Column Volume Research Articles

Chromatographic separation of hemoglobin variants using robust molecularly imprinted polymers

Devising a robust, efficient and cost effective hemoglobin (Hb) purification strategy is one of the key challenges in the development of Hb-based blood substitutes. The aim of this study was to use molecularly imprinted polymers (MIPs) as a novel and efficient chromatographic resin to selectively recognize and purify different Hb variants. The results showed that the Hb-MIP material developed here could selectively recognize and purify various Hb directly from either crude E. coli extracts or human body fluids, such as blood plasma and cerebrospinal fluid (CSF), in one-step. The dynamic binding capacity at 10% breakthrough was around 7.4 mg mL−1resin for adult Hb (HbA) and fetal Hb (HbF). This chromatographic material also allowed identification of changes related to amino acid substitutions on the Hb protein surface. For instance, when an additional lysine residue was introduced, the HbA αY42K mutant eluted later in an Hb-MIP column than wildtype HbA. Additional negative charges on the protein surface, such as aspartate, mitigated the interaction between the protein and imprinted polymers, and therefore an αA19D-αA12D HbF mutant eluted earlier, at −2.7 column volumes compared to wildtype HbF.

Pickering Emulsion-Templated Encapsulation of Ionic Liquids for Contaminant Removal.

Ionic liquids (ILs) have received attention for a diverse range of applications, but their liquid nature can make them difficult to handle and process and their high viscosities can lead to suboptimal performance. As such, encapsulated ILs are attractive for their ease of handling and high surface area and have potential for improved performance in energy storage, gas uptake, extractions, and so forth. Herein, we report a facile method to encapsulate a variety of ILs using Pickering emulsions as templates, graphene oxide (GO)-based nanosheets as particle surfactants, and interfacial polymerization for stabilization. The capsules contain up to 80% IL in the core, and the capsule shells are composed of polyurea and GO. We illustrate that capsules can be prepared from IL-in-water or IL-in-oil emulsions and explore the impact of monomer and IL identity, thereby accessing different compositions. The spherical, discrete capsules are characterized by optical microscopy, scanning electron microscopy, infrared spectroscopy, Raman spectroscopy, thermogravimetric analysis, and 1H NMR spectroscopy. We illustrate the application of these IL capsules as a column material to remove phenol from oil, demonstrating ≥98% phenol removal after passage of >170 column volumes. This simple method to prepare capsules of IL will find widespread use across diverse applications.

Prediction of lab and manufacturing scale chromatography performance using mini-columns and mechanistic modeling

Chromatography is a cornerstone of biologics downstream purification processes, and there is an ever increasing demand for improved speed and efficiency in process development. Scale-down models are used in process development to optimize operating conditions and study process robustness while expending as little time and material as possible. The advent of automated liquid handling systems and miniature columns has taken the efficiency of process development to another level by allowing up to eight column runs in parallel with column volumes under 1 ml. As expected, results between these miniature columns and typical lab/manufacturing scale columns can deviate due to scale dependent and/or configuration dependent differences. Regulatory guidelines do not require an exact match in scale-down and large scale data, but do require that small scale models account for scale effects, be representative of the commercial process, and be scientifically justified. Therefore, it is important to gain insight into what causes differences between scales and account for them during development. Mechanistic models can be used to understand the physics of the process (fluid flow, mass transfer, etc.) as a function of scale, and provide explanation for deviations that may be observed. We have used mechanistic modeling to study the factors leading to differences in pool sizes observed between scales, and to make predictions on lab scale pool sizes from miniature column data. Results indicate that changes in mass transfer parameters, specifically axial dispersion, between scales leads to the observed differences in pool size. Additionally, we have studied the effect of system differences between automated liquid handling systems and conventional preparative chromatography systems on elution pool volume. This work provides new insight into the fundamental differences observed between scales and overcomes the challenge of enabling the use of miniature column chromatography as a scale-down model for process characterization.

