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This study reviews the recent applications of enhanced separation methods employed in forensic analysis utilizing gas chromatography, liquid chromatography, and supercritical fluid chromatography published between 2015 to 2020, except papers previously covered in relevant review articles. Applications of enhanced chromatographic separation methods to arson investigations, environmental forensics, sexual assault investigations, drug analysis, and toxicology are discussed. Future directions for enhanced chromatographic separation methods in forensic science are also explored., (© 2021 Wiley-VCH GmbH.)

Even at low concentrations in environmental waters, some viruses are highly infective, making them a threat to human health. They are the leading cause of waterborne enteric diseases. In agriculture, plant viruses in irrigation and runoff water threat the crops. The low concentrations pose a challenge to early contamination detection. Thus, concentrating the virus particles into a small volume may be mandatory to achieve reliable detection in molecular techniques. This paper reviews the organic monoliths developments and their applications to concentrate virus particles from waters (waste, surface, tap, sea, and irrigation waters). Free-radical polymerization and polyaddition reactions are the most common strategies to prepare the monoliths currently used for virus concentration. Here, the routes for preparing and functionalizing both methacrylate and epoxy-based monoliths will be shortly described, following a revision of their retention mechanisms and applications in the concentration of enteric and plant viruses in several kinds of waters., (© 2021 Wiley-VCH GmbH.)

In this study, a sensitive, selective, and environmentally friendly analytical method for direct extraction and preconcentration of iodine was developed. Iodine, as an iodate ion or iodide ion, was simultaneously extracted and preconcentrated by gel electromembrane microextraction (G-EME) and analyzed for total iodine by ion chromatography. The total iodine was determined by combining the peak areas of both iodate and iodide ions. Under the optimized conditions, linear calibration for iodine using a mixture of iodate and iodide ions was obtained from 10 to 100 µg L -1 (r 2  > 0.996). The detection limit was 7.0 µg L -1 . Recoveries of spiked iodine (as iodate) in the samples were greater than 90%. The method was applied for the determination of iodine in dietary supplements and fortified food samples, i.e., iodine-enriched eggs. Our developed method could be directly applied for the determination of iodine in different matrix samples including eggs without a pretreatment step., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Background: RT-qPCR technique is the current world-wide method used for the early detection of SARS-CoV2 RNA in the suspected clinical samples. Viral RNA extraction is the key pre-analytical step for SARS-CoV2 detection which often achieved using commercial RNA-extraction kits. However, due to the COVID-19 pandemic, bulk production and the supply chains for the commercial RNA-extraction kit have been seriously compromised. The shortage of commercial RNA-extraction kit is even more acute in developing country. Furthermore, use of one-off design RNA-columns can generate plastic wastes that have an environmental pollution effect., Methods and Results: To address these issues, in this study, we used warm alkaline solution containing Triton X-100 for the complete removal of the residual SARS-CoV2 RNA from the used RNA-binding silica column. Columns regenerated using the alkaline solution have the viral RNA purification capability that is comparable to the fresh silica columns. We also demonstrated that RNA-binding silica columns can be regenerated and reused for a minimum of five-times., Conclusions: Therefore, the use of the RNA-column regeneration method may benefits several SARS-CoV2 diagnostic laboratories throughout the world by cutting down the requirement of commercial RNA-purification column., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

The effect of adding ethoxylated sorbitan ester surfactants (Tweens®) to poly(ethylene glycol) diacrylate-based monolithic recipes was investigated. Five different Tweens® have been evaluated to investigate the exact role of non-ionic surfactants in poly(ethylene glycol) diacrylate-based monolith preparations. These monoliths were characterized by scanning electron microscopy, infrared spectroscopy, and nitrogen physisorption analysis. Different morphological features, and surface areas were observed when different types of Tween® were included in the recipe; Tween® 20 and 85 showed small globules, while Tween® 40, 60 and 80 exhibited larger globular structures with different sizes and degrees of coalescence. The different Tween®-based monoliths were investigated for the chromatographic separation of mixtures consisting of hydroxybenzoic acids and alkylbenzenes. These columns were mechanically stable, except for Tween® 80. The highest methylene selectivity and the best overall performance were achieved by Tween® 60. The efficiency was increased by increasing the concentration of the Tween® 60 and the amount of poly(ethylene glycol) diacrylate M n 700 in the recipes up to 30 wt%, each. Further increases in either Tween® 60 or poly(ethylene glycol) diacrylate M n 700 led to formation of non-permeable columns. The optimized column was successfully used for separation of mixtures of nonsteroidal anti-inflammatory and sulfa drugs, with a maximum efficiency of 60,000 plates/m., Competing Interests: Declaration of Competing Interest The authors declare no competing financial interest., (Copyright © 2021 Elsevier B.V. All rights reserved.)

A computational fluid dynamics method was used for prediction of flow behavior and band profiles of small- and macro-molecule compounds eluting in extra-column volumes (ECV) of an Äkta chromatographic system. The model compounds were: acetone, bovine serum albumin and an antibody. The construction of ECV was approximated by different types of geometries, starting from the simplest two-dimensional (2D) arrangement consisting of a straight capillary tube, and ending with a three-dimensional system (3D), which accounted for the flow path curvature of individual elements of ECV, including: injection loop capillary, multi-way valve, connecting capillary and detector cell. The accuracy of the model predictions depended on the flow path length and the eluent flowrate. 2D-geometry models reproduced pretty well the shapes of band profiles recorded at the lowest eluent flowrate used, but they failed for increased flowrates. The 3D-geometry model was found to be sufficiently accurate for all conditions investigated. It was exploited to analyze band broadening in the individual ECV elements. The simulation results revealed that the flow behavior in the injection loop capillaries strongly influenced the shape of band profiles, particularly at higher eluent velocities. This was attributed to the formation of Dean vertices triggered by centrifugal forces in curved parts of the eluent flow path., 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 © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Twin-column recycling chromatography with a solvent gradient (TCRC-SG) was investigated with the equilibrium-dispersive chromatography model. The solvent gradient caused by constant addition of a modifier between the two columns created a band compression effect to counterbalance band broadening, so that the target component band neither broadened nor shrunk. Meanwhile, band compression accelerated the separation but prevented excessive separation. Increasing the volume fraction of weak solvent in the modifier and reducing the modifier flowrate enhanced band compression and improved the separation. The effect of column efficiency (number of theoretical plates: 500-1500) on the separation was not significant. According to the separation behavior, a simple operation scheme is proposed to automatically control column switching without needing to determine the adsorption isotherm and designing operating conditions in advance. In comparison with simulated moving bed, TCRC-SG had a higher feed throughput, but consumed more solvent. The results showed that TCRC-SG is favorable for preparative separation., 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 © 2021. Published by Elsevier B.V.)

