Hydrophobic Interaction Column Research Articles

The role of intermolecular interactions on monoclonal antibody filtration through virus removal membranes.

The removal of viruses by filtration is a critical unit operation to ensure the overall safety of monoclonal antibody (mAb) products. Many mAbs show very low filtrate flux during virus removal filtration, although there are still significant uncertainties regarding both the mechanisms and antibody properties that determine the filtration behavior. Experiments were performed with three highly purified mAbs through 3 different commercial virus filters (Viresolve ® Pro, Viresolve® NFP, and Pegasus™ SV4) with different pore structures and chemistries. The flux decline observed during mAb filtration was largely reversible, even under conditions where the filtrate flux with the mAb was more than 100-fold smaller than the corresponding buffer flux. The extent of flux decline was highly correlated with the hydrodynamic diameter of the mAb as determined by dynamic light scattering. The mAb with the lowest filtrate flux for all 3 membranes showed the largest attractive intermolecular interactions and the greatest hydrophobicity, with the latter determined by binding to a butyl resin in an analytical hydrophobic interaction chromatography column. These results strongly suggest that the flux behavior is dominated by reversible self-association of the mAbs, providing important insights into the design of more effective virus filtration processes and in the early identification of problematic mAbs / solution conditions. This article is protected by copyright. All rights reserved.

Preliminary purification and expression of Asian malaria vaccine candidate recombinant erythrocyte-binding antigen 140

Background: Causing an estimated 219 million cases in 2017, malaria is a life-threatening mosquito-borne disease caused by Plasmodium parasites, with P. falciparum being the most deadly. P. falciparum antigens involved in erythrocyte recognition and invasion during the merozoite stage are likely targets of protective immune responses. Natural antibody responses against the Erythrocyte Binding Antigen 140 (EBA-140) ligand have been found in individuals in malaria-endemic areas. EBA-140, a member of the Pf erythrocyte binding antigen (EBA) family, is one of the prospective candidates for malaria vaccine development. Methods and materials: In the study, the Philippine malaria parasite strain, FCH/4, was identified using its sequences in Genbank. Erythrocyte-binding antigen 140 (EBA-140) was selected by in silico screening of putative vaccine candidates in P. falciparum using the genomic database PlasmoDB and was then synthesized as codon-optimized full-length EBA-140. This 3284 bp gene was cloned into peT30-a(+) vector, transformed and expressed in E. coli BL21(DE3) cells. The EBA-140 gene insert was confirmed via restriction enzyme digestion and its protein 3D conformation was predicted using I-TASSER. Protein expression was optimized at 28 °C induction for 17 h using 0.3 mM IPTG. Recombinant EBA-140 in the native lysate was initially run on a weak anion-exchanger and then on a hydrophobic interaction column for purification. Results: The recombinant EBA-140 was constitutively expressed in the cytosol and highly expressed in the inclusion bodies upon induction at 28 °C for 17 h using 0.3 mM IPTG. The expressed rEBA-140 was then purified by several optimization steps of anion exchange chromatography, producing a 51.9% pure protein. To further purify the protein, hydrophobic interaction chromatography was employed. Conclusion: Recombinant EBA-140 was successfully expressed and purified optimally using anion exchange chromatography. The hydrophobic interaction chromatography method as polishing step is continuously being optimized to purify the 32% hydrophobic rEBA-140. To further confirm the specificity and identity of the purified fractions, Western blot and peptide mass fingerprinting methods are being optimized. Since no vaccine is yet available for malaria, characterization studies of immunoreactive Plasmodium antigens are of significant importance.