Characterization of a centrifugal partition chromatographic column with spherical cell design

A new centrifugal partition chromatography (CPC) column with spherical cells, insignificant duct (channel) volume, and without PTFE plates between the disks was evaluated. Due to the reduced duct volume, almost the entire column volume is available for the separation. This allows operation at higher pressure drop and thus increased rotational speed and flow rates. The new column was characterized in terms of stationary phase retention, column efficiency and resolution as a function of flow rate and rotational speed. The separation performance was compared to that of two conventional twin-cell CPC columns of the same total volume. The new column leads to an increased productivity especially at higher flow rates and outperforms columns with smaller cells in terms of higher separation factors and reduced purity or recovery requirements.

Packing quality, protein binding capacity and separation efficiency of pre-packed columns ranging from 1 mL laboratory to 57 L industrial scale

Pre-packed chromatography columns are routinely used in downstream process development and scale-down studies. In recent years they have also been widely adopted for large scale, cGMP manufacturing of biopharmaceuticals. Despite columns being qualified at their point of manufacture before release for sale, the suitability of pre-packed chromatography columns for protein separations at different scales has not yet been demonstrated. In this study, we demonstrated that the performance results obtained with small scale columns (0.5 cm diameter × 5 cm length, 1 mL column volume) are scalable to production sized columns (60 cm diameter × 20 cm length, 57 L column volume). The columns were characterized with acetone and blue dextran pulses to determine the packing density and packed bed consistency. Chromatography performance was evaluated with breakthrough curves including capacity measurements and with separation of a ternary protein mixture (lysozyme, cytochrome C and RNase A) with a step gradient. The equilibrium binding capacity and dynamic binding capacity were equivalent for all columns. The step gradient separation of the ternary protein mixture displayed similar peak profiles when normalized in respect to column volume and the eluted protein pools had the same purities for all scales. Scalable performance of pre-packed columns is demonstrated but as with conventionally packed columns the influence of extra column volume and system configurations, especially buffer mixing, must be taken into account when comparing separations at different scales.

Ionic Liquids as Modifying Agents for Protein Separation in Centrifugal Partition Chromatography

AbstractAqueous two‐phase systems (ATPSs) can be applied in centrifugal partition chromatography (CPC) to separate biomolecules. Two ionic liquids (ILs) served as modifying agents to tune partition coefficients in the separation of two model proteins, myoglobin and lysozyme. Myoglobin was not affected by the ILs, while the partition coefficient of lysozyme was lowered with addition of IL. High stationary phase retention was achieved with ATPSs in a CPC column with cell size and geometry specially designed for unstable biphasic systems. When ILs were used as modifying agents, no notable decrease in stationary phase retention was observed. The injection volume was increased to 20 % of the total column volume and process simulations indicate that a further increase in the injection volume is possible.

Calculation of a packed column for absorption of hydrogen sulfur from gases formed by separation of crude oil

According to the well-known calculating algorithm for the packed absorption column, the main technological parameters and geometric dimensions of the apparatus for hydrogen sulfide containing gases, formed during the separation of crude oil, are determined. A 2,5-n solution of monoethanolamine is selected as the absorbent. Comparative results of the calculations are showing, that the working pressure in the column should be 4 atm, since with their lower values, the flow rate of the absorbent and the size of the column are increasing. The increase in working pressure is impractical, since it will require a transition from centrifugal and compresses pumps to piston pumps. ∏ The obtained parameters were compared for an absorption column in which the flow structure of the gas and liquid phases corresponds to ideal displacement, with the calculation results when the flow structure in the gas phase corresponds to the ideal displacement mode (as in the standard calculation algorithm) and in the liquid phase to ideal mixing. It is shown that, with the Peclet number of longitudinal diffusion pe<40 the height and volume of the column increases by 10 and more percent and should be taken into account when packed columns, intended for absorption processes, are designed. At pe=30 the height and volume of the nozzle in the column increases by 27%. Another feature of the modelling and calculation of devices is a spike in concentration, meaning, that the lower part of the working line will cross the equilibrium. Calculations in thisparticular case show that at ≈ 11 the working line crosses the equilibrium and theoretically column height and volume →∞.