Efficiently tunable capture of the glycosylated/phosphorylated proteins is critical to meet the need of in-depth glycoproteome and phosphoproteome studies. Reported here is a new bifunctional polymer monolithic column by introducing benzeneboronic acid and phosphonic acid onto monolithic column (denoted as poly (EDMA-co-VPBA-co-VPA) monolith) for tunable and specific enrichment of glycoproteins and phosphoproteins via switching different mobile phases. Based on boronate affinity and immobilized metal affinity, the as-prepared poly (EDMA-co-VPBA-co-VPA) monolith exhibited superior performance in selective separation of small molecules and biomacromolecules containing cis-diol/phosphate groups or not. And the frontal chromatography analysis showed that the binding capacity of the poly (EDMA-co-VPBA-co-VPA) monolith towards horseradish peroxidase (HRP, glycoprotein) or β-casein (phosphoprotein) is four-fold higher than that of bovine serum albumin (BSA, non-glycosylated/phosphorylated protein). Furthermore, combined with mass spectrometry identification, the successful application in specific enrichment of glycopeptides/phosphopeptides from tryptic digests of HRP/β-casein and direct capture of low abundant endogenous phosphopeptides from human serum proved great practicability in complex samples. This study provides a novel insight for fabricating the monolithic columns with multifunctionalization to facilitate further post-translational modification (PTM)-proteomics development., 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 © 2021 Elsevier B.V. All rights reserved.)

Simultaneously reducing the bed-height and increasing the area of cross-section, while keeping the bed-volume the same, would substantially reduce the pressure drop across a process chromatography column. This would minimize problems such as resin compaction and non-uniformity in column packing, which are commonly faced when using soft chromatographic media. However, the increase in macroscale convective dispersion due to the increase in column diameter, and the resultant loss in resolution would far outweigh any potential benefit. Cuboid-packed bed devices have lower macroscale convective dispersion compared to their equivalent cylindrical columns. In this paper, we discuss how and why a flat cuboid chromatography device having a short bed-height gives better protein separation, at a significantly lower pressure drop, than a taller column having the same bed-volume. First, we explored this option based on computational fluid dynamic (CFD) simulations. Depending on the flow rate, the pressure drop across the flat cuboid device was lower than that in the tall column by a factor of 6.35 to 6.4 (i.e. less than 1/6 th the pressure). The CFD results also confirmed that the macroscale convective dispersion within the flat cuboid device was significantly lower. Head-to-head separation experiments using a 1 mL flat cuboid device having a bed-height of 10 mm, and a 1 mL tall column having a bed-height of 25.8 mm, both packed with the same chromatographic media, were carried out. The number of theoretical plates per unit bed-height was on an average, around 2.5 time times greater with the flat cuboid device, while the total number of theoretical plates in the two devices were comparable. At any given superficial velocity, the height equivalent of a theoretical plate in the tall column was on an average, higher by a factor 2.5. Binary protein separation experiments showed that at any given flow rate, the resolution obtained using the flat cuboid device was significantly higher than that obtained with the tall column. This work opens up the possibility of designing and developing short bed-height chromatography devices for carrying out high-resolution biopharmaceutical purifications, at very low pressures., 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 © 2021. Published by Elsevier B.V.)

Multiple-open-tubular columns enabling transverse diffusion (MOTTD) are made of straight, parallel, and cylindrical flow channels separated by a mesoporous stationary phase. In Part 1, a model of band broadening along MOTTD columns accounting for longitudinal diffusion, the trans-channel velocity bias, and mass transfer resistance in the stationary phase was proposed and validated. In this Part 2, the model is completed by considering the impact of short-range inter-channel velocity biases on the MOTTD plate number. These velocity biases are caused by the wide distribution of the channel diameters. Different ratios, ρ, of the average inner diameter, 2c >, of the flow channels to their closest center-to-center distance d (d= 5 μm, ρ= 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, and 0.9) with a relative standard deviation (RSD) increasing from 0 to 50% are considered. The zone retention factor k 1 was increased from 1 to 25. The complete model of band broadening is validated after adjustment to dispersion data obtained by 1) the lattice-Boltzmann method for modeling fluid flow, 2) a random-walk particle-tracking (RWPT) technique to address advective-diffusive transport, and 3) by considering two distinct populations of flow channels (inner radii r c,1 =c >(1-RSD) and r c,2 =c >(1+RSD)) arranged at the nodes of a hexagonal compact array. The completed model of band broadening in MOTTD columns reveals that the RSD of the channel diameters has only a moderate impact on the optimum plate number of MOTTD columns: the relative increase of the minimum plate height do not exceed 30% even for the largest RSDs. However, when the mass transfer of the analyte is governed by its slow rate of transverse diffusion across the MOTTD column, the plate height can be increased by up to 100% at high average velocities. Regarding the best trade-off between analysis speed and column performance at a fixed pressure drop of 400 bar, irrespective of the zone retention factor and RSD of the distribution of the channel diameters, the fastest analyses are recommended for MOTTD columns having a small structural parameter ρ. In contrast, for the longest analysis times, the largest values of ρ are required to maximize the performance of MOTTD columns., 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 © 2021 Elsevier B.V. All rights reserved.)

The adoption of process analytical technologies by the biopharmaceutical industry can reduce the cost of therapeutic drugs and facilitate investigation of new bioprocesses. Control of critical process parameters to retain critical product quality attributes within strict bounds is important for ensuring a consistently high product quality, but developing the sophisticated analytical technologies required has proven to be a major challenge. Here, we demonstrate a new optical technique for continuous monitoring of protein species as they are eluted from a chromatographic column, even when they fully co-elute with other protein species, without making any assumption about or peak-fitting to the elution profile. To achieve this, we designed and constructed a time-resolved intrinsic fluorescence lifetime chromatograph, and established an analytical framework for deconvolving and quantifying distinct but co-eluting protein species in real time. This proof-of-concept technology has potentially useful applications as a process analytical technology and more generally as an analytical technique for label-free quantification of proteins in mixtures., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: J.E.H., S.A., G.A., and P.A.D. are co-authors on a patent application which could increase in value if the methods and ideas described in this paper find widespread application., (Copyright © 2021. Published by Elsevier B.V.)

