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A continuously regenerated cationic impurity removal device (CR-CRD) has been fabricated and applied for ion chromatography (IC). The removal of cationic impurities is realized by electrodialytically replacing the cationic impurities with hydronium ions. The device is configured in a sandwich structure and the central eluent channel is respectively isolated from both electrodes by stacked cation exchange membranes and a bipolar membrane (BPM) plus stacked anion exchange membranes. The eluent channel is packed with cation exchange resins in hydronium form and their continuous regeneration can be achieved by electrodialysis. A desirable feature of the device is gas-free, and no degasser is required. It showed sufficient ability to remove cationic impurities, as indicated by > 99.9 % removal of 10 mL of 1 mM LiOH solution injected (∼10 μmol) or continuous removal of 1 mM LiOH solution at the flow rate of 1 mL/min (1 μmol/min). A useful application was for sample pretreatment in nuclear power industry, by eliminating strong matrix interference of the sample containing LiOH (1 mM) and boric acid (2000 mg/L) with trace anion analysis., Competing Interests: Declaration of competing interest The authors declare no competing financial interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Novel grafted anion exchangers with covalently bonded hyperbranched functional layers were prepared and evaluated for the separation of monovalent standard inorganic anions and oxyhalides. Preparation of base coating included grafting highly polar N-vinylformamide to the ethylvinylbenzene-divinylbenzene (EVB-DVB) substrate surface in highly polar solvent (methanol) with subsequent hydrolysis of grafted amide polymer in basic media, which resulted in preparation of polymer chains with multiple primary amino groups. Those amino groups were used as attachment points for forming hyperbranched anion-exchange layers using 1,4-butanediol diglycidyl ether and primary mono- or diamine (methylamine or 1,3-diaminopropane, respectively). The effects of hyperbranching reaction cycles number on selectivity were evaluated which revealed that selectivity and capacity can be controlled independently for the covalently bonded stationary phases in contrast to electrostatically bonded phases. It was demonstrated that unlike for electrostatically bonded phases, the intentional increase of crosslink by using primary diamine instead of primary monoamine doesn't cause the shift of selectivity coefficients. It was also shown that crosslink distribution throughout the hyperbranched layer is an important factor determining selectivity of hyperbranched anion exchangers., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Polyallylamine (PAA) has been utilized as a salt tolerant anion exchange chromatography ligand in downstream processing of biopharmaceuticals. We have developed novel MMC resins based on PAA polymer ligand partially modified with hydrophobic butyl or phenyl group. The resulting hydrophobic modified PAA ligand reduced HCP level to 12% (21-23 ppm) under 6 mS/cm in a flow-through polishing step of mAb, while not modified PAA ligand showed only 79% (145 ppm). We also found that structure of hydrophobic groups in the ligand mainly influenced on mAb yield. That is 25% increase of phenyl group modification ratio reduces mAb yield from 95% to 90%. On the other hand, modification with butyl group kept mAb yield more than 95%. The optimized ligand structure displayed a wide operational conductivity range. Extended purification studies of mAb using the MMC resin in the flow-through polishing step were carried out under optimized pH and conductivity condition as determined in a DOE study. The study revealed that the MMC resin was effective for developing one-step flow-through polishing workflow for mAb purification. In addition, the MMC flow-through polishing step could be directly coupled with a specified CEX chromatography step to efficiently remove mAb aggregates from 2.3% to <1.0% to achieve a biopharmaceutical-grade quality and a high yield of mAb (>93%) with a high loading capacity around 1000 mg/mL-resin. This new MMC resin will be useful in future mAb manufacturing platforms comprising of a robust and cost-effective flow-through polishing step., (Copyright © 2021. Published by Elsevier B.V.)

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., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

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. ., (© 2021. The Author(s).)