Chitosan based composite sorbents for arsenic removal

The possibilities of using chitosan and its composites with transition metal oxides for arsenic removal from solutions with low concentrations are discussed. Methods for the formation of composite sorbents based on chitosan and molybdenum in a composition with a carbon fiber providing improved physical and chemical properties with respect to the recovered component are considered. The sorption properties of the obtained materials under dynamic conditions for the purification of solutions from arsenic are compared. It is shown that the preliminary modification of the carbon fiber with chitosan by ionic gelation using sulfate ion and then by adsorption with molybdate ion leads to the production of an efficient sorption material that provides the purification of 1600 column volumes of an arsenic solution at an initial concentration of 105 μg L–1 to a maximum permissible concentration of 50 μg L–1. The calculation methods show that the gel ability of sulfate and molybdate ions is approximately the same.

Organic source of vanadium. preparation and physicalchemical characteristic

The complex was obtained at room temperature: 10% water solution of SPI was mixed with 25% solution of vanadium salt (VOSO4×хH2O) in ratio 10:1 (in dry matter). The reaction was kept during 1 h at constant mixing with pH kept at 7.0- 7.1 with 1.0 M NaOH. The concentration of vanadium was determined in dry product by means of inductively coupled plasma mass-spectrometry. The chromatograms of SPI and V-SPI were obtained by means of size-exclusion high-pressure liquid chromatography, and then were integrated by weight method in the range of free till full column volume. The obtained complex of vanadium with SPI enzymatic hydrolysate (V-SPI) was water-soluble and contained 15.8 mg of vanadium per gram of product dry weight. Analysis of the molecular weight distribution of the peptide fractions of the original SPI enzymatic hydrolysate and the V-SPI complex showed that more than 87% of the vanadium complex was in peptide fractions with molecular weights more than 4.1 kD, including more than 75% of vanadium contained in fractions with molecular weights from 14.6 to 4.1 kD. The experimental evaluation in vivo will be the next stage of this research. The complex bioavailability and its effects on carbohydrate and lipid metabolism of Wistar rats with obesity will be evaluated.

Coupling between Land⁻Ocean⁻Atmosphere and Pronounced Changes in Atmospheric/Meteorological Parameters Associated with the Hudhud Cyclone of October 2014.

India is vulnerable to all kinds of natural hazards associated with land, ocean, biosphere, atmosphere, and snow/glaciers. These natural hazards impact large areas and the population living in the affected regions. India is surrounded by ocean on three sides and is vulnerable to cyclonic activities. Every year cyclones hit the east and west coasts of India, affecting the population living along the coasts and infrastructure and inland areas. The extent of the affected inland areas depends on the intensity of the cyclone. On 12 October 2014, a strong cyclone “Hudhud” hit the east coast of India that caused a high degree of devastation along the coast. The impact of this cyclone was seen up to the Himalayan region. Detailed analysis of satellite and ground data show a strong coupling between land-ocean-atmosphere associated with the Hudhud cyclone. The contrast between land and ocean temperature was found to be closely related with the formation of the cyclone in the ocean and its movements towards land. Pronounced changes in the ocean, land, atmospheric, and meteorological parameters with the development of the cyclone and its landfall have been observed. Changes in total column ozone (TCO), relative humidity (RH), and volume mixing ratio of CO (CO VMR), water mixing ratio (H2O MMR), surface latent heat flux (SLHF), and aerosol optical properties derived from satellite data show characteristic behavior of the Hudhud cyclone.

Effective filter screening approach with fractionation for enabling inline filtration of protein A eluate.