The dynamics of adsorption of the Leu-Leu stereoisomers in a chromatographic column packed with the Chirobiotic R chiral stationary phase bearing grafted antibiotic ristocetin A was studied by means of measurement and analysis of van Deemter plots. Similar measurements were carried out with weakly retained Gly-Gly for the sake of comparison. The bulk diffusion coefficients of the investigated dipeptides were also determined. It is found that the van Deemter plots of both the Leu-Leu stereoisomers and Gly-Gly have an uncommon convex-upward shape. Besides, the van Deemter B coefficients for the Leu-Leu stereoisomers, but not for Gly-Gly, have unusually high values. It is suggested that a high transcolumn contribution to eddy dispersion, which turned out to be enantioselective, accounts for these findings. Adsorption kinetics of all the dipeptides considered is relatively slow, the adsorption rate constant (k ads ) being of order of magnitude 20-60 s -1 . k ads does not depend on the configuration of Leu-Leu stereoisomers, although their affinity toward the chiral selector depends on this factor. This supports the above hypothesis that eddy dispersion is mainly responsible for the observed peculiarities in the dynamic behavior of dipeptides, and adsorption kinetics has secondary importance in this phenomenon., 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. Published by Elsevier B.V.)

Apremilast is an anti-inflammatory agent. It has been a flourishing molecule in the field of dermatology. In the year 2014, Apremilast got its approval for treatment of psoriatic arthritis. Presently it is known to treat a number of other conditions, including atopic dermatitis and plaque psoriasis. Apremilast a phthalimide derivative, is non-hygroscopic in nature. It is practically insoluble in water. Apremilast acts by inhibiting the activity of phosphodiesterase 4 (PDE4), an intracellular enzyme. Analytical method plays a key role to understand the physio-chemical properties of a drug molecule. Because of poor solubility and low permeability, analytical method development and formulation becomes challenging. Till date, there are no standard test methods available to analyze Apremilast. So, a critical review of the analytical techniques of Apremilast was carried out. The literature search was done by screening the papers reporting analytical techniques of Apremilast from year 2014 to 2019. Methodologies particularly UV spectroscopy, HPTLC, HPLC, X-ray diffraction, NMR, LC-MS were collected and reviewed. Interminable efforts are made by the researchers to develop simple, accurate, robust and cost-effective methods of analysis. In pharmaceutical research, this information will aid in the development of new delivery systems. The review will prove beneficial and advantageous pre-formulation studies and will guide the formulation development.

An analysis of the relationship between the number of plates measured with a small molecule tracer and the breakthrough curve of a strongly bound protein in short laboratory chromatography columns (1-5 cm) considering flow nonuniformity is presented. For practical conditions, while axial dispersion has only a small impact on the breakthrough curve, radial flow nonuniformity has a profound effect. Radial parabolic velocity profiles lead to tailing tracer peaks and broader breakthrough curves. Profiles where the velocity varies radially only in a thin region near the column wall lead to fronting tracer peaks and early breakthrough when the velocity at the wall is higher than the average and to tailing peaks and tailing breakthrough curves when the velocity at the wall is lower than the average. Experiments conducted in laboratory minicolumns (0.5-1 cm diameter, 0.5-1 ml volume) show tracer peaks and protein breakthrough curves that are consistent with higher velocities at the wall. The model presented in this work provides a tool to model experimental breakthrough data and to assess the degree of flow uniformity required to obtain meaningful dynamic binding capacity measurements using minicolumns in a high-throughput lab setting., (© 2020 American Institute of Chemical Engineers.)

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.

The VariCol process is a variant of the conventional simulated moving bed (SMB) process, distinguished by the asynchronous shifting of the inlet and outlet ports of the chromatographic column train. This feature allows for a more flexible operation in column utilization and can also achieve higher separation performances. However, to take full benefit out of it, the operating parameters, such as the strategy for port switching, must be optimal. in this paper, a novel methodology for optimizing those parameters, based on a single NLP (non-linear programming), is proposed. The main advantage of this approach is that it significantly reduces the complexity of the original MINLP (mixed-integer non-linear programming) formulation currently discussed in the literature. The proposed optimization problem is built, considering that the average column configuration of three zones provides the necessary and sufficient information to describe the VariCol process. Several optimization scenarios for the enantioseparation of 1,1´-bi-2-naphthol and aminoglutethimide were considered to evaluate the proposed methodology and to compare the performance of VariCol and SMB processes. The results have shown that with the single NLP approach, it is possible to explore the optimal solution in all the VariCol process domains with less computational effort than other optimization strategies reported in the literature. That is a great advantage, especially in the context of real-time applications., 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.)

Buffer preparation and storage requires a significant facility footprint in large scale bioprocessing and together with the costs of supply chain management can have a substantial economic impact. In-line buffer mixing in chromatography is commonly performed by blending different buffer solutions using at least two pumps and a static or dynamic mixer. We developed a device for an in-line gradient delivery of buffering agents directly from solids to be applied for chromatographic separation processes. A solid feeding device with a screw conveyor and a hold tank for the solids was designed and a miniaturized system was 3D printed. The coefficient of variation for the precision of the solid feeding of 5 different buffering agents was below 5% even for very small solid flow rates necessary for lab-scale chromatography. Stability was demonstrated by a constant linear solid feed at a very low dosing rate of 0.05 g.min -1 over 24 hours. We demonstrated the suitability for chromatography by directly connecting the system to a standard chromatography workstation for protein chromatography. The solids were fed into a miniaturized continuously stirred tank reactor connected to an ÄKTA purification system. The performance of the in-line gradient delivery of buffering agents directly from solids was compared to conventional in-line buffer mixing. We were able to achieve highly linear gradients for elution using only one pump of a chromatographic system, generating the gradient by the direct addition of solids avoiding the necessity of additional pumps and hold tanks. By direct conditioning of buffers and the addition of solids a simple, just in time, at site preparation of buffers was possible. The design of the feeding unit for solid addition for buffer preparation is easily scalable and adaptable to work with or as a replacement for already existing in-line dilution or conditioning units., 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.)

This work proposes an experimental method for the estimation of the phase ratio of reversed-phase C8 columns by employing the equation log(k)=alog(K om )+log(Φ), where k is the retention factor, K om is the octane-mobile phase partition coefficient, a is a proportionality constant and Φ is the phase ratio (defined as volume ratio of the stationary phase to the mobile phase). The immiscible liquid octane and mobile phase are chosen as the surrogate model for the C8 stationary phase and mobile phase of the chromatographic system. The octane-mobile phase is used for measuring the partition coefficient K om of six compounds of the homologous series of linear alkylbenzenes, viz. benzene, toluene, ethylbenzene, propylbenzene, butylbenzene and pentylbenzene. The distribution of a compound between the octane and mobile phase is proposed to simulate the partitioning process in the chromatography. The retention factor k of each compound is measured using the same mobile phase for two C8 columns (Zorbax Eclipse XDB-C8 and Symmetry C8). The set of data of k and K om is fitted to the above linear equation to give the best-fit values of a and log(Φ) for each column and various mobile phase compositions (methanol-water or acetonitrile-water). The regression analyses have coefficients of determination r 2 > 0.992. This observed linear relationship can therefore be expressed as k=K om a Φ. The experimental values of Φ for the C8 columns are in the range of 0.206 to 0.842, with a from 0.544 to 0.811, respectively., 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. Published by Elsevier B.V.)