A high-performance polyacid ion exchange (IEX) nanofiber membrane was used in membrane chromatography for the recovery of lysozyme from chicken egg white (CEW). The polyacid IEX nanofiber membrane (P-BrA) was prepared by the functionalization of polyacrylonitrile (PAN) nanofiber membrane with ethylene diamine (EDA) and bromoacetic acid (BrA). The adsorption performance of P-BrA was evaluated under various operating conditions using Pall filter holder. The results showed that optimal conditions of IEX membrane chromatography for lysozyme adsorption were 10% (w/v) of CEW, pH 9 and 0.1 mL/min. The purification factor and yield of lysozyme were 402 and 91%, respectively. The adsorption process was further scaled up to a larger loading volume, and the purification performance was found to be consistent. Furthermore, the regeneration of IEX nanofiber membrane was achieved under mild conditions. The adsorption process was repeated for five times and the adsorption capacity of adsorber was found to be unaffected., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The development of ion chromatography has made remarkable progress in the past few decades, and it is now widely used for the analysis of common ions and organic compounds. Ion chromatography has many advantages, such as fast, high sensitivity, good selectivity and support for simultaneous analysis of multiple ionic compounds. In order to meet the high requirements of material analysis, new packing materials for ion chromatography with higher sensitivity and selectivity have been developed. In this paper, a lot of knowledge of ion chromatography is reviewed, and the development of ion chromatographic packings in recent years, especially in the last five years, is summarized., Competing Interests: Declaration of Competing Interest None., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Chromatographic separation serves as "a workhorse" for downstream process development and plays a key role in the removal of product-related, host-cell-related, and process-related impurities. Complex and poorly characterized raw materials and feed material, low feed concentration, product instability, and poor mechanistic understanding of the processes are some of the critical challenges that are faced during the development of a chromatographic step. Traditional process development is performed as a trial-and-error-based evaluation and often leads to a suboptimal process. A high-throughput process development (HTPD) platform involves the integration of miniaturization, automation, and parallelization and provides a systematic approach for time- and resource-efficient chromatographic process development. Creation of such platforms requires the integration of mechanistic knowledge of the process with various statistical tools for data analysis. The relevance of such a platform is high in view of the constraints with respect to time and resources that the biopharma industry faces today.This protocol describes the steps involved in performing the HTPD of chromatography steps. It describes the operation of a commercially available device (PreDictor™ plates from GE Healthcare). This device is available in 96-well format with 2 or 6 μL well size. We also discuss the challenges that one faces when performing such experiments as well as possible solutions to alleviate them. Besides describing the operation of the device, the protocol also presents an approach for statistical analysis of the data that are gathered from such a platform. A case study involving the use of the protocol for examining ion exchange chromatography of the Granulocyte Colony Stimulating Factor (GCSF), a therapeutic product, is briefly discussed. This is intended to demonstrate the usefulness of this protocol in generating data that are representative of the data obtained at the traditional lab scale. The agreement in the data is indeed very significant (regression coefficient 0.93). We think that this protocol will be of significant value to those involved in performing the high-throughput process development of the chromatography process.

High-throughput process development is more and more used in chromatography. Limitations are the tools provided by the manufacturers. Here, we describe a method to select ion exchange chromatographic media using a 96-well filter microplate.

Membrane chromatography is gradually emerging as an alternative to conventional column chromatography. It alleviates some of the major disadvantages associated with the latter, including high-pressure drop across the column bed and dependence on intraparticle diffusion for the transport of solute molecules to their binding sites within the pores of separation media. In the last decade, it has emerged as a method of choice for final polishing of biopharmaceuticals, in particular, monoclonal antibody products. The relevance of such a platform is high in view of the constraints with respect to time and resources that the biopharma industry faces today.This protocol describes the steps involved in performing HTPD of a membrane chromatography step. It describes the operation of a commercially available device (AcroPrep™ Advance filter plate with Mustang S membrane from Pall Corporation). This device is available in 96-well format with a 7 μL membrane in each well. We will discuss the challenges that one faces when performing such experiments as well as possible solutions to alleviate them. Besides describing the operation of the device, the protocol also presents an approach for statistical analysis of the data that are gathered from such a platform. A case study involving the use of the protocol for examining ion-exchange chromatography of the Granulocyte Colony Stimulating Factor (GCSF), a therapeutic product, is briefly discussed. This is intended to demonstrate the usefulness of this protocol in generating data that are representative of the data obtained at the traditional lab scale. The agreement in the data is indeed very significant (regression coefficient 0.9866). We think that this protocol will be of significant value to those involved in performing high-throughput process development of membrane chromatography.

A stirred fluidized bed (SFB) ion exchange chromatography was successfully applied in the direct recovery of recombinant enhanced green fluorescent protein (EGFP) from the unclarified Escherichia coli homogenate. Optimal conditions for both adsorption and elution processes were determined from the packed-bed adsorption systems conducted at a small scale using the clarified cell homogenate. The maximal adsorption capacity and dissociation constant for EGFP-adsorbent complex were found to be 6.3 mg/mL and 1.3 × 10 -3  mg/mL, respectively. In an optimal elution of EGFP with 0.2 M of NaCl solution (pH 9) and at 200 cm/h, the recovery percent of the EGFP was approximately 93%. The performances of SFB chromatography for direct recovery of EGFP was also evaluated under different loading volumes (50-200 mL) of crude cell homogenate. The single-step purification of EGFP by SFB recorded in a high yield (95-98%) and a satisfactory purification factor (~3 folds) of EGFP from the cell homogenate at 200 rpm of rotating speed., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Over the past several years, hyphenation of native (nondenaturing) liquid chromatography (nLC) methods, such as size exclusion chromatography (SEC), ion exchange chromatography (IEX), and hydrophobic interaction chromatography (HIC) with mass spectrometry (MS) have become increasingly popular to study the size, charge, and structural heterogeneity of protein drug products. Despite the availability of a wide variety of nLC-MS methods, an integrated platform that can accommodate different applications is still lacking. In this study, we described the development of a versatile, sensitive, and robust nLC-MS platform that can support various nLC-MS applications. In particular, the developed platform can tolerate a wide range of LC flow rates and high salt concentrations, which are critical for accommodating different nLC methods. In addition, a dopant-modified desolvation gas can be readily applied on this platform to achieve online charge-reduction native MS, which improves the characterization of both heterogeneous and labile biomolecules. Finally, we demonstrated that this nLC-MS platform is highly sensitive and robust and can be routinely applied in protein drug characterization.

