Acquisition of enzymatic progress curves in real time by quenching-free ion exchange chromatography.

We describe a quenching-free, 'online' ion exchange chromatography (oIEC) method for the quantitative analysis of enzymatic reactions in real-time. We show that separate quenching of the ongoing reaction performed conventionally is not required, since enzymatic reactions are interrupted upon immobilization of the reaction compounds by binding to the stationary phase of the ion exchange column. The reaction mix samples are directly injected into the column, thereby improving data consistency and allowing automation of the process. The method allows reliable and efficient acquisition of enzymatic progress curves by automatic loading of aliquots of an ongoing reaction at predefined timepoints. We demonstrate the applicability of this method for a variety of enzymatic reactions. SUBJECT: Enzymatic assays and analysis.

Combination of strong anion exchange liquid chromatography with microchip capillary electrophoresis sodium dodecyl sulfate for rapid two-dimensional separations of complex protein mixtures.

Two-dimensional separations provide a simple way to increase the resolution and peak capacity of complex protein separations. The feasibility of a recently developed instrumental approach for two-dimensional separations of proteins was evaluated. The approach is based on the general principle of two-dimensional gel electrophoresis. In the first dimension, semi-preparative strong anion exchange high-performance liquid chromatography is utilized and fractions are collected by means of a fraction collector. They are subsequently analyzed in the second dimension with microchip capillary electrophoresis sodium dodecyl sulfate. Microchip capillary electrophoresis provides the necessary speed (approximately 1 min/fraction) for short analysis. In this study, three different samples were investigated. Different constructs of soluble guanylyl cyclase were expressed in Sf9-cells using the baculovirus expression system. Cell lysates were analyzed and the resulting separations were compared. In our experimental setup, the soluble guanylyl cyclase was identified among hundreds of other proteins in these cell lysates, indicating its potential for screening, process control, or analysis. The results were validated by immunoblotting. Samples from Chinese hamster ovary cell culture before and after a purification step were investigated and approximately 9% less impurities could be observed. The separation patterns obtained for human plasma are closely similar to patterns obtained with two-dimensional gel electrophoresis and a total of 218 peaks could be observed. Overall, the approach was well applicable to all samples and, based on these results, further directions for improvements were identified. .

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.

On-chip pressure generation using a gel membrane fabricated outside of the microfluidic network.

On-chip generation of pressure gradients via electrokinetic means can offer several advantages to microfluidic assay design and operation in a variety of applications. In this article, we describe a simple approach to realizing this capability by employing a polyacrylamide-based gel structure fabricated within a fluid reservoir located at the terminating end of a microchannel. Application of an electric field across this membrane has been shown to block a majority of the electroosmotic flow generated within the open duct yielding a high pressure at the channel-membrane junction. Experiments show the realization of higher pressure-driven velocities in an electric field-free separation channel integrated to the micropump with this design compared to other similar micropumps described in the literature. In addition, the noted velocity was found to be less sensitive to the extent of Debye layer overlap in the channel network, and therefore more impressive when working with background electrolytes having higher ionic strengths. With the current system, pressure-driven velocities up to 3.6 mm/s were realized in a 300-nm-deep separation channel applying a maximum voltage of 3 kV at a channel terminal. To demonstrate the separative performance of our device, a nanofluidic pressure-driven ion-chromatographic analysis was subsequently implemented that relied on the slower migration of cationic analytes relative to the neutral and anionic ones in the separation channel likely due to their strong electrostatic interaction with the channel surface charges. A mixture of amino acids was thus separated with resolutions greater than those reported by our group for a similar analysis previously.

A parallel demonstration of different resins' antibody aggregate removing capability by a case study.

Aggregation is a major concern for therapeutic monoclonal antibody (mAb), as aggregates reduce drug efficacy and safety. In addition to aggregation control, aggregate removal by downstream processing is crucial. Hydrophobic and mixed-mode resins are widely used for aggregate removal in different cases, but they are seldom compared side by side. In this study, the aggregate removing capability of eight resins belonging to different chromatographic types was demonstrated by a case study. This work, by providing multiple options for aggregate removal, allows more flexibility to be gained in downstream processing.