A comparative study on the leishmanicidal activity of the L-amino acid oxidases BjussuLAAO-II and BmooLAAO-II isolated from Brazilian Bothrops snake venoms

This study aims to examine whether two L-amino acid oxidases isolated from Bothrops snake venom (SV-LAAOs) were cytotoxic to Leishmania (Leishmania) amazonensis and Leishmania (Viannia) braziliensis, two causative agents of leishmaniasis, which is an endemic disease in tropical and subtropical countries. The SV-LAAOs BjussuLAAO-II and BmooLAAO-II were isolated from Bothrops jararacussu and Bothrops moojeni venom, respectively, through a three-step chromatography process that used molecular exclusion, hydrophobic interaction, and affinity columns. BmooLAAO-II is a new SV-LAAO isoform that we isolated in this study. The purified BjussuLAAO-II and BmooLAAO-II had high L-amino acid oxidase-specific activity: 3481.17 and 4924.77 U/mg/min, respectively. Both SV-LAAOs were strongly cytotoxic to the two Leishmania species, even at low concentrations. At the same concentration, BjussuLAAO-II and BmooLAAO-II exerted different cytotoxic effects on the parasites. We reported for the first time that the SV-LAAOs suppressed cell proliferation and altered the mitochondrial membrane potential of the two Leishmania species. Surprisingly, BjussuLAAO-II increased the intracellular reactive oxygen species production only in L. (L.) amazonensis, while BmooLAAO-II increased the intracellular reactive oxygen species production only in L. (V.) braziliensis, indicating that these SV-LAAOs had a certain specificity of action.

Adaptive methodology to determine hydrophobicity of nanomaterials in situ.

The hydrophobicity of nanoparticles (NPs) is a key property determining environmental fate, biological partitioning and toxicity. However, methods to characterize surface hydrophobicity are not uniformly applied to NPs and cannot quantify surface changes in complex environments. Existing methods designed to evaluate the hydrophobicity of bulk solids, chemicals, and proteins have significant limitations when applied to NPs. In this study, we modified and evaluated two methods to determine the hydrophobicity of NPs, hydrophobic interaction chromatography (HIC) and dye adsorption, and compared them to the standard octanol-water partitioning protocol for chemicals. Gold, copper oxide, silica, and amine-functionalized silica NPs were used to evaluate methods based on their applicability to NPs that agglomerate and have surface coatings. The octanol water partitioning and HIC methods both measured Au NPs as hydrophilic, but despite having a small size and stable suspension, NPs could not be fully recovered from the HIC column. For the dye adsorption method, hydrophobic (Rose Bengal) and hydrophilic (Nile Blue) dyes were adsorbed to the NP surface, and linear isotherm parameters were used as a metric for hydrophobicity. CuO was determined to be slightly hydrophilic, while SiO2 was hydrophilic and Ami-SiO2 was hydrophobic. The advantages and limitations of each method are discussed, and the dye adsorption method is recommended as the most suitable for application across broad classes of nanomaterials. The dye assay method was further used to measure changes in the surface hydrophobicity of TiO2 NPs after being suspended in natural water collected from the Alsea Rivers watershed in Oregon. TiO2 NPs adsorbed Rose Bengal when suspended in ultrapure water, but adsorbed Nile Blue after being incubated in natural water samples, demonstrating a shift from hydrophobic to hydrophilic properties on the outer surface. The dye adsorption method can be applied to characterize surface hydrophobicity of NPs and quantify environmental transformations, potentially improving environmental fate models.

Online coupling of analytical hydrophobic interaction chromatography with native mass spectrometry for the characterization of monoclonal antibodies and related products