Development of a chromatographic method in bioanalysis is a challenging and complex procedure with many pitfalls and often unexpected reversals that can require several months to accomplish. Even an experienced analytical team must contend many limitations mainly in connection with the strict requirements imposed on current clinical research. These restrictions typically persist throughout the whole development process, from clinical trial assignment, across optimization of extraction of biological materials and chromatographic separation, to validation and data interpretation. This paper describes questions and their possible answers raised during the pre-analytical phase such as use of modern sample preparation techniques in clinical methods, application of internal standards, as well as selection of stationary phases and detection techniques in the analytical phase. Validation problems and interpretation of results are demonstrated with three typical examples of characteristics to be considered, i.e. recovery, matrix effect, and limit of detection vs. lower limit of quantification.

A modified method for the Craig scheme solution applied for a chromatographic column has been proposed and the corresponding implicit code is presented. The new approach improves the mass conservation problem frequently reported during the numerical solution of the Equilibrium Dispersive model with Craig scheme. The modified code has been successfully verified in gradient chromatography conditions by comparison with the reliable solutions of the Equilibrium Dispersive model by the Orthogonal Collocation on Finite Elements (OCFE). The errors obtained with the modified Craig method are less than about 1% in the case of retention times, and less than about 11% in the case of apparent number of theoretical plates, comparing to the OCFE solutions., Competing Interests: Declaration of Competing Interest All authors declare that they have no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Multi-column periodic counter-current chromatography (PCC) has been developed for continuous antibody capture, but the complexity of continuous processes makes experimental optimization time consuming and costly. In this work, with twin-column continuous system as a typical case, mathematical models were established and used to evaluate the impacts of operating parameters for process development. The model fitted well with the experimental breakthrough curves and process performance under varying protein concentrations and residence times. Three important operating parameters, residence time for interconnected feeding (RT C ), breakthrough percentage control for interconnected feeding (s) and disconnected feeding time (t DC ), were evaluated systematically. The profiles of productivity and resin capacity utilization showed three phases as a function of RT C , which resulted in different optimization strategies towards s and t DC . Based on the model prediction, a working window of RT C and s can be determined for process development. Finally, a model-based design approach was proposed to determine the optimum operating conditions and to design a suitable continuous process for high productivity and capacity utilization. With the model-based design approach developed, the best performance of 12.8 g/L/h productivity and 91.9% capacity utilization was found for MabSelect SuRe resin under 1 mg/mL feeding IgG concentration at RT C  = 2 min, s = 65% and t DC  = 26 min., Competing Interests: Declaration of Competing interests 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. Published by Elsevier B.V.)

We discuss how the efficiency of a chromatography device could be enhanced by incorporating a new feature which ensures flow uniformity. The overall flow of fluid within the device, which has a cuboid shape, could be visualized as a combination of two z patterns. The device is therefore designated as cuboid z 2 . The reason for flow uniformity is explained using a simple mathematical model, and also based on computational fluid dynamic (CFD) simulations. The cuboid z 2 device outperformed its equivalent conventional cylindrical column in terms of separation efficiency metrics such as the number of theoretical plates, the reduced plate height, and attributes of non-interacting tracer solute peaks. The results discussed in this paper clearly indicate the suitability of the cuboid z 2 device for carrying out high-resolution chromatographic separations. The z 2 flow enhancing feature would also be broadly applicable to any process which requires uniform flow in three-dimensional porous media. In addition to superior fluidic attributes, the cuboid z 2 chromatography device has other advantages such as compactness and scalability., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: A provisional US patent application for the flow feature examined in this study has been filed., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Recent studies have demonstrated that box-shaped or cuboid packed-bed chromatographic devices represent an efficient alternative to conventional cylindrical columns for high-resolution preparative protein separations. This has been attributed to the greater uniformity of flow within these devices. However, for a more complete explanation, it is important to understand how the system hydrodynamics affects band broadening during the transport of proteins through these devices. In this study, we present first principle mathematical models to capture this interplay. These models were validated by flow-through and bind-and-elute experiments carried out using a colored protein as tracer. Control experiments were also carried out using equivalent commercial columns, i.e. having same bed height and cross-sectional area, and packed with same media. The trends observed in the experiments matched those predicted by the models, though there were deviations in the absolute values. These deviations are explained in terms of non-idealities that exist in the experimental set-up, as well as in terms of factors that were not considered in the model. The models discussed in this paper are not only useful for understanding the workings of the cuboid packed-bed device, but are also useful tools for designing, optimizing and scaling-up such devices., 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 © 2019 Elsevier B.V. All rights reserved.)

Ultraperformance convergence chromatography is an environmentally friendly analytical technique for dramatically reducing the use of organic solvents compared to conventional chromatographic methods. In this study, a rapid and sensitive ultraperformance convergence chromatography method was firstly established for quantification of thymol and carvacrol, two positional isomers of a major bioactive in the volatile oil of Thymi herba , the dried leaves and flowers of Thymus mongolicus or Thymus przewalskii , known in China as "Dijiao." Using a Trefoil TM CEL1 column, thymol and carvacrol were separated in less than 2.5 min and resolution was enhanced. The method was validated with respect to precision, accuracy, and linearity according to the National Medical Products Administration guidelines. The optimized method exhibited good linear correlation ( r = 0.9998-0.9999), excellent precision (relative standard deviations (RSDs) < 1.50%), and acceptable recoveries (87.29-102.89%). The limits of detection for thymol and carvacrol were 1.31 and 1.57 ng/L, respectively, while their corresponding limits of quantification were 2.63 and 3.14 ng/L. Finally, the quantities of the two compounds present in 16 T. mongolicus and four T. przewalskii samples were successfully evaluated by employing the developed method. It is hoped that the results of this study will serve as a guideline for the quality control of Thymi herba .

Continuously growing demand for plant derived therapeutic molecules obtained in a sustainable and eco-friendly manner favors biotechnological production and development of innovative extraction techniques to obtain phytoconstituents. What is more, improving and optimization of alternative techniques for the isolation of high value natural compounds are issues having both social and economic importance. In this critical review, the aspects regarding plant biotechnology and green downstream processing, leading to the production and extraction of increased levels of fine chemicals from both plant cell, tissue, and organ culture or fresh plant materials and the remaining by-products, are discussed.