A novel two-dimensional liquid chromatography system for the simultaneous determination of three monoterpene indole alkaloids in biological matrices.

The present paper describes a novel two-dimensional liquid chromatography (2D-LC) system, which is comprised of a first-dimensional ion exchange chromatography (IEX1) column, trap column, and second-dimensional reversed-phase chromatography (RP2) column system. The biological sample is separated by the first-dimensional LC using an IEX column to remove interferences. The analytes are transferred to the trap column after heart-cutting. Then, the analytes are transferred to the second-dimensional LC using an RP2 column for further separation and ultraviolet detection. This 2D-LC system can offer a large injection volume to provide sufficient sensitivity and exhibits a strong capacity for removing interferences. Here, the determination of three monoterpene indole alkaloids (MIAs; gelsemine, koumine, and humantenmine) from Gelsemium in biological matrices (plasma, tissue, and urine) was used this 2D-LC system. After a rapid and easy sample preparation method based on protein precipitation, the sample was injected into the 2D-LC. The method was developed and validated in terms of the selectivity, LOD, LOQ, linearity, precision, accuracy, and stability. The sample preparation time for the three MIAs was 15 min. The LOD for these compounds was 10 ng/mL, which was lower than the developed HPLC methods. The results showed that this method had good quantitation performance and allowed the determination of gelsemine, koumine, and humantenmine in biological matrices. The method is rapid, exhibits high selectivity, has good sensitivity, and is low-cost, thus making it well-suited for application in the pharmaceutical and toxicological analysis of Gelsemium. Graphical abstract.

Determination of d-myo-inositol phosphates in 'activated' raw almonds using anion-exchange chromatography coupled with tandem mass spectrometry.

BACKGROUND: Activated almonds are raw almonds that have been soaked in water for 12-24 h at room temperature, sometimes followed by a 24 h drying period at low temperature (50 ± 5 °C). This treatment is thought to enhance the nutrient bioavailability of almonds by degrading nutrient inhibitors, such as phytic acid or d-myo-inositol hexaphosphate (InsP6 ), through the release of phytase or passive diffusion of InsP6 into the soaking water. Over a wide pH range, InsP6 is a negatively charged compound that limits the absorption of essential nutrients by forming insoluble complexes with minerals such as iron and zinc. It is hypothesized that hydrating the seed during soaking triggers InsP6 degradation into lower myo-inositol phosphates with less binding capacity. RESULTS: Anion-exchange chromatography coupled with tandem mass spectrometry was used to quantify myo-inositol mono-, di-, tris-, tetra-, penta-, and hexaphosphates (InsP1-6 ) in raw pasteurized activated almonds. At least 24 h of soaking at ambient temperature was required to reduce InsP6 content from 14.71 to 14.01 µmol g-1 . CONCLUSIONS: The reduction in InsP6 is statistically significant (P < 0.05) after 24 h of activation, but only represents a 4.75% decrease from the unsoaked almonds. © 2018 Society of Chemical Industry.

Increasing the Separation Capacity of Intact Histone Proteoforms Chromatography Coupling Online Weak Cation Exchange-HILIC to Reversed Phase LC UVPD-HRMS.

Top-down proteomics is an emerging analytical strategy to characterize combinatorial protein post-translational modifications (PTMs). However, sample complexity and small mass differences between chemically closely related proteoforms often limit the resolution attainable by separations employing a single liquid chromatographic (LC) principle. In particular, for ultramodified proteins like histones, extensive and time-consuming fractionation is needed to achieve deep proteoform coverage. Herein, we present the first online nanoflow comprehensive two-dimensional liquid chromatography (nLC×LC) platform top-down mass spectrometry analysis of histone proteoforms. The described two-dimensional LC system combines weak cation exchange chromatography under hydrophilic interaction LC conditions (i.e., charge- and hydrophilicity-based separation) with reversed phase liquid chromatography (i.e., hydrophobicity-based separation). The two independent chemical selectivities were run at nanoflows (300 nL/min) and coupled online with high-resolution mass spectrometry employing ultraviolet photodissociation (UVPD-HRMS). The nLC×LC workflow increased the number of intact protein masses observable relative to one-dimensional approaches and allowed characterization of hundreds of proteoforms starting from limited sample quantities (∼1.5 µg).