Native mass spectrometry (native MS) has seen tremendous development and an increase in application over the past decade for the study of proteins and protein complexes. Although conventionally performed using a static nanospray emitter in an offline fashion, native MS has been increasingly applied in hyphenated methods, where a wide variety of separation techniques are directly coupled to online native MS detection. Those new developments have greatly expanded the utility of native MS in protein biopharmaceutical characterization. Analytical hydrophobic interaction chromatography (HIC) method, although frequently used for the characterization of monoclonal antibodies (mAbs) and antibody-drug-conjugates (ADCs), has rarely been explored for online coupling with native MS. This is largely due to the high salt concentrations used in HIC analysis that are not compatible with direct MS detection. In this study, we overcame this challenge via an innovative makeup and splitting flow design and successfully achieved online coupling of analytical HIC separation with native MS detection. The development and experimental setup of this HIC-MS method is outlined in detail to elucidate how this design could tackle the high salt concentrations used in HIC separation and ultimately achieve both good chromatographic resolution and MS data quality. Subsequently, the utility of this HIC-MS method was demonstrated in three different applications, where a mAb mixture, mAb molecular variants resulting from PTMs, and a Cys-based ADC mimic were all readily characterized in detail. Unlike previously reported HIC-MS methods, this newly developed method utilizes an analytical scale HIC column with conventional ligand so that the achieved separation profile is highly comparable to those obtained by a standard HIC-UV method. As a result, this HIC-MS method not only provides an alternative approach for in-depth characterization of mAbs and related products during their development but could also be readily applied to assist peak assignment and identity elucidation for the HIC-UV method used in quality control.

Chromatographic behavior of bivalent bispecific antibodies on hydrophobic interaction chromatography columns

The elution behavior of bivalent bispecific antibodies (BiSAb) comprising an immunoglobulin G framework genetically fused to a pair of single chain variable fragments (scFvs) was studied on hydrophobic interaction chromatography (HIC) columns using ammonium sulfate gradients. Each of the BiSAb molecules studied exhibited a three-peak elution behavior regardless of the location of scFv attachment to the framework IgG. Collecting and re-injecting each of the isolated peaks and eluting with the same gradient resulted in the same three-peak profile indicating that the behavior is reversible. Analogous behavior was observed for HIC resins with different functional ligands, matrix structures, and particle sizes. Residence time, operating temperature, and hold time were shown to affect the elution behavior. While three peaks were obtained at short residence times and room temperature, residence times longer than about 27 min or operating at 45 °C resulted in a single merged peak indicating that the underlying mechanism occurs on time scales comparable to that of chromatographic separation. Holding the protein on the resins prior to elution enriched the late eluting peak indicating that multiple binding states formed on the chromatographic surface are responsible for this behavior. Tryptophan auto-fluorescence measurements show that stronger binding forms have increased solvent exposure indicating that surface-catalyzed conformational changes play a role. A model was developed to describe the interplay of chromatographic separation and slow conformational changes.

A systematic and methodical approach for the efficient purification of recombinant protein from silkworm larval hemolymph

The silkworm, Bombyx mori, is a promising expression system for the production of recombinant proteins, but the purification of these proteins is not easy because of the large amount of host proteins present. To investigate purity, recovery and scale-up ability of the purification of recombinant proteins expressed in silkworm larval hemolymph without any affinity tags, we used mCherry, a red fluorescence protein, as a model. The host cell proteins could be greatly reduced using a three-step chromatography protocol consisting of hydrophobic interaction chromatography (HIC), size exclusion chromatography (SEC) and heparin chromatography after heat pretreatment. The thermal treatment had the greatest impact on the removal of host cell extracellular proteins and increasing purity. There were still some minor traces of host cell proteins in the purified sample, which showed that the purification of recombinant proteins from the silkworm hemolymph was still challenging. The proposed protocol and affinity tag purification reduced the overall protein content by 99.84% and 99.95%, respectively, while the amount of DNA was reduced by 98.41% and 99.53%, respectively. Purities of our proposed protocol based on SDS-PAGE and capillary electrophoresis (CE) analyses were 85.45% and 43.60%, respectively, while those of Strep-tag affinity purification were 100% or 63.69%, respectively. Using densitometry, the overall recovery was calculated was 5.78%, which was higher than 4.09% using Strep-tag affinity purification. This proposed protocol, mainly based on thermal treatment, HIC, SEC and HiTrap Heparin HP column chromatography, is applicable to an upscalable purification for the silkworm expression system without employing affinity tag chromatography process.