The species Karenia mikimotoi is a common nearshore red tide alga that can secrete hemolytic exotoxin and ichthyotoxin, which can induce the death of fish and shellfish, causing severe economic losses. In this study, loop-mediated isothermal amplification (LAMP) was employed in combination with the lateral flow dipstick (LFD) visual detection method to establish the LAMP-LFD rapid detection method for K. mikimotoi. The internal transcribed spacer ITS1-5.8S-ITS2 of K. mikimotoi was used as the target sequence and was amplified with specific primers designed in this study. The results indicated that the amplification optimal reaction conditions for LAMP in this paper were for 20 min at 65 °C. Moreover, LAMP had excellent specificity, showing negative results for other common red tide causing algal species. In field samples, we successfully reduced the total time, with only 23 min needed from LAMP amplification to LFD result display, which was shorter than that of conventional PCR. Consequently, LAMP-LFD should be useful for rapid field detection of low-density K. mikimotoi and for the early prevention of red tide induced by such algae., (Copyright © 2019. Published by Elsevier B.V.)

Antibody-drug conjugate (ADC) in vitro potency has been shown to be dependent on drug load, with higher drug load providing lower IC 50 values. However, in vivo potency is affected by intrinsic biological effects as well, such as plasma clearance, dose-limiting toxicity, etc. Developing a preparative HIC process for ADC purification to isolate species with a specific drug loading involves several steps including conjugation optimization, resin selection, solubility studies gradient screening, and step gradient development (buffer selection). In this chapter, the rationale and general considerations for developing a preparative hydrophobic interaction chromatography (HIC) method are described for isolation of an example ADC with specific drug load, e.g., two monomethyl auristatin E (MMAE) payloads (E2).

Developing nanofibrous aerogels with high porosity, robust underwater mechanical strength, and rich adsorption ligands, has been considered as one of the most promising strategies for preparing the next generation of high-efficiency and high-throughput chromatographic media; yet great challenges still remain. Herein, a novel type of highly phosphorylated nanofibrous aerogels (PNFAs) is fabricated, for the first time, by combining electrospinning, cryogenic induced phase separation regulation, and in situ phosphorylation modification. The PNFAs exhibit outstanding underwater superelasticity and excellent compression fatigue resistance (∼0% plastic deformation after 1000 compression cycles), as well as favorable shape-memory property. Besides, the PNFAs also can be bent and compressed even in the ultracold liquid nitrogen without obvious plastic deformation, further highlighting their robust structural stability. Benefiting from the superelastic, interconnected, and highly phosphorylated 3D nanofibrous frameworks, the PNFAs possess a superb protein adsorption capability of 3.3 × 10 3 mg g -1 and a large liquid flux of 1.5 × 10 4 L m -2 h -1 , which are superior to the commercial and previously reported fiber-based chromatographic media. Moreover, the PNFAs also exhibit superior performance stability, easy assembly, and outstanding applicability, highlighting their potential actual application. The successful preparation of such fascinating PNFAs may not only provide a new option for the current protein adsorption and purification engineering, but also could open up some new perspectives for further design and development of next-generation nanofibrous aerogel-based chromatographic media for various bioseparation applications.

Exosomes are one class of extracellular vesicles (30-150 nm diameter) that are secreted by cells. These small vesicles hold a great deal of promise in disease diagnostics, as they display the same protein biomarkers as their originating cell. On a cellular level, exosomes are attributed to playing a key role in intercellular communication, and may eventually be exploited for targeted drug delivery. In order for exosomes to become useful in disease diagnostics, and as burgeoning drug delivery platforms, they must be isolated efficiently and effectively without compromising their structure. Plasma from peripheral blood is an excellent source of exosomes, as it is easily collected and the process does not normally cause undue discomfort to the patient. Unfortunately, blood plasma content is complex, containing abundant amounts of soluble proteins and aggregates, making exosomes extremely difficult to isolate in high purity from plasma. Most current exosome isolation methods have practical challenges including being too time-consuming and labor intensive, destructive to the exosomes, or too costly for use in clinical settings. To this end, this study examines the use of poly(ethylene terephthalate) (PET) capillary-channeled polymer (C-CP) fibers in a hydrophobic interaction chromatography (HIC) protocol to isolate exosomes from a human plasma sample. Initial results demonstrate the ability to isolate exosomes with comparable yields and size distributions and on a much faster time scale when compared to traditional isolation methods, while also alleviating concomitant proteins and other impurities. As a demonstration of the potential quantitative utility of the approach, a linear response (particles injected on-column vs peak area) using a commercial exosome standard was established using a standard UV absorbance detector. Based on the calibration function, the concentration of the original human plasma sample was determined and subsequently confirmed by NTA measurement. The potential for scalable separations covering sub-milliliter spin-down solid phase extraction tips to the preparative scale is anticipated., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Developing a mechanistic understanding of protein dynamics and conformational changes at polymer interfaces is critical for a range of processes including industrial protein separations. Salting out is one example of a procedure that is ubiquitous in protein separations yet is optimized empirically because there is no mechanistic description of the underlying interactions that would allow predictive modeling. Here, we investigate peak narrowing in a model transferrin-nylon system under salting out conditions using a combination of single-molecule tracking and ensemble separations. Distinct surface transport modes and protein conformational changes at the negatively charged nylon interface are quantified as a function of salt concentration. Single-molecule kinetics relate macroscale improvements in chromatographic peak broadening with microscale distributions of surface interaction mechanisms such as continuous-time random walks and simple adsorption-desorption. Monte Carlo simulations underpinned by the stochastic theory of chromatography are performed using kinetic data extracted from single-molecule observations. Simulations agree with experiment, revealing a decrease in peak broadening as the salt concentration increases. The results suggest that chemical modifications to membranes that decrease the probability of surface random walks could reduce peak broadening in full-scale protein separations. More broadly, this work represents a proof of concept for combining single-molecule experiments and a mechanistic theory to improve costly and time-consuming empirical methods of optimization., Competing Interests: The authors declare no competing interest.