Dual Electrolytic Eluent Generation for Oligosaccharides Analysis Using High-Performance Anion-Exchange Chromatography.

The research on oligosaccharides is growing and gaining in importance at a rapid pace. The efforts to understand their bioactivity and to develop new products based on oligosaccharides in biotherapeutics and food industry require effective and reliable tools for analysis of oligosaccharides. Here we present a dual electrolytic eluent generation platform for the analysis of oligosaccharides by high-performance anion-exchange liquid chromatography (HPAE) in both analytical and capillary column formats. The system consists of one eluent generator producing methanesulfonic acid (MSA) connected in series with a second eluent generator producing potassium hydroxide (KOH). Through manipulating the concentration output of both eluent generators, chromatographic performance comparable to that obtained using the conventional sodium acetate/sodium hydroxide (NaOAc/NaOH) eluents is achieved using the electrolytically generated potassium methanesulfonate/potassium hydroxide (KMSA/KOH) eluent. This platform utilizes deionized water as the only carrier stream through a single isocratic pump, overcomes the various drawbacks associated with manually prepared NaOAc/NaOH eluents, and offers an easy to use, simplified operation solution for oligosaccharides profiling with increased precision and accuracy.

Specific and robust ion chromatographic determination of hypothiocyanite in saliva samples.

The enzymatic system in saliva, consisting of salivary peroxidase (SPO), hydrogen peroxide (H2O2), and thiocyanate (SCN-), produces hypothiocyanite (OSCN-) as a high effective antibacterial compound. OSCN- is of great importance for the natural non-specific antibacterial resistance in the oral cavity. However, no analytical method currently exists to selectively quantify OSCN- in saliva samples. A robust and specific analytical method for the determination of OSCN- was developed based on ion chromatography with combined UV and electrochemical detection. Calibration was achieved by calculating a derived calibration factor based on the known ratio of molar extinction coefficients of SCN- and OSCN-. Thus, the specific quantification of OSCN- in saliva samples is possible, as demonstrated here. The median value of 200 saliva samples was determined to be 0.56 mg L-1 (median), with a maximum of 3.9 mg L-1; the minimum value was below the detection limit (< 0.09 mg L-1). The recovery rate in individual saliva samples was 95 ± 8%.

Boronate Affinity Chromatography Accurately Measures HbA1c also in Patients with End-Stage Renal Disease - Performance Evaluation of the A1c HPLC Analyzer.

BACKGROUND: Boronate affinity chromatography is widely used, and the method has lately been improved and designed for HbA1c measurements. We report performance evaluation of the affinity chromatography HbA1c HPLC analyzer. METHODS: Within- and between-run imprecision was assessed based on the results of a series of measurements in three different EDTA blood samples and in control materials. HbA1c levels were measured and compared in 349 EDTA blood samples and 50 samples from patients with end-stage renal disease (ESRD) using the Premier Hb9210 analyzer (affinity chromatography) and the D-10 Hemoglobin Testing System (ion-exchange chromatography). RESULTS: The within- and between-run imprecision CVs ranged from 0.72% to 2.01%. Median HbA1c level measured by the Premier Hb9210 was significantly lower (6.4% [46 mmol/mol] vs. 6.6% [49 mmol/mol], p < 0.001). The Passing-Bablok agreement test yielded a slope of 1.0 (95% CI: 1.0 to 1.0) and intercept of -0.1 (95% CI: -0.1 to -0.1). Correlation coefficient and the mean difference amounted to 0.992 and -0.13% (95% CI: -0.11 to -0.15), respectively. Similar results were obtained for HbA1c levels < 7% [< 53 mmol/mol] and ≥ 7% [≥ 53 mmol/mol]. In ESRD patients, median HbA1c level measured by the Premier Hb9210 was also significantly lower (6.0% [42 mmol/mol] vs. 6.5 [48 mmol/mol], p < 0.001) with the mean difference equal to -0.52% (95% CI: -0.59 to -0.46). CONCLUSIONS: Although the Premier Hb9210 gave lower HbA1c levels, good results agreement with the D-10 Hemoglobin Testing System was found. Analytical performance found for HbA1c measurements in ESRD patients was similar. The Premier Hb9210 analyzer is suitable for routine HbA1c testing in clinical practice.