Purification of Human S100A12 and Its Ion-induced Oligomers for Immune Cell Stimulation.

In this protocol, we describe a method to purify human calcium-binding protein S100A12 and its ion-induced oligomers from Escherichia coli culture for immune cell stimulations. This protocol is based on a two-step chromatography strategy, which comprises protein pre-purification on an anion-exchange chromatography column and a subsequent polishing step on a hydrophobic-interaction column. This strategy produces S100A12 protein of high purity and yield at manageable costs. For functional assays on immune cells eventual remnant endotoxin contamination requires careful monitoring and further cleaning steps to obtain endotoxin-free protein. The majority of endotoxin contaminations can be excluded by anion-exchange chromatography. To deplete residual contaminations, this protocol describes a removal step with centrifugal filters. Depending on the available ion-strength S100A12 can arrange into different homomultimers. To investigate the relationship between structure and function, this protocol further describes ion-treatment of S100A12 protein followed by chemical crosslinking to stabilize S100A12 oligomers and their subsequent separation by size-exclusion chromatography. Finally, we describe a cell-based assay that confirms the biological activity of the purified protein and confirms LPS-free preparation.

Accelerating Biomanufacturing by Modeling of Continuous Bioprocessing—Piloting Case Study of Monoclonal Antibody Manufacturing

An experimental feasibility study on continuous bioprocessing in pilot-scale of 1 L/day cell supernatant, that is, about 150 g/year product (monoclonal antibody) based on CHO (Chinese hamster ovary) cells for model validation is performed for about six weeks including preparation, start-up, batch, and continuous steady-state operation for at least two weeks stable operation as well as final analysis of purity and yield. A mean product concentration of around 0.4 g/L at cell densities of 25 × 106 cells/mL was achieved. After perfusion cultivation with alternating tangential flow filtration (ATF), an aqueous two-phase extraction (ATPE) followed by ultra-/diafiltration (UF/DF) towards a final integrated counter-current chromatography (iCCC) purification with an ion exchange (IEX) and a hydrophobic interaction (HIC) column prior to lyophilization were successfully operated. In accordance to prior studies, continuous operation is stable and feasible. Efforts of broadly-qualified operation personal as well as the need for an appropriate measurement and process control strategy is shown evidently.

Caracterización estructural y funcional de una fosfolipasas A2 homóloga K49 del veneno de Bothrops barnetti, por aproximación proteómica.

Las Fosfolipasas A2 (PLA2) son enzimas que intervienen en señales de transducción, activando distintos procesos como la contracción de músculo liso, activación de la cascada de inflamación e intervención en procesos digestivos del intestino; procesando lípidos para su posterior absorción. Dentro del grupo de fosfolipasas existe un subgrupo conocido como “PLA2 homólogas”, las cuales no ejercen actividad catalítica debido a la mutación de un aminoácido en el sitio activo; pero aunque estas toxinas carecen de acción lipolitica, producen efectos biológicos similares a sus homólogas catalíticamente activas, entre los cuales se destacan la miotoxicidad e inflamación.En la presente investigación se buscó encontrar una PLA2 homóloga K49 en el veneno de Bothrops barnetti peruana; mediante una columna de interacción hidrofóbica se obtuvieron diversas enzimas, las cuales por análisis de actividad pro-inflamatoria y electroforesis SDS-PAGE fue posible aislar una muestra de aproximadamente 14 kDa con actividad fosfolipásica en relación al resto, la cual se denominó BbTX-II. Mediante espectrometría de masas se determinó la masa molecular promedio de 14242 Da. y se realizó una caracterización de la estructura primaria, obteniendo la secuencia de 3 péptidos; finalmente se realizó un estudio de homología secuencial determinando que la muestra es una PLA2 homóloga K49. Paralelamente se realizó una caracterización biológica de la muestra para determinar el efecto miotóxico; evaluando los mediadores envueltos en la migración leucocitaria y en el efecto inflamatorio (IL-1, IL-6 y TNFα), observando un aumento de los mediadores dentro de las primeras 3 horas; determinando así un efecto local de la muestra