Filtration of protein A eluates inline with a chromatography column is a common challenge for monoclonal antibody (mAb) purification due to the high system backpressure during elution, which can result in system shut down or require a decreased elution flow rate. The inability to filter inline not only poses a risk for process deviations, but can also lead to tank constraints and microbial ingress risk. Here, we evaluated and described a novel approach for identifying filters for inline filtration of protein A eluates at pilot scale. We fractionated the protein A eluates into 0.25 column volume fractions to screen filters under constant pressure or constant flow conditions. We observed that filtration properties for eluate fractions are significantly different from the offline eluate, and the conventional filter sizing study using elution pool is not able to predict inline filtration behavior. Through the submicron particle counts and size distribution in pre- and post-filtration samples, we determined that both attributes contribute to the high pressure across the filters. A successful proof-of-concept experiment on a column 10cm in diameter inline with the filter train selected validated this fractionation method, and the approach was applied to a different mAb molecule to confirm effectiveness.

Effect of the relative degree of foulant “hydrophobicity” on membrane fouling

Foulant hydrophobicity has been regarded as a key factor in membrane fouling. While hydrophobic/philic fractionation using DAX (or XAD) resin column has been extensively utilized in fouling studies, the critical retention factor (k’cr, the ratio of water volume to column volume) has been long neglected which can alter the definition and relative degree of “hydrophobicity” of the obtained fractions and affect fouling results. To this end, this study compared fouling behavior of a membrane bioreactor (MBR) supernatant fractionated at varied k’cr, and found that not the distinctively strong “hydrophobic” (k’cr > 50) or “hydrophilic” (k’cr < 5) fractions, but rather the nominally moderate “hydrophobic/philic” fractions had the greatest contribution to overall fouling. The joint influence of foulant “hydrophobicity” and coexisting factors (molecular weight, carboxylic group, UV/fluorescence properties and polysaccharide/protein/humic contents) was elucidated via multivariate statistical analysis. Therefore, special attention should be placed on the nominally moderate “hydrophobic/philic” fractions for fouling control.

Extraction from aqueous solutions ions of iron and lead by filter loading, created on the basis of steel melting slag

The aim of the present paper is investigation of removal of Fe3+ and Pb2+ ions from aqueous media by material on the basis of steel melting slag. It has been established that the process of water purification from Fe3+ and Pb2+ ions by this material proceeds by a mixed mechanism both due to the formation of slightly soluble silicates and also due to the formation of hydroxides. When the filtration rate varies from 2 to 10 column volumes per hour, the percentage of sorption remains within 91,5–99,8%. The most optimal filtration rate is 6 column volumes per hour. Material on the basis of steel melting slag absorbs of Fe (III) up to 5 mg-eq/g and Pb (II) 0.64–1.27 mg-eq/g.

Preparation and characterization of large-format macroporous cryogel disks for use in affinity chromatography and biotechnological applications.

We have prepared and evaluated larger format phage-bound epoxy-cryogel columns in order to increase the yield of bound target. Freezing thermograms showed that larger column formats (2.5-5cm diameter) are not usable due to irregular polymerization phenomena. Preparing thin disks of 0.5cm height with similar diameter proved to be an excellent alternative. Disks could be stacked and run in a chromatographic setup. In this way, we could increase the matrix volume, ligand-binding capacity, and finally the yield of bound target. By increasing the column volume about sevenfold, we observed a 12-fold increase of ligand density and a sevenfold increase in the yield of protein recovery in a column where phages were attached without spacer and a 10- to 34-fold increase in a spacer column, depending on the spacer used.

Retention characteristics of some antibiotic and anti-retroviral compounds in hydrophilic interaction chromatography using isocratic elution, and gradient elution with repeatable partial equilibration

The separation of some zwitterionic, basic and neutral antibiotic and antiretroviral compounds was studied using hydrophilic interaction chromatography (HILIC) on bare silica, bonded amide and urea superficially porous phases. The differences in the selectivity and retentivity of these stationary phases were evaluated for compounds with widely different physicochemical properties (logD −3.43 to 2.41 at wwpH 3.0). The mobile phase was acetonitrile-ammonium formate buffered at low wwpH. Compounds containing quinolone and serine groups showed poor peak shapes on all columns, attributed to metal-oxide interactions with system metals. Peak shapes were improved by addition of citrate buffers. Gradient elution, particularly with regard to column equilibration, was also studied due to the large differences in retention factors observed under isocratic conditions. Full equilibration in HILIC was slow for both ionogenic and neutral solutes, requiring as much as ∼40 column volumes. However, highly repeatable partial equilibration, suitable for gradient elution, was achieved in only a few minutes. Pronounced selectivity differences in the separations were shown dependent on the partial equilibration time.