On-Line Ion Exchange Liquid Chromatography as a Process Analytical Technology for Monoclonal Antibody Characterization in Continuous Bioprocessing.

Combining process analytical technology (PAT) with continuous production provides a powerful tool to observe and control monoclonal antibody (mAb) fermentation and purification processes. This work demonstrates on-line liquid chromatography (on-line LC) as a PAT tool for monitoring a continuous biologics process and forced degradation studies. Specifically, this work focused on ion exchange chromatography (IEX), which is a critical separation technique to detect charge variants. Product-related impurities, including charge variants, that impact function are classified as critical quality attributes (CQAs). First, we confirmed no significant differences were observed in the charge heterogeneity profile of a mAb through both at-line and on-line sampling and that the on-line method has the ability to rapidly detect changes in protein quality over time. The robustness and versatility of the PAT methods were tested by sampling from two purification locations in a continuous mAb process. The PAT IEX methods used with on-line LC were a weak cation exchange (WCX) separation and a newly developed shorter strong cation exchange (SCX) assay. Both methods provided similar results with the distribution of percent acidic, main, and basic species remaining unchanged over a 2 week period. Second, a forced degradation study showed an increase in acidic species and a decrease in basic species when sampled on-line over 7 days. These applications further strengthen the use of on-line LC to monitor CQAs of a mAb continuously with various PAT IEX analytical methods. Implementation of on-line IEX will enable faster decision making during process development and could potentially be applied to control in biomanufacturing.

Effect of pore size on performance of monolithic tube chromatography of large biomolecules.

Effect of pore size on the performance of ion-exchange monolith tube chromatography of large biomolecules was investigated. Radial flow 1 mL polymer based monolith tubes of different pore sizes (1.5, 2, and 6 µm) were tested with model samples such as 20 mer poly T-DNA, basic proteins, and acidic proteins (molecular weight 14 000-670 000). Pressure drop, pH transient, the number of binding site, dynamic binding capacity, and peak width were examined. Pressure drop-flow rate curves and dynamic binding capacity values were well correlated with the nominal pore size. While duration of the pH transient curves depends on the pore size, it was found that pH duration normalized on estimated surface area was constant, indicating that the ligand density is the same. This was also confirmed by the constant number of binding site values being independent of pore size. The peak width values were similar to those for axial flow monolith chromatography. These results showed that it is easy to scale up axial flow monolith chromatography to radial flow monolith tube chromatography by choosing the right pore size in terms of the pressure drop and capacity.

Application of monolithic chromatographic supports in virus research.

Key properties of monolithic chromatographic supports, make them suitable for separation and/or concentration of large biomolecules, especially virus particles and viral genomes. One by one, the studies that have been completed so far, contributed to the knowledge that monolith chromatography has hardly any limitation to be applied in virus research. Viruses of different sizes, possessing icosahedral structure and symmetrical morphology, as well as rod-shaped or filamentous viruses with helical structure, even enveloped ones, all of them could be successfully managed by means of monolith chromatography. Same is true for viral genomes, primarily when being distinct from other nucleic acid forms present in a host cell. This review is exclusively focused on viruses. It describes the application of monolith chromatography to different problematics within the virus research field. The reviewed achievements offer new possibilities and trigger new aspects in virology.