A two-membrane electrodialytic carbonate eluent generator (EDG) for ion chromatography (IC) is described. It is in a sandwich-like configuration, in which the central eluent channel is spatially isolated from two outer regenerant channels by stacked cation exchange membranes (CEMs) and anion exchange membranes (AEMs). A platinum screen electrode is placed in each of two outer regenerant channels. The electrode at the CEMs side is set as an anode with respect to the electrode at the AEMs side being cathode. Potassium carbonate and/or bicarbonate solution is pumped into the regenerant channel as feed solution. The electromigration of carbonate and/or bicarbonate and potassium from feed solution respectively through AEMs and CEMs will form a gas-free eluent. With this configuration, ion transport behavior through AEMs was explored. The device demonstrated good reproducibility, as indicated by the relative standard deviation of retention time of less than 0.08%., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Multidimensional protein identification (MudPIT), developed in the Yates Laboratory 20 years ago, is regarded as a powerful tool for proteomics research. Due to its remarkable online separation advantages, MudPIT has been widely used to facilitate discoveries in the field of proteomics research. However, it has one major disadvantage: the process of eluting peptides during strong cation exchange introduces salts, of different concentrations, into the mass spectrometer. Considering the sensitivity of the new generation of high-resolution mass spectrometers, developing a new version of MudPIT that could eliminate the introduction of salts in the elute would be a significant advancement to current technology. Herein, we developed a new, clean version of MudPIT called parallel channels-multidimensional protein identification technology (PC-MudPIT) to overcome this issue. In this design, the original biphasic trapping column was replaced by two parallel analytical column channels. We successfully averted the salt contamination yet retained all the other advantages of MudPIT. A total of 8161 and 7359 protein groups were identified from A549 whole cell lysate using PC-MudPIT and classic MudPIT, respectively. Moreover, we discovered the additional advantage that, in online mode, PC-MudPIT can also be used for an enrichment process of phosphopeptide identification. We identified a total 11453 phosphopeptides using PC-MudPIT and 7729 phosphopeptides using offline TiO 2 enrichment followed by classic MudPIT. These advances indicate the possibility of other innovative applications of PC-MudPIT technology in deep proteome exploration.

Background: The ADAMS™ HA-8180V is the 8th generation of a fully automated ion-exchange HPLC system from ARKRAY, and the first to be released onto the US market. We evaluated the HA-8180V, for routine hemoglobin A1c measurement in comparison with the Roche Cobas c501, the Tosoh G8 analyzer for normal hemoglobin, and with the Trinity analyzer for hemoglobin variants., Methods: The analytical performance (linearity, precision, carryover, and sample stability) was assessed based on the Clinical and Laboratory Standards Institute (CLSI) and manufacturer guidelines. A comparison of the HA-8180V against two major analytical methods was performed for 100 whole blood samples. HA-8180V variant mode was also compared against the Trinity ultra2 A1c analyzer for 50 samples containing hemoglobin variants (HbC 14, HbS 14, HbD 12, and HbE 10)., Results: The within-run and total CVs were <0.01 and 0.75% at low HbA1c concentration and 0.46 and 0.63% at high HbA1c concentration, respectively. Linearity was shown in the concentration range 3.4-18.1% HbA1c, carryover was 0.00%, and stability values were excellent. Method comparison demonstrated a high concordance between methods., Conclusion: The eighth generation ADAMS HA-8180V A1c analyzer demonstrated high analytical performance adequate for routine clinical use., (© American Association for Clinical Chemistry 2020. All rights reserved. For permissions, please contact journals.permissions@oup.com.)

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.

Biomedical analytical methods often rely on indirect measurements, such as immunoassays, which can lack effective metrological traceability. In the nephelometric determination of ceruloplasmin (Cp), an important protein whose circulating level is altered in Wilson's disease (WD), the anti-Cp antibody used is not specific for the biologically active holoprotein so the assay can overestimate the concentration of Cp due to the presence of the apoprotein. By providing quantitation using elemental standards, the use of strong anion exchange chromatography (SAX) coupled to triple quadrupole inductively coupled plasma mass spectrometry (ICP-MS-MS) can overcome the drawbacks of methods for the measurement of metalloproteins reliant on immunoassays. In the current study, a SAX-ICP-MS-MS method for Cp quantification was designed and tested in samples of blood serum of WD patients and healthy controls. Using standards based on a copper-EDTA complex for calibration, the method provides relatively simple quantification of Cp with the limit of detection of 0.1 μg L -1 (limit of quantification 0.4 μg L -1 ). The method was also used to investigate the copper species separated by using a 30 kDa cut-off ultrafiltration device. The so-called "exchangeable" copper fraction is considered as an alternative clinical biomarker of WD. Using the designed speciation approach, it was shown that the ultrafiltration method can overestimate the "exchangeable" copper fraction due to a removal of copper from Cp. This was confirmed by comparing the enzymatic activity of the fractions. Thus, the specificity of the "exchangeable" copper test can be ensured only under strict maintenance of ultrafiltration conditions., (Copyright © 2019 Elsevier B.V. All rights reserved.)

This rapid and efficient method to prepare highly purified recombinant adeno-associated viruses (rAAVs) is based on binding of negatively charged rAAV capsids to an anion-exchange resin that is pH dependent., (© 2020 Cold Spring Harbor Laboratory Press.)