One of the most critical aspects of chromatographic analysis is effective data acquisition and processing. Typical approaches include software platforms designed for specific instruments or commercial data acquisition hardware boards, both of which require expensive licenses to use and operate. To increase the access and affordability of chromatographic data acquisition, especially for systems in which software control has become obsolete or must be written in-house, an open-source digital stripchart recorder has been developed. This system is built upon a Raspberry Pi single-board computer and a plug-in printed circuit board with the necessary integrated circuits for data acquisition. Using an open-source software called Processing, a complete user interface to control the system was developed that enables the acquisition, filtering, and processing of chromatographic data. The system performs comparably to more expensive platforms, with calculated values for peak area, retention time, and plate count all within 3% of the values calculated by a widely used commercial chromatography data software package., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The optimization of chromatography ligands for the purification of biopharmaceuticals is highly demanded to meet the needs of the pharmaceutical industry. In the case of monoclonal antibodies (mAbs), synthetic ligands comprising multiple types of interactions (multimodal) provide process and economic advantages compared to protein-based affinity ligands. However, optimizing the operation window of these ligands requires the development of effective high-throughput screening platforms. Here, a novel microfluidics-based methodology to perform rapid and multiplexed screening of various multimodal ligands relative to their ability to bind different target molecules is demonstrated. The microfluidic structure comprises three individual chambers (≈8 nL each) packed with different types of chromatography beads in series with the feed flow. An artificial mixture composed of immunoglobulin G (IgG) and bovine serum albumin, labeled with different thiol-reactive neutral fluorescent dyes, is used as a model to quantitatively optimize the performance (yield and purity) of the separation. This approach can potentially be used as a predictive analytical tool in the context of mAb purification, allowing low consumption of molecules and providing results in <3 min. Furthermore, this versatile approach can potentially be extended not only with respect to the number of different resins and target molecules, but also for parallel analysis of multiple conditions., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The main aim of this study was to define the optimal adsorption and elution conditions for the purification of human immunoglobulin G (IgG) by mixed-mode chromatography using the multimodal resin Capto MMC. To this end, Central Composite Experimental Design (ED) was performed for both the adsorption and desorption stages. In the first case, the conditions were systematically studied in batch mode while in the latter case, these were performed in column. For both studies, the experimental design was conducted using high-purity human IgG samples. Buffer pH and concentration as well as the salt concentration were the parameters under study in the ED. Adsorption kinetics and equilibrium experiments were performed under the best conditions defined in the ED (phosphate buffer 60 mmol/L, pH 6.75, no salt). The equilibrium experimental data were fit to the Langmuir equation, with maximum uptake q max equal to 549.2 mg/g. The q max value found for IgG in Capto MMC was quite high as compared to other chromatographic techniques that employ single modes of interaction. Regarding elution, the best conditions were obtained with acetate buffer (56.40 mmol/L), pH 5.2 and 0.2 mol/L NaCl. An ultimate recovery of 46.96% for high-purity IgG was achieved. Thus, the effectiveness of Capto MMC for IgG adsorption and recovery could be confirmed. Moreover, electrophoretic runs in the human serum indicated that although co-elution of HSA and IgG proteins occurs, substantial HSA removal and a high IgG recovery were achieved in the elution step., (Copyright © 2019 Elsevier B.V. All rights reserved.)

We analyse a relationship between the bulk microstructure of randomly packed beds, which we quantify through chord length distribution (CLD) analysis of the interparticle void space, and the associated flow heterogeneity, as expressed by the longitudinal dispersion coefficient at a Péclet number of Pe = 10. A random collection of physically reconstructed packings is complemented with a systematic set of computer-generated packings of monosized spheres, for which the packing-generation algorithm has been carefully adjusted to realize a monotonic variation of the bed porosity and microstructural heterogeneity. The most relevant difference in the morphology between these computer-generated and the physically reconstructed packings are structural defects present in the real packings, such as particle oligomers and larger voids as well as contaminations and particle debris. These defects influence the pore space morphology and introduce additional structural heterogeneity. Hydrodynamic dispersion coefficients for all packings are derived by implementing the lattice-Boltzmann method to simulate fluid flow and a random-walk particle tracking technique to record the transport of passive, point-like tracers in the flow fields. We propose a morphological descriptor, σ/μ, based on statistical parameters of a CLD (standard deviation σ and mean chord length μ) that can be used to predict the dispersion coefficient in packed beds, independent from the underlying particle size distribution, packing-generation protocol, bed porosity, and the occurrence of structural defects., (Copyright © 2019 Elsevier B.V. All rights reserved.)

In the present work, mixed-mode chromatography adsorbents grafted with dextran of different molecular weight were prepared. Compared to non-grafted adsorbent (BA-6B), the morphology of dextran-grafted adsorbents (BA-6B-Ts) did not change significantly, while the water content (ω), porosity (P) and pore volume (V) decreased. Static adsorption, salt tolerance and dynamic adsorption behaviors of adsorbents BA-6B-Ts against BSA/IgG were studied. Compared to adsorbent BA-6B, adsorbents BA-6B-Ts had higher saturated adsorption capacity (Q m ) toward BSA/IgG, especially for BA-6B-T20, the Q m values toward BSA and IgG increased by 32.8% and 39.5%, respectively. Adsorbents BA-6B-Ts showed enhanced salt tolerance toward BSA, and relatively weaker salt tolerance toward IgG. The relative adsorption capacity of BA-6B-T20 was 66.7% at 0.1 mol/L NaCl and 39.2% at 0.5 mol/L NaCl, higher than those of BA-6B. Moreover, for both BSA and IgG, the effective pore diffusivity (D e ) of BA-6B-Ts was higher than that of BA-6B. D e values of BA-6B-T20 toward BSA and IgG increased by 77% and 139% compared to BA-6B, respectively. Dynamic binding capacities (DBCs) of BA-6B-Ts were also improved. DBC of BSA and IgG on BA-6B-T20 were 25.6% and 46.5% higher than those on BA-6B. The possible mechanisms for the above difference in adsorption behavior of dextran-grafted adsorbents toward BSA/IgG were discussed, providing some insights into protein adsorption and mass transport for dextran-grafted MMC resins., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Rationale: Porous graphic carbon chromatography (PGC) has a different mechanism in the retention of tryptic peptides compared with reversed-phase chromatography and in this study we show that coupling PGC with tandem mass spectrometry offer advantages for the quantitation of phosphorylation stoichiometry and characterization of site-specific glycosylation., Methods: Digests of protein standards (horse myoglobin, bovine fetuin and β-casein) were analyzed with a capillary liquid chromatography/tandem mass spectrometry (LC/MS/MS) system by coupling an Agilent 1100 HPLC system to a Synapt G2-Si HDMS (Waters). Peptides were separated using a HyperCarb PGC column (300 μm i.d. × 100 mm) packed with 3 μm particles. MS/MS data were collected in data-dependent mode and three MS/MS scans were acquired after the full MS scan. RAW data were transformed to .mgf by PLGS (Waters) and searched against the Swissprot database by Mascot. Chromatograms and MS/MS spectra of identified compounds were extracted with Masslynx (Waters) and imported to Origin for analysis. Glycan composition and peptide sequence were manually annotated., Results: PGC/MS/MS enabled accurate quantitation of the stoichiometry of specific phosphorylation sites from β-casein by efficient separation of the phosphopeptide and its non-phosphorylated counterpart, which cannot be achieved by reversed-phase chromatography. PGC/MS/MS also enabled comprehensive characterization of protein sialoglycosylation as isomeric glycopeptides with different combinations of α2-3- and α2-6-linked sialic acids can be separated and the ratios of each combination were verified by exoglycosidase digestion., Conclusions: PGC has demonstrated superior separation of peptides with phosphorylation and glycosylation and can be used as an alternative in the proteomic characterization of post-translational modifications (PTMs) by polar groups., (© 2019 Her Majesty the Queen in Right of Canada. Rapid Communications in Mass Spectrometry © 2019 John Wiley & Sons, Ltd.)