Self-Sufficient Flow-Biocatalysis by Coimmobilization of Pyridoxal 5′-Phosphate and ω-Transaminases onto Porous Carriers

We expanded the application of self-sufficient heterogeneous biocatalysts containing coimmobilized ω-transaminases and pyridoxal 5′-phosphate (PLP) to efficiently operate packed-bed reactors in continuous flow. Using a ω-transaminase from Halomonas elongata coimmobilized with PLP onto porous methacrylate-based carriers coated with polyethylenimine, we operated a packed-bed reactor continuously for up to 50 column volumes at 1.45 mL × min–1 in the enantioselective deamination of model amines (α-methylbenzyl amine), yielding >90% conversion in all cycles without exogenous addition of cofactor. In this work, we expanded the concept of self-sufficient heterogeneous biocatalysts to other ω-transaminases such as the ones from Chromobacterium violaceum and Pseudomonas fluorescens. We found that enzymes with lower affinities toward PLP present lower operational stabilities in flow, even when coimmobilizing PLP. Furthermore, ω-transaminases coimmobilized with PLP were successfully implemented for the continuous sy...

Development and modeling of two-dimensional fast protein liquid chromatography for producing nonstructural protein-free foot-and-mouth disease virus vaccine

Concerns for the use of non-purified or incompletely purified inactivated foot-and-mouth disease (FMD) vaccine, like difficulties for differentiation vaccinated from infected animals, can be a motivation in order to develop methods based on size exclusion chromatography (SEC). In this study, a two dimensional size exclusion chromatography (2D-SEC) system was successfully constructed using two different SEC column media to achieve a high-throughput purification system for the cell culture-derived foot and mouth diseases virus (FMDV). A mathematical model was also utilized to predict and to get a better insight into the separation process. Column and the packing particles characteristics such as column void volume, total column volume, particle porosity and accessible particle porosity was acquired experimentally. Retention times and elution profile of two different molecules, blue dextran and bovine serum albumin, were used for evaluating the capability of SEC media for separating two critical impurities (residual DNA (rDNA) and non-structural protein (NSP)) from active ingredient of vaccine (FMDV particle). Experiments were carried out with two different commercial columns (XK 26/60) and (XK 16/100) and with four different packing media superdex 200 prep grade, sephacryl S-500 HR, Sephacryl S-400 HR and Sephacryl S-300HR. The mathematical model was first validated by experimental chromatographic data of different SEC media and was then used to propose the best 2D-SEC system for downstream processing of the FMDV vaccine. The loading capacity of the constructed 2D-SEC sample was increased to 12.5% of total column volume and the purity of the final product was more than 90%. The entire purification process was performed with 77% FMDV recovery and 79.1% virus yield. Based on the high-performance size exclusion chromatography (HPSEC), the purity of the final NSP-free FMDV was about 90% and over 94.6% of host cell DNA was removed. Analyses of the purified FMDV by HPSEC, transmission electron microscopy (TEM) and dynamic light scattering (DLS) indicated that the final product had spherical shape with mean size about 30 nm and their structure remained intact.

Evaluation of continuous nonaffinity capture chromatography for a recombinant enzyme-optimization for a changing perfusion feedstream and comparison to batch processing.