It has been known that anion exchangers prepared by grafting poly(ethyleneimine) (PEI) onto Sepharose FF (PEI-Sepharose) at ionic capacities (IC) over 600 mmol/L show both high protein adsorption capacity and uptake kinetics, and charge reversal of PEI-Sepharose by modification with succinic anhydride can produce protein cation exchangers of high capacity and uptake rate. Previously, a Charge Reversal-then-Reduction procedure (route A) was studied for preparation of cation exchangers of different IC values from PEI-Sepharose. In this work, we proposed a new route, that is, Charge Reduction-then-Reversal route (route B), to develop cation exchangers of different IC values from PEI-Sepharose FF with an IC of 700 mmol/L (FF-PEI-L700) as the starting resin. The two kinds of cation exchangers (route A, PEI-L700-CRn; route B, PEI-Rm-Cn) are compared for lysozyme (Lys) adsorption and chromatography. The two modification routes result in the difference in the ligand structures that significantly affect protein adsorption equilibrium and kinetics. Route A introduces long electroneutral groups that hinder protein adsorption and reduce equilibrium capacity. Moreover, charge reversal by reaction with succinic anhydride could cause diamide formation, which reduces remaining carboxyl groups or the IC. In the charge-reduced FF-PEI-Rm resins of the lowest IC (394 mmol/L) prepared in route B, the diamide formation was little due to the lack of primary and secondary amine groups, so its charge reversal makes a higher-IC cation exchanger. This makes PEI-Rm-Cn show a higher IC (589 mmol/L) than PEI-L700-CRn (463 mmol/L) in which D e /D 0 jumps about four times. The differences in the adsorption equilibrium and kinetics make the two kinds of resins behave distinctly in dynamic adsorption and chromatography. Namely, PEI-Rm-Cn resins display obviously higher dynamic binding capacities than PEI-L700-CRn resins in the IC range studied. For instance, the DBC (at 10% breakthrough) of PEI-R590-C680 (192 mg/mL) is 33% higher than that of PEI-L700-CR680 (144 mg/mL). This is proved by the purification of Lys from chicken egg white solution, in which the PEI-R590-C680 column purified Lys with a recovery yield 14% higher than the PEI-L700-CR680 column. This research thus demonstrated that Charge Reduction-then-Reversal route is superior over Charge Reversal-then-Reduction route in fabricating a high-capacity cation exchanger from PEI-Sepharose., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Novel biopharmaceutical products, such as vaccines and viral vectors, play a significant role in the development of innovative therapeutic, prophylactic, and clinical applications. However, several challenges are posed when manufacturing these products. The diversity of cell lines and the different physical and chemical properties of these biologicals require the use of different production and processing technologies. Alternative purification strategies that can improve the purification yield, such as continuous chromatography, are regarded nowadays as enabling technologies to overcome some of the bottlenecks in biomanufacturing. This chapter offers a shortcut approach to implement a semi-continuous chromatography purification of hepatitis C virus-like particles produced in insect cells with recombinant baculovirus. Although the purification is based on ion exchange chromatography, the present methodology can be extended to other types of chromatography.

We developed temperature-responsive mixed-mode columns packed with poly(N-isopropylacrylamide) (PNIPAAm)-modified beads and poly(2-acrylamido-2-methylpropane sulfonic acid) (PAMPS)-modified beads in various ratios. The PNIPAAm-modified silica beads and PAMPS-modified silica beads were prepared by surface-initiated atom transfer radical polymerization of N-isopropylacrylamide and 2-acrylamido-2-methylpropane sulfonic acid, respectively. We confirmed polymer modification of the silica beads by CHN elemental analysis, FTIR, zeta-potential measurements, and SEM. To determine the column separation efficiency, we examined the elution behaviors of cold medicine active ingredients and monoamines from each column. Analyte separation occurred on columns with PNIPAAm to PAMPS ratios of 1:20 and 1:10, whereas a column containing only PNIPAAm-modified beads did not retain the analytes. The analytes were retained on the columns through hydrophobic and electrostatic interactions with PNIPAAm and PAMPS, respectively. The separation performance improved with increasing column temperature because of dehydration of PNIPAAm and enhancement of hydrophobic interactions at elevated temperatures. The mixed-mode columns will be useful for separating basic bioactive compounds because the retention of analytes can be modulated by changing the column temperature and the composition., (Copyright © 2019 Elsevier B.V. All rights reserved.)