Two novel extraction chromatographic resins (EC), termed as RL-1 and RL-2, were prepared by impregnating two benzene-centered tripodal iglycolamide ligands (Bz-T-DGA) containing different spacer groups where the ligands are termed as L-1 and L-2, respectively. They were employed for the uptake of actinide and fission product ions, viz. Am 3+ , Eu 3+ , UO 2 2+ , Np 4+ , Pu 4+ , Sr 2+ , and Cs + , from acidic feeds. Weight distribution coefficient (K d ) values were measured by the batch method and the loaded metal ions were back extracted using a 0.01 M EDTA solution at pH 4. Kinetic modeling of the sorption data of Am(III) on both resins suggested pseudo-second order rate kinetics with rate constants of 1.68 × 10 -6 and 2.47 × 10 -6 g/cpm.min for the resins containing L-1 and L-2, respectively. Sorption isotherm studies indicated the Langmuir monolayer chemisorption phenomenon with Eu(III) experimentally determined saturation uptake capacities of 6.02 ± 0.11 and 5.49 ± 0.14 mg per g of RL-1 and RL-2 resins, respectively. As the batch uptake study results appeared encouraging, column studies were also carried out using both resins. The resin reusability data indicated a marginal change in the K d values for the RL-1 resin up to three repeat runs beyond which a steady decrease of the K d value was seen. On the other hand, in the case of RL-2 a steady decrease in the K d values was observed for three repeat runs beyond which there was marginal change., (Copyright © 2019 Elsevier B.V. All rights reserved.)

An integrated, parallel-plate microfluidic device is engineered to interrogate and fractionate cells based on their adhesivity to a substrate surface functionalized with adhesive ligand in a tightly controlled flow environment to elucidate associated cell-intrinsic pathways. Wall shear stress levels and endothelial presentation of E-selectin are modeled after the inflamed vasculature microenvironment in order to simulate in vitro conditions under which in vivo hematogenous metastasis occurs. Based on elution time from the flow channel, the collection of separate fractions of cells-noninteracting and interacting-at high yields and viabilities enables multiple postperfusion analyses, including flow cytometry, in vivo metastasis modeling, and transcriptomic analysis. This platform enables the interrogation of flow-regulated cell molecular profiles, such as (co)expression levels of natively expressed selectin ligands sLe x , CD44, and carcinoembryonic antigen, and cancer stem cell marker CD24. This additionally reveals E-selectin adhesivity exhibited by metastatic human colon carcinoma cells to be a transient phenotype. Facile and rapid, this methodology for unbiased, label free sorting of large populations of cells based on their adhesion in flow represents a method of studying flow-regulated adhesion in vitro for the identification of molecular drug targets for development as antimetastatic cancer therapeutics., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Human African trypanosomiasis (HAT), or sleeping sickness, is a neglected tropical disease that is fatal if untreated, caused by Trypanosoma brucei gambiense and T. brucei rhodesiense. In its 2012 roadmap, WHO targeted HAT for elimination as a public health problem in 2020 and for zero transmission in 2030. Diagnosis of HAT is a multistep procedure comprising of clinical suspicion, confirmation, and stage determination. Suspects are identified on clinical signs and/or on screening for specific antibodies. Parasitological confirmation of suspects remains mandatory to avoid unnecessary toxic drug administration. The positive predictive value of the antibody detection tests is low. Simple parasite detection techniques, microscopic examination of lymph node aspirate, or stained thick blood films lack sensitivity, whereas in T. brucei gambiense patients, the number of blood trypanosomes may be very low. Parasite concentration techniques are therefore indispensable. Half a century ago, Sheila Lanham discovered a technique to separate trypanosomes from the blood of infected rodents, based on anion exchange chromatography with diethyl amino ethyl (DEAE) cellulose, a weak anion exchanger. Between pH 6-9, trypanosome surface is less negatively charged than that of blood cells. When blood is poured on top of a DEAE cellulose column, blood cells are retained, whereas parasites pass the column together with the elution buffer. The result is a pure suspension of trypanosomes that retain their morphology and infectivity. Because cell surface charges vary among trypanosome and mammal species, the optimal buffer pH and ionic strength conditions for different combinations of host and trypanosome species were established. Lanham's technique revolutionized the diagnosis of HAT. It is indispensable in the production of the Card Agglutination Test for Trypanosomiasis (CATT), the most used field test for screening in T. brucei gambiense HAT foci and essential to confirm the diagnosis in suspected people. Lumsden and colleagues developed the mini anion exchange centrifugation technique (mAECT). After adaptation for field conditions, its superior diagnostic and analytical sensitivity compared to another concentration technique was demonstrated. It was recommended as the most sensitive test for demonstrating trypanosomes in human blood. At the beginning of the 21st century, the mAECT was redesigned, allowing examination of a larger volume of blood, up to 0.35 ml with whole blood and up to 10 ml with buffy coat. The plastic collector tube in the new kit is also used for detection of trypanosomes in the cerebrospinal fluid. Unfortunately, mAECT also has some disadvantages, including its price, the need to centrifuge the collector tube, and the fact that it is manufactured on a noncommercial basis at only two research institutes. In conclusion, 50 years after Sheila Lanham's discovery, CATT and mAECT have become essential elements in the elimination of HAT., Competing Interests: NO authors have competing interests.