This work presents the optimization and critical evaluation of continuous capture chromatography in the downstream process of a recombinant enzyme. For the upstream manufacturing of this molecule, a perfusion process was implemented due to benefits for product quality and productivity. This process is, however, characterized by low titer and significant changes over the course of the harvest duration in terms of active enzyme concentration and impurity content. We evaluated the feasibility and benefits of a continuous capture operation. This case study illustrates the design approach that can be utilized to address challenges presented by a changing feedstream, and the statistical measures that can be employed to characterize and optimize the operating space under material and time constraints. Process economic modeling in conjunction with Monte Carlo simulations indicate that even for a nonaffinity capture step utilizing a relatively cheap ion-exchange resin, the smaller column volume used in a continuous set-up results in cost savings compared to the batch process. We compare this option to the scenario of repeated processing using a small capture column in batch mode. Our analysis establishes that continuous processing becomes economically attractive for processes where only a small portion of the potential column lifetime can be utilized or for column steps with slow mass transport and shallow breakthrough curves. In cases where column breakthrough is sharp and resin lifetime is relatively short, continuous processing may offer an improvement over traditional batch processing, but much of the productivity and cost savings can be realized through repeated column cycling. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 2018 © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1195-1204, 2018.

Characterization of mechanical parameters of microbeads by means of analytical centrifugation

Microcapsules, microspheres and microbeads are widely used in the food industry, pharmacy, agriculture, cosmetics, construction, textile industry and printing to protect immobilized substances against harsh conditions or to mask bad taste, as well as to enable and control the release of substances e.g. active pharmaceutical ingredients. The knowledge of the mechanical properties is a key parameter in development, application and control of processing. The paper focuses on the experimental determination of the stiffness of alginate beads (about 400 μm) in terms of bulk compressional behaviour by using a commercial available multisample analytical centrifuge (LUMiSizer). The basic principle consists in applying an increasing centrifugal force on an alginate bead column in a measuring cell and to record the height of the bead column (volume) in dependence on the applied force during centrifugation. The packing/compression of beads is quantified by the decreased column volume in relation to the applied force. It was shown that beads made of alginate of high α-l-guluronic acid (G) content are more stable compared to alginate types of high ß-d-mannuronic acid (M) content. In contrast, gelling cations and their concentrations have less influence on the mechanical behaviour. The described ensemble method is sensitive, reproducible, easy to perform and, due to the simultaneous analysis of up to 12 samples, time saving. The new method is especially suitable for quality control during microsphere production.

Purification and enrichment of polycyclic aromatic hydrocarbons in environmental water samples by column clean-up coupled with continuous flow single drop microextraction

A novel method for the determination of sixteen polycyclic aromatic hydrocarbons (PAHs) in environmental water samples was developed using column clean-up (CCU) coupled with continuous flow single drop microextraction (CF-SDME) prior to the determination by gas chromatography-mass spectrometry (GC–MS). In this method, purification, extraction and enrichment were carried out in a single step, which reduced the effect of most organic interferents on the determination of target analytes, greatly simplified the operation process and shortened the whole pre-treatment time. The CCU-CF-SDME system was first used, and shortcomings such as long extraction time, instability of suspended microdrop of the traditional SDME system were overcome. The influences of several experimental parameters, including the type and amount of column packing material, the type and volume of extraction solvent, the flow rate of sample solution, extraction temperature, pH and ionic strength of sample solution, on the extraction efficiency were investigated. Under the optimal experimental conditions, the good linearities were obtained in the concentration range of 0.02–10 μg L−1 with correlation coefficients ranging from 0.9912 to 0.9995 for all the analytes. The limits of detection and quantification of the analytes were in the range of 0.0012–0.0101 μg L−1 and 0.0041–0.0336 μg L−1, respectively. The pre-concentration factors in the range of 777–978 for the PAHs were obtained. The proposed method was applied on the analysis of environmental water samples, and the recoveries of target analytes were in the range of 81.8–105.8% with the relative standard deviations ranging from 0.5% to 6.4%. The results showed that the developed method was a simple, quick and feasible method for the determination of PAHs in environmental water samples.