Can the Afinion HbA1c Point-of-Care instrument be an alternative method for the Tosoh G8 in the case of Hb-Tacoma?

BACKGROUND: Hb-variant interference when reporting HbA1c has been an ongoing challenge since HbA1c was introduced to monitor patients with diabetes mellitus. Most Hb-variants show an abnormal chromatogram when cation-exchange HPLC is used for the determination of HbA1c. Unfortunately, the Tosoh G8 generates what appears to be normal chromatogram in the presence of Hb-Tacoma, yielding a falsely high HbA1c value. The primary aim of the study was to investigate if the Afinion HbA1c point-of-care (POC) instrument could be used as an alternative method for the Tosoh G8 when testing for HbA1c in the presence of Hb-Tacoma. METHODS: Whole blood samples were collected in K2EDTA tubes from individuals homozygous for HbA (n = 40) and heterozygous for Hb-Tacoma (n = 20). Samples were then immediately analyzed with the Afinion POC instrument. After analysis, aliquots of each sample were frozen at -80 °C. The frozen samples were shipped on dry ice to the European Reference Laboratory for Glycohemoglobin (ERL) and analyzed with three International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) and National Glycohemoglobin Standardization Program (NGSP) Secondary Reference Measurement Procedures (SRMPs). The Premier Hb9210 was used as the reference method. RESULTS: When compared to the reference method, samples with Hb-Tacoma yielded mean relative differences of 31.8% on the Tosoh G8, 21.5% on the Roche Tina-quant Gen. 2 and 16.8% on the Afinion. CONCLUSIONS: The Afinion cannot be used as an alternative method for the Tosoh G8 when testing for HbA1c in the presence of Hb-Tacoma.

High throughput determination of cleaning solutions to prevent the fouling of an anion exchange resin.

Effective cleaning of chromatography resin is required to prevent fouling and maximize the number of processing cycles which can be achieved. Optimization of resin cleaning procedures, however, can lead to prohibitive material, labor, and time requirements, even when using milliliter scale chromatography columns. In this work, high throughput (HT) techniques were used to evaluate cleaning agents for a monoclonal antibody (mAb) polishing step utilizing Fractogel(®) EMD TMAE HiCap (M) anion exchange (AEX) resin. For this particular mAb feed stream, the AEX resin could not be fully restored with traditional NaCl and NaOH cleaning solutions, resulting in a loss of impurity capacity with resin cycling. Miniaturized microliter scale chromatography columns and an automated liquid handling system (LHS) were employed to evaluate various experimental cleaning conditions. Cleaning agents were monitored for their ability to maintain resin impurity capacity over multiple processing cycles by analyzing the flowthrough material for turbidity and high molecular weight (HMW) content. HT experiments indicated that a 167 mM acetic acid strip solution followed by a 0.5 M NaOH, 2 M NaCl sanitization provided approximately 90% cleaning improvement over solutions containing solely NaCl and/or NaOH. Results from the microliter scale HT experiments were confirmed in subsequent evaluations at the milliliter scale. These results identify cleaning agents which may restore resin performance for applications involving fouling species in ion exchange systems. In addition, this work demonstrates the use of miniaturized columns operated with an automated LHS for HT evaluation of chromatographic cleaning procedures, effectively decreasing material requirements while simultaneously increasing throughput. Biotechnol. Bioeng. 2016;113: 1251-1259. © 2015 Wiley Periodicals, Inc.

A new approach for trace analysis of guanidine compounds in surface water with resorcinarene-based ion chromatography columns.