A new optimization strategy for purification of alkaloids from Rhizoma Corydalis using preparative liquid chromatography was developed, featuring a selective separation of different types of alkaloids into different parts by a reversed-phase/weak cation-exchange mixed-mode column (named C18WCX) at first. The total alkaloids of Rhizoma Corydalis were divided into four fractions with fraction III and IV corresponding to the tertiary type medium bases and the quaternary type strong bases, respectively. For fraction III, a conventional C18 column was used to isolate tertiary alkaloids using acetonitrile and 0.1% phosphoric acid (adjusted with triethylamine to pH 6.0) as mobile phases. High selectivity and symmetrical peak shapes of tertiary alkaloids were obtained, resulting in six main tertiary alkaloids isolated in a single run. As strong bases, quaternary alkaloids often suffer from serious peak tailing problem on conventional C18 columns. Therefore, a silica-based strong cation-exchange (SCX) column was used for purification of fraction IV. On the SCX column, good peak shapes in high sample loading were achieved. Five main quaternary alkaloids were isolated and identified from the fraction in one-step. The procedures presented effective for the preparative isolation and purification of alkaloids from Rhizoma Corydalis., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Ion exchangers of high binding capacity and uptake rate are desired for high performance purification of proteins by ion exchange chromatography. For this purpose, we have herein developed a new anion exchanger by grafting N,N-Dimethylaminopropyl acrylamide (DMAPAA) onto Sepharose FF by atom transfer radical polymerization. Six DMAPAA-grafted Sepharose FF resins of the same grafting density but different chain lengths (ionic capacities, ICs) were prepared, and named as FF-pDMAPAAn (n denotes IC value, 65-831 mmol/L). Bovine serum albumin (BSA) adsorption and its chromatography were studied to evaluate the anion exchange resins. It was found that the equilibrium protein adsorption capacity (q m ) increased rapidly from 91 mg/mL at IC = 65 mmol/L till reaching a peak value (368 mg/mL) at IC = 458 mmol/L and then decreased slightly with further increasing IC (chain length) to 831 mmol/L. The uptake rate, D e /D 0 , defined as the ratio of effective diffusion coefficient (D e ) to free diffusivity of the protein (D 0 ), also presented similar changes as a function of IC. Namely, D e /D 0 increased from 0.25 to 2.45 in the IC range of 65 to 458 mmol/L, kept almost unchanged from 458 to 573 mmol/L and then decreased slightly with further increase of IC to 831 mmol/L. Therefore, FF-pDMAPAA458 presented the best performance of the six resins for BSA adsorption, possessing very high q m and D e /D 0 values (368 mg/mL and 2.45, respectively). The resin was thus extensively characterized in the effect of ionic strength and column chromatography. The q m and D e /D 0 of FF-pDMAPAA458 were much higher than other anion exchangers reported in literatures and kept over 230 mg/mL and 2.3, respectively, at salt concentrations up to 100 mmol/L. Therefore, the dynamic binding capacity of the FF-pDMAPAA458 column was remarkably higher than Q Sepharose FF and other two typical polymer-grafted anion exchangers reported in literatures in the salt concentration range, and kept as high as 180-220 mg/mL in the superficial flow velocity rang of 150-1350 cm/h. Taken together, the results demonstrated that FF-pDMAPAA resins of appropriate ionic capacities are promising for high-performance protein chromatography., (Copyright © 2019 Elsevier B.V. All rights reserved.)

An electrodialytic potassium hydroxide eluent generator (EDG) well-suited to small-bore (2 mm i.d.) ion chromatography (sIC) system is described. The basic configuration of EDG cartridge is much similar to that of conventional counterpart except lower void volume, which was measured to be ˜9 μL relative to ˜ 31 μL of conventional one. The corresponding dispersion volume was computed to be ˜58 μL and ˜106 μL, respectively. Lower void volume will improve the sharpness of a step gradient (or steeper rise curve), which is helpful to gradient elution. The device can bear the pressure up to at least 20.5 MPa and the effective KOH concentration that can be generated is ranging from 0.15 mM to 100 mM at the typical flow rate of sIC (0.4 mL/min). High purity of the produced KOH eluent and excellent running reproducibility of the device were observed, as indicated by the typical suppressed background (0.36 μS/cm) and <0.11% (or <0.23%) of the relative standard deviation of the retention times for common inorganic anions achieved under isocratic (or gradient) mode., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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.

We developed a new apparatus for the routine production of 64 Cu in clinical use. The apparatus has many disposable parts that stabilize the product quality (such that there is a low deviation of the concentrations of impurity metals in the product) and reduce the work load of preparation for routine production. We also developed a new evaporator using near-infrared heaters for disposable use. We conducted a production test using the new apparatus and evaluated product quality. The product yield was 6.3 ± 0.32 GBq (end of bombardment) (N = 4), the product quality in terms of the concentrations of impurity metals (Cu 2+ , Ni 2+ , Fe 3+ , Zn 2+ , Mn 2+ ) was as good as that usually achieved, likely on the order of parts per billion, and the preparation time was reduced from 2 days to 1 day., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

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., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A fast, precise, and accurate method that can simultaneously determine 7 anions in whole blood was established by on line dialysis-double suppression ion chromatography. Performance parameters which could affect the determination of anions were optimized, including the selection of protein precipitant in samples, the amount of filtrate discarded, selection of eluent flow rate, influence of the Ag-Na column on experimental results, influence of ethylenediamines on ClO 2 - , and investigation of nitrogen drying. Finally, 3.6 mmol/L sodium carbonate was selected as eluent, with a flow rate of 0.8 mL/min, to separate the 7 anions. Blood and alcohol (v/v, 1:4) were used to precipitate the proteins in blood. The 7 anions reached an adequate recovery rate when the first 2 mL of filtrate from the C18 column was discarded. The recovery rate at LLOQ, low, medium, and high concentrations was 80-120%. The correlation coefficients (r 2 ) of the calibration curves of the targeted anions ranged from 0.9975 to 0.9998. The limit of detection (LOD) was 0.309-7.71 μg/L. This method has simple pretreatment, high accuracy, and good reproducibility and selectivity, and is suitable for the separation and determination of anions in blood., (Copyright © 2018. Published by Elsevier B.V.)