Colistin is a multicomponent polypeptide antibiotic consisting mainly of colistin A and colistin B, produced by selected strains of Bacillus polymyxa var. Colistinus. Only recently, the prodrug of colistin, colistimethate sodium, is widely used as last resort antibiotic for infections caused by resistant gram-negative bacteria. Colistin having been discovered several years ago, has not subjected to the drug development and regulatory approval processes that are applied today. However, pharmacological and pharmacokinetic information are necessary for its optimal use thus, during the last decades several studies are carried out in order to shed light on this issue. In the current review, the analytical methodologies of colistin assessment in biological material are summarized and the analytical challenges are critically discussed and critical aspects of the determinations such as the method of detection, the sample pretreatment methodology etc. are compared. Furthermore, critical quality aspects of the assessment methodologies such as the sensitivity of the currently developed methodologies are presented. Lastly, some future trends that should be incorporated in the determination pipeline of modern drugs are suggested., (Copyright © 2018 Elsevier B.V. All rights reserved.)

For performing point-of-care molecular diagnostics, magnetic immunoassays constitute a promising alternative to established enzyme-linked immunosorbent assays (ELISA) because they are fast, robust and sensitive. Simultaneous detection of multiple biomolecular targets from one body fluid sample is desired. The aim of this work is to show that multiplex magnetic immunodetection based on magnetic frequency mixing by means of modular immunofiltration columns prepared for different targets is feasible. By calculations of the magnetic response signal, the required spacing between the modules was determined. Immunofiltration columns were manufactured by 3D printing and antibody immobilization was performed in a batch approach. It was shown experimentally that two different target molecules in a sample solution could be individually detected in a single assaying step with magnetic measurements of the corresponding immobilization filters. The arrangement order of the filters and of a negative control did not influence the results. Thus, a simple and reliable approach to multi-target magnetic immunodetection was demonstrated.

Previously, we have reviewed in this journal (Arakawa, T., Kita, Y., Curr. Protein Pept. Sci., 15, 608-620, 2014) the interaction of arginine with proteins and various applications of this solvent additive in the area of protein formulations and downstream processes. In this special issue, we expand the concept of protein-solvent interaction into the analysis of the effects of solvent additives on various column chromatography, including mixed-mode chromatography. Earlier in our research, we have studied the interactions of such a variety of solvent additives as sugars, salts, amino acids, polymers and organic solvents with a variety of proteins, which resulted in mechanistic understanding on their protein stabilization and precipitation effects, the latter known as Hofmeister series. While such a study was then a pure academic research, rapid development of genetic engineering technologies and resultant biotechnologies made it a valuable knowledge in fully utilizing solvent additives in manipulation of protein solution, including column chromatography., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Multimodal or mixed-mode chromatography can be used to separate various proteins, including antibodies. The separation quality and efficiency have been improved by the addition of solutes, especially arginine. This review summarizes the mechanism underlying the effects of arginine on protein elution in multimodal chromatography with neutral, anionic or cationic resin ligands; the mechanism has been investigated using experiments and molecular dynamics simulations. Arginine is effective in facilitating protein elution compared to salts and protein denaturants such as guanidine and urea. The unique elution effect of arginine can be explained by the interplay among arginine, proteins and the resin ligands. Arginine exhibits multiple binding modes for the ligands and further affinity for protein aromatic residues through its guanidinium group. These properties make arginine versatile for protein elution in multimodal chromatography. Taking into account that arginine is an aggregation suppressor for proteins but not a protein denaturant, arginine is a promising protein-eluting reagent for multimodal chromatography., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

TOYOPEARL particles are cross-linked hydroxylated methacrylic polymers available in different pore and particle sizes. They are conjugated with different ligands to generate ion-exchange, hydrophobic interaction and affinity resins. They have excellent physical and chemical properties. A mixed-mode resin, TOYOPEARL MX-Trp-650M, is made of this particle with tryptophan conjugated via N-terminal amino group and hence has both hydrophobic/aromatic side chain and carboxyl group. In this review, I will summarize the properties of the TOYOPEARL particles and MX-Trp-650M resin and application of this resin for purification of proteins and in some detail the separation of disulfide (SS)- scrambled oligomers of insulin-like growth factor-1 (IGF-1). For this particular application, the intact IGF-1 was used to examine binding and elution conditions of TOYOSCREEN MX-Trp-650M column. Strong binding was obtained at pH 4.0, at which arginine, but not NaCl, resulted in elution. Both NaCl and arginine resulted in elution at pH 6.5. In addition, a pH gradient from 4.0 to 8.5 was effective. When applied to SS-scrambled IGF-1 oligomers, both pH and arginine gradient exhibited an efficient separation of the oligomers., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Mixed mode chromatography resins with salt tolerance, large design space and orthogonal selectivity requires a slightly more complex development than traditional resins. It is important to screen several ligands and several binding and elution conditions. This allows taking full advantage of these resins. High-Throughput Screening (HTS) for Process Development should be done with the help of Design of Experiment (DoE). It could be performed in filter plates or Robocolumns, and assisted by liquid handling automated workstation. Modeling of the results allows the choice of optimal parameters that can then be validated and scaled up. All this leads to a better knowledge and robustness of the purification step., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

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

Proteins often generate structure isoforms naturally or artificially due to, for example, different glycosylation, disulfide scrambling, partial structure rearrangement, oligomer formation or chemical modification. The isoform formations are normally accompanied by alterations in charged state or hydrophobicity. Thus, isoforms can be fractionated by reverse-phase, hydrophobic interaction or ion exchange chromatography. We have applied mixed-mode chromatography for fractionation of isoforms for several model proteins and observed that cation exchange Capto MMC and anion exchange Capto adhere columns are effective in separating conformational isoforms and self-associated oligomers., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

The needs for purified nucleic acids for preparative and analytical applications have increased constantly, demanding for the development of new and more efficient methods for their recovery and isolation. DNA molecules harbour some intrinsic chemical properties that render them suitable for chromatographic separations. These include a negatively charged phosphate backbone as well as a hydrophobic character originating mainly from the major groove of DNA which exposes the base pairs on the surface of the molecule. In addition, single stranded DNA often allows for a free exposure of the hydrophobic aromatic bases. In this review, multimodal chromatography (MMC) has been evaluated as an alternative tool for complex separations of nucleic acids. MMC embraces more than one kind of interaction between the chromatographic ligand and the target molecules. These resins have often proved superior to conventional single-mode chromatographic materials for DNA isolation, including, e.g., the purification of plasmid DNA from crude cell lysates and for the preparation of DNA fragments before or after a polymerase chain reaction (PCR)., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Mixed mode chromatography offers a diversity of ligands, each providing a new selectivity. This allows the design of novel purification processes with reduced column steps. Structure of ligands is based on both hydrophobic and ionic groups. Thanks to its salt tolerance, crude extracts or post-IEX samples can be loaded directly without conditioning. The selectivity could be enhanced by modulating elution parameters or by using additives. More importantly, mixed mode chromatography could be as effective as affinity chromatography for mAb purification processes. Mixed mode chromatography opens the way to short and economical processes., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)