Trace levels of pharmaceuticals have been detected in surface water and may pose a health risk to humans and other organisms. New chromatographic materials will help identify and quantify these contaminants. We introduce a new ion chromatographic (IC) material designed to separate cationic pharmaceuticals and report its ability to separate a group of guanidine compounds. Guanidine moieties are strongly basic and protonated under acid conditions, and therefore can potentially be separated on the newly designed stationary phase and detected by ion exchange chromatography. The new column packing material is based on glutamic acids bonded to resorcinarene moieties that in turn are bound to divinylbenzene macroporous resin. Detection limits in the range of 5-30 µg L(-1) were achieved using integrated pulsed amperometric detection (IPAD) for guanidine (G), methylguanidine (MG), 1,1-dimethylbiguanide (DMG), agmatine (AGM), guanidinobenzoic acid (GBA) and cimetidine (CIM). Suppressed conductivity (CD) and UV-vis detection resulted in limits of detection similar to IPAD, in the range of 2-66 µg L(-1), but were not able to detect all of the analytes. Three water sources, river, lake, and marsh, were analyzed and despite matrix effects, sensitivity for guanidine compounds was in the 100 µg L(-1) range and apparent recoveries were 80-96%. The peak area precision was 0.01-2.89% for IPAD, CD and UV-vis detection.

Minicircle DNA purification using a CIM® DEAE-1 monolithic support.

Minicircle DNA is a new biotechnological product with beneficial therapeutic perspectives for gene therapy because it is constituted only by the eukaryotic transcription unit. These features improve minicircle DNA safety and increase its therapeutic effect. However, being a recently developed product, there is a need to establish efficient purification methodologies, enabling the recovery of the supercoiled minicircle DNA isoform. Thus, this work describes the minicircle DNA purification using an anion exchange monolithic support. The results show that with this column it is possible to achieve a good selectivity, which allows the isolation of the supercoiled minicircle DNA isoform from impurities. Overall, this study shows a promising approach to obtain the minicircle DNA sample with adequate quality for future therapeutic applications.

Use of polymeric membranes for purification of an E. coli expressed biotherapeutic protein.

Polymers have had a significant impact on the field of bioseparations in the past few decades. Most recently, membrane chromatography has emerged as an efficient alternative to the conventional packed-bed chromatography by eliminating the diffusion-related limitations associated with the traditional resin beads. In this article, we examine six membrane adsorbers for purification of granulocyte colony-stimulating factor (GCSF), an Escherichia coli-based biotherapeutic. These adsorbers differ either in their base matrix or in the surface chemistry. The role of interactions between the filter surfaces and the protein molecules in effecting these separations is the focus of the article.

[Lipoprotein Analysis -Measurement of Lipoprotein Fractions and Anion-Exchange High-Performance Liquid Chromatography].

Many clinical studies, including Framingham Heart study, have demonstrated that increased LDL choles- terol and decreased HDL cholesterol are associated with the risk of coronary heart disease (CHD), and lipo- protein evaluation is of importance in the prevention and treatment of CHD risk. Lipoprotein analysis methods by high performance liquid chromatography (HPLC) are divided broadly into 2 categories (HPLC with gel-filtration column and with anion-exchange column), and both methods can determine lipid levels of fractionated serum lipoproteins in a small volume of around 10 pL within 30 min. In contrast, with the gel- filtration HPLC method by Gaussian approximation following the particle sizes, the anion-exchange HPLC method elutes lipoproteins following the ion intensity of the lipoprotein particle surface and the hydrophobic property, and determines cholesterol levels of separated lipoproteins without overlapping lipoprotein frac- tions. We have established a method with anion-exchange chromatography using a 1.0-mL injection volume that 5.2 min takes to assay one sample. The within-day and between-day assay coefficients of variation of lipoprotein cholesterol values were 0.33-4.31 and 2.37-9.19%, respectively. Previous studies generating fractionated lipoprotein cholesterol data using anion-exchange HPLC revealed the clinical significance in a variety of settings and, in Japan, this rapid anion-exchange HPLC assay was approved as a medical examina- tion covered by health insurance for medical service fees in 2013, and the anion-exchange HPLC method for lipoprotein fraction cholesterol measurement has the potential to offer further clinical benefit. [Review].