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 (InsP 6 ), through the release of phytase or passive diffusion of InsP 6 into the soaking water. Over a wide pH range, InsP 6 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 InsP 6 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 (InsP 1-6 ) in raw pasteurized activated almonds. At least 24 h of soaking at ambient temperature was required to reduce InsP 6 content from 14.71 to 14.01 µmol g -1 ., Conclusions: The reduction in InsP 6 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., (© 2018 Society of Chemical Industry.)

In this work, a mesoporous Fe 3+ type ion exchanger (DOW-3) was used to adsorb the geniposide (GS) involved in gardenia yellow pigment waste water. To test its viability, the mass transfer zone (MTZ) in dynamic adsorption column that plays an important role in the future scale-up of the GS adsorption process was studied systematically. Simulation of the MTZ behavior under various operation conditions in a packed adsorption column enabled the predictions of breakthrough curves of GS for the mesoporous adsorbent. Several modes of mass transport and processes taking place simultaneously, such as permeation, convection, dispersion and adsorption equilibrium were incorporated in the proposed model. Modeling process firstly affected the behavior of MTZ when it formed and moved throughout the column, and then had a much greater impact on the shape of breakthrough curve in the simulation. The dynamics of MTZ were studied under different adsorption conditions such as various column heights, flow rates and inlet concentrations. Moreover, the post-processing technique and 3D visualization of MTZ simulation results were capable of demonstrating the expanding and progressing of the MTZ related to the adsorption isotherm. The basic findings of MTZ dynamics will be very useful for process scale up. Additionally, the effective molecule docking results obtained from this study, help us fully understand the contribution of various molecular forces in the adsorption mechanism., (Copyright © 2018. Published by Elsevier Ltd.)

The extracellular matrix of the bone and dentin contains several non-collagenous proteins (NCPs). One category of NCPs is termed the SIBLING (small integrin-binding ligand, N-linked glycoprotein) family, which includes osteopontin (OPN), bone sialoprotein (BSP), dentin matrix protein 1 (DMP1), dentin sialophosphoprotein (DSPP), etc. These proteins have abundant phosphoserines, aspartic acids, and glutamic acids. In this protocol, we describe the extraction of NCPs from the bone and dentin matrices using guanidine-HCl/EDTA and the isolation of polyanionic SIBLINGs from NCPs using ion-exchange fast protein liquid chromatography (FPLC) to separate the differentially charged proteins into different fractions through a gradient elution by NaCl.

Background: Mixed-mode chromatography is becoming an important tool for downstream process purification, as it provides the selectivity and robustness unmatched by conventional singlemode chromatographic methods. The joint action of multiple functionalities present on the ligands of mixed-mode chromatography matrices effectively enhances the separation of target molecules from impurities., Material and Methods: Using Nuvia cPrime as an example, we elucidate the separation principles of hydrophobic cation exchange mixed-mode chromatography and its difference from traditional strong cation exchangers. We have developed a Nuvia cPrime based polish purification step specifically for the removal of a major process contaminant, which has an isoelectric point similar to that of the target monoclonal IgM molecule. Additional purification was accomplished using a second mixed-mode chromatography column packed with Ceramic Hydroxyapatite., Conclusion: The monoclonal IgM prepared with this new process fully retained its biological activity and was free of high molecular weight aggregates, a product quality that was not achievable in previous attempts using traditional ion exchange or hydrophobic interaction chromatography., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

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., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: Retention mechanism of proteins in hydroxyapatite chromatography (HAC) was investigated by linear gradient elution experiments (LGE)., Materials and Methods: Several mobile phase (buffer) solution strategies and solutes were evaluated in order to probe the relative contributions of two adsorption sites of hydroxyapatite (HA) particles, C-site due to Ca (metal affinity) and P-site due to PO4 (cation-exchange). When P-site was blocked, two basic proteins, lysozyme (Lys) and ribonuclease A(RNase), were not retained whereas cytochrome C(Cyt C) and lactoferrin (LF) were retained and also retention of acidic proteins became stronger as the repulsion due to P-site was eliminated. The number of the binding site B values determined from LGE also increased, which also showed reduction of repulsion forces., Conclusion: The selectivity (retention) of four basic proteins (RNase, Lys, Cyt C, LF) in HAC was different from that in ion-exchange chromatography. Moreover, it was possible to tune the selectivity by using NaCl gradient., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

The present chapter describes an accurate and user-friendly method for determining amino acid composition of wheat gluten proteins and their gliadin and glutenin fractions. The method consists of hydrolysis of the peptide bonds in 6.0 M hydrochloric acid (HCl) solution at 110 °C for 24 h, followed by evaporation of the acid and separation of the free amino acids by high-performance anion-exchange chromatography with integrated pulsed amperometric detection (HPAEC-IPAD). In contrast to conventional methods, the analysis requires neither pre- or post-column derivatization nor a time-consuming oxidation or derivatization step prior to hydrolysis. Correction factors account for incomplete release of Val and Ile even after hydrolysis for 24 h and for losses of Ser during evaporation. Gradient conditions including an extra eluent allow multiple sequential sample analyses without risk of Glu accumulation on the anion-exchange column which otherwise would result from high Gln levels in gluten proteins.

