phenylpropanolamine to epoxysilane-modified silica. This support was characterized with regard to its physical and chromatographic properties and has a surface coverage of 150 μmol g−1. The reversed-phase and the anion-exchange behaviour of the new stationary phase were investigated by injection of acidic, neutral and basic drugs and inorganic anions. The retention of the tested compounds was manipulated by modifying the pH and proportion of organic modifier in the mobile phase in isocratic mode. The mixed-mechanism column provided a flexible and versatile method for the simultaneous separation of neutral, acidic and basic compounds.

Determination of Pharmaceuticals by Multi-Phase Chromatography: Combined Reversed Phase and Ion Exchange in One Column

Abstract Separations of various classes of pharmaceuticals using several HPLC retention modes simultaneously are described. The drugs including barbiturates, anti-inflammatories, cephalosporins and sulfa antibiotics are separated by a combination of reversed phase and ion exchange on a new multi-phase HPLC column. This column contains two types of particles, a macroporous substrate particle, which has adsorptive properties, and a monolayer of ion exchange latex particles which is attached to the substrate. The reversed phase and ion exchange retention modes operate independently and therefore can be controlled independently through selection of eluant components and gradient conditions. This approach to multi-mode chromatography is applied to accomplish assay reduction and on-line sample preparation. The determination of drugs in biomedical matrices such as urine, serum and saliva are discussed. Advantages of multi-mode chromatography over ion-pair reversed phase chromatography are discussed.

Separation of peptides by multimode chromatography on a column packed with vinyl alcohol copolymer gel

Several mechanisms of peptide separation in high-performance liquid chromatography were observed to occur on the Asahipak ® GS-320 packed with vinyl alcohol copolymer. Neutral rather than acidic mobile phases were employed as they were found to result in higher retention of many peptides on the GS-320. For neutral peptides, the retention volume corresponded to the Rekker's hydrophobic fragmental constant, with a correlation coefficient of 0.71. Peptides with acidic residues eluted early, as an effect of ionic exclusion; those with basic residues were retained longer, owing to an ion-exchange effect. The ionic interactions were shown to involve the car☐ylic group present on the gel polymer. The net result was found to be excellent separation of hydrophilic as well as hydrophobic peptides, related to differences in their isoelectric points. The combination of these complex mechanisms, together with the size-exclusion effect of the GS-320 gel for separation of proteins and other large molecules and for analysis with a mobile phase high in acetonitrile content, makes possible high-resolution isocratic analysis of peptides, which cannot be achieved on octadecylsilica or ion-exchange columns.

Multi-mode liquid chromatography of polystyrenes on Partisil PAC

Multi-mode liquid chromatography of polystyrenes on Partisil PAC