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).

Protein A chromatography is quite commonly used for capture of monoclonal antibodies from the clarified cell culture broths. Protein A resins are expensive and economic feasibility demands that the resin be reused for 50-300 cycles. Resin reuse is, however, accompanied by resin fouling, impacting both the binding and mass transfer characteristics of the resin. In the present study, we attempt to model the variations in binding and mass transfer characteristics of a commercially available Protein A resin, mAbSelect SuRe ™ , as a function of resin's reuse. Simplified linear driving force modeling and kinetic modeling of Protein A chromatography step elution cycling data has been successfully used to predict resin performance up to 100 cycles based on fouling data up to 50 cycles. Fouling factor for Protein A resin has been empirically modeled as a function of binding and mass transfer characteristics of resin and the resin's reuse using a combination of Buckingham's π theorem and statistical analysis. The proposed empirical model enables reliable prediction of performance of Protein A resin as well as offers an improved understanding of the underlying mechanism behind the decline in resin performance during fouling., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The wide spread of infectious diseases have provoked the scientists to develop new types of vaccines. Among the different types of vaccines, the recently discovered plasmid DNA vaccines, have gained tremendous attentions in the last few decades as a modern approach of vaccination. The scientific interest in plasmid DNA vaccines is attributed to their prominent efficacy as they trigger not only the cellular immune response but also the humoral immune responses. Moreover, pDNA vaccines are easily to be stored, shipped and produced. However, the purification of the pDNA vaccines is a crucial step in their production and administration, which is usually conducted by different chromatographic techniques. This review summarizes the most recent chromatographic purification methods provided in the literature during the last five years following our last review in 2013, including affinity chromatography, hydrophobic interaction chromatography, ion exchange chromatography, multimodal chromatography, sample displacement chromatography and miscellaneous chromatographic methods., (Crown Copyright © 2018. Published by Elsevier B.V. All rights reserved.)

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.

Nucleotides and their sugar conjugates are fundamental molecules in life, participating in processes of DNA/RNA composition, cell wall build-up, glycosylation reactions, and signal conduction. Therefore, the quantification of these compounds in biological samples significantly benefits the understanding of their functions. However, nucleotides and nucleotide sugars are extremely hydrophilic, causing bad retention and peak symmetry on regular C18 chromatographic columns. To solve this problem, ion-pair (IP) chromatography, ion-exchange (IE) chromatography and hydrophilic interaction chromatography (HILIC) were applied, of which differentiated mechanisms were utilized to increase the retention of the analytes on the stationary phases. IP-HPLC and HILIC were convenient for coupling with many kinds of detectors (ultraviolet, UV or mass spectrometry, MS). Combining these two kinds of techniques, the advantages of better separation and retention were increased, while disadvantages like irreversible adsorption by stationary phases were greatly decreased. Due to the high concentrations of nonvolatile buffer salts used, IE-HPLC was not suitable for MS detectors. Protein precipitation and solid phase extraction were the common methods for sample treatment in the analysis of nucleotides and nucleotide sugars. By carefully optimizing the LCUV or LCMS conditions, high sensitivities could be achieved, and the methods could be applied to the analysis of many kinds of biological samples (cells, tissues, plants, bacteria, etc.). Developing new analyzing techniques may help the utilization of nucleotides and nucleotide sugars in the diagnosis and therapy of diseases., (Copyright © 2018 Elsevier B.V. All rights reserved.)

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.

In this study, we developed an anion exchanger-disposable pipette extraction (DPX) method to detect the residual concentrations of eight neonicotinoid insecticides (dinotefuran, acetamiprid, clothianidin, thiacloprid, imidachloprid, imidaclothiz, nitenpyram, and thiamethoxam) and eight insect growth regulators (IGRs; triflumuron, cyromazine, buprofezin, methoxyfenozide, tebufenozide, chromafenozide, fenoxycarb, and RH 5849) in Chinese honey samples collected from different floral sources and different geographical regions using liquid chromatography tandem mass spectrometry (LC-MS/MS). QAE Sephadex A-25 was used as the anion exchanger in the DPX column for the purification and cleanup of honey samples. Analytes were eluted with a mixture of acetonitrile and 0.1 M HCl, and the elution was subjected to LC analysis. This method was thoroughly validated for its reproducibility, linearity, trueness, and recovery. Satisfactory recovery of pesticides was obtained ranging from 72% to 111% with intraday RSDs (n = 5) of 1%-10%. High linearity (R 2  ≥ 0.9987) was observed for all 16 pesticides. Limits of detection and quantification for all 16 compounds ranged from 0.3 to 3 μg/kg and from 1 to 10 μg/kg, respectively. Pesticide residues (9-113 μg/kg) were found in Chinese honey samples. The anion exchanger-DPX method was effective for removing sugars and retaining target analytes. Moreover, this method was highly reliable and sensitive for detecting neonicotinoids and IGRs in different floral sources of honey and will be applicable to matrixes with high sugar content., (Copyright © 2018 Elsevier B.V. All rights reserved.)

We discuss the reported capacities of available packed ion exchange columns and the different methods used for their measurement. We outline basic considerations related to both packed and open tubular columns based on ion exchange latex particles. There is a large body of information covering the retention behavior of packed ion exchange columns based on ion exchange latex particles. We propose a parameter γ iex , which is the ion exchange capacity of a column (packed or open tubular) per unit liquid volume present in the column (including accessible volume within pores) and show that the retention factor for any given ion is directly related to γ iex . On this basis, if based on the same type of latex, the behavior of one type of column can be reasonably predicted from the known behavior of the other, even when the absolute capacities differ by more than 5 orders of magnitude., (Copyright © 2018 Elsevier B.V. All rights reserved.)

A comprehensive two-dimensional liquid chromatography (LCxLC) system consisting of twelve columns in the second dimension was developed for comprehensive analysis of intact proteins in complex biological samples. The system consisted of an ion-exchange column in the first dimension and the twelve reverse-phase columns in the second dimension; all thirteen columns were monolithic and prepared inside 250 µm i.d. capillaries. These columns were assembled together through the use of three valves and an innovative configuration. The effluent from the first dimension was continuously fractionated and sequentially transferred into the twelve second-dimension columns, while the second-dimension separations were carried out in a series of batches (six columns per batch). This LCxLC system was tested first using standard proteins followed by real-world samples from E. coli. Baseline separation was observed for eleven standard proteins and hundreds of peaks were observed for the real-world sample analysis. Two-dimensional liquid chromatography, often considered as an effective tool for mapping proteins, is seen as laborious and time-consuming when configured offline. Our online LCxLC system with increased second-dimension columns promises to provide a solution to overcome these hindrances., (Copyright © 2018 Elsevier B.V. All rights reserved.)

In recent papers we have discussed the optimization of design and operating conditions for cuboid packed-bed device for chromatographic separations. The efficiency metrics used in these studies included the number of theoretical plates per unit bed height as well as attributes of flow-through and eluted peaks. These studies were carried out using equivalent columns as benchmarks. The cuboid packed-bed devices consistently outperformed the columns in terms of the above metrics. The current study examines how well, or indeed if at all these superior efficiency metrics translate to superiority in multi-component protein separation. Cation exchange resin was examined in the current study using appropriate multi-component model protein system which was chosen with close isoelectric points to make the separation challenging. Effects of operating and experimental parameters such as flow rate, loop size and linear gradient length on separation performance were systematically investigated. Separation metrics examined included peak width, tailing factor, asymmetry factor and resolution of separated protein peaks. The results obtained showed that the cation exchange cuboid packed-bed device significantly outperformed its equivalent commercial column (e.g., the number of theoretical plates per unit bed height was 8636/m for the cuboid packed-bed device as opposed to 1480/m for the column at a flow rate of 0.5 mL/min). The difference in efficiency was particularly high at lower flow rate and when shorter gradients were employed. The results suggest that the cuboid packed-bed devices could potentially have promising application in preparative separations such as biopharmaceutical purifications., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The enzymatic system in saliva, consisting of salivary peroxidase (SPO), hydrogen peroxide (H 2 O 2 ), 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%.

We develop a new two-dimensional (2D) high performance liquid chromatography (HPLC) approach for intact protein analysis. Development of 2D HPLC has a bottleneck problem - limited second-dimension (second-D) separation speed. We solve this problem by incorporating multiple second-D columns to allow several second-D separations to be proceeded in parallel. To demonstrate the feasibility of using this approach for comprehensive protein analysis, we select ion-exchange chromatography as the first-dimension and reverse-phase chromatography as the second-D. We incorporate three second-D columns in an innovative way so that three reverse-phase separations can be performed simultaneously. We test this system for separating both standard proteins and E. coli lysates and achieve baseline resolutions for eleven standard proteins and obtain more than 500 peaks for E. coli lysates. This is an indication that the sample complexities are greatly reduced. We see less than 10 bands when each fraction of the second-D effluents are analyzed by sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE), compared to hundreds of SDS-PAGE bands as the original sample is analyzed. This approach could potentially be an excellent and general tool for protein analysis., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Budding yeast Dmc1 is a member of the RecA family of strand exchange proteins essential for homologous recombination (HR) during meiosis. Dmc1 mediates the steps of homology search and DNA strand exchange reactions that are central to HR. To achieve optimum activity, Dmc1 requires a number of accessory factors. Although methods for purification of Dmc1 and many of its associated factors have been described (Binz, Dickson, Haring, & Wold, 2006; Busygina et al., 2013; Chan, Brown, Qin, Handa, & Bishop, 2014; Chi et al., 2006; Cloud, Chan, Grubb, Budke, & Bishop, 2012; Nimonkar, Amitani, Baskin, & Kowalczykowski, 2007; Van Komen, Macris, Sehorn, & Sung, 2006), Dmc1 has been particularly difficult to purify because of its tendency to aggregate. Here, we provide an alternative and simple high-yield purification method for recombinant Dmc1 that is active and responsive to stimulation by accessory factors. The same method may be used for purification of recombinant Rdh54 (a.k.a. Tid1) and other HR proteins with minor adjustments. We also describe an economical and sensitive D-loop assay for strand exchange proteins that uses fluorescent dye-tagged, rather than radioactive, ssDNA substrates., (© 2018 Elsevier Inc. All rights reserved.)

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.

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., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)