On-line coupling of hydrophobic interaction column with reverse phase column -charged aerosol detector/mass spectrometer to characterize polysorbates in therapeutic protein formulations

Polysorbates are complex mixtures of over a thousand components with a wide range of hydrophobicity. This paper describes a methodology for characterization of heterogeneity and stability of polysorbates in therapeutic protein formulation. The method utilizes on-line coupling of a hydrophobic interaction chromatography (HIC) column with reverse phase liquid chromatography (RPLC) and charged aerosol detection (CAD)/mass spectrometer (MS). The addition of a low concentration of formic acid and organic solvent (e.g. 0.05% formic acid and 3% acetonitrile) in the mobile phase enables the use of the HIC column to separate small molecule excipients (including major components of polysorbates) and the large protein molecules by a mixed mechanism of size exclusion chromatography (SEC) and RPLC. The protein and the charged excipients, which elute early from the HIC column, are directed by a switching valve to waste. The polysorbates and other neutral excipients, which elute later from the HIC column, are directed to the RPLC column for further separation. The separated polysorbate components are detected by CAD, and characterized by MS. This method has been used to characterize the degradation of polysorbate 80 (PS80) in placebo and protein formulations. Our studies have revealed different degradation behavior of PS80 in placebo vs. protein formulation.

Preparation and characterization of a nanovaccine of pVAX1-NH36 for leishmaniasis

Leishmaniasis is an endemic disease mainly in very poor countries. This disease may cause only a cutaneous lesion but also a fatal visceral disease. Vaccines of plasmid DNA (pDNA) are an alternative of conventional treatments of the disease since are considered safer, cheaper and easier to produce than conventional vaccines. However, it is being shown that naked plasmids are very susceptible to degradation and do not enter the cells efficiently. Liposomes may be used for pDNA administration due to their biocompatibility, biodegradability and low immunogenicity. However, they also present problems in reaching the correct cells, such as opsonization, aggregation and phagocytosis. Pegylation of liposomes has proven to increase circulation time and reduce cytotoxicity. This work aimed at the development of a process to prepare lipidic nanoparticles (NPs) loaded with plasmid pVAX1-NH36 for application as leishmaniasis nanovaccine. NPs were prepared by thin film technique using l-α-phosphatidylcholine, octadecylamine and cholesterol in a molar ratio of 7:2:1. An experimental setup was used to analyze the effect of PEG% and molar charge ratio (R) on the physicochemical properties and pDNA carry capacity of the NPs. In the preparation of the nanovaccines 0, 2.5 or 5 mol% of PEG2000-PE was used. The molar charge ratio of pVAX1 to phospholipids was varied between 1 and 11 for each type of nanovaccine. The initial experiments showed that R = 1 and R = 5 nanovaccines present instability and were discarded for further studies. Dynamic light scattering (DLS) analysis showed pDNA-NPs size with an average diameter of 120–160 nm depending on R and PEG2000-PE% and a polydispersity index (PDI) between 0.06-0.16. The Z potentials determined by electrophoretic light scattering were in the range of 15–40 mV. The PEGylated particles showed a PDI and Z potential values more adequate for nanovaccine preparation. The PEGylated and non-PEGylated nanovaccines were visualized by transmission electron microscopy (TEM) and presented a rather spherical shape which is also a desired property. To determine the stability of the nanovaccines their pDNA content was estimated in samples obtained from the reaction solutions at days 0, 1, 2, 7 and 14, measuring the pDNA concentration in the solution by high-performance liquid chromatography on a hydrophobic interaction column (HPLC-HIC). All the samples presented a stability greater than 84% in the period of study. A mixed order lumped parameter model was successfully fitted (with r2 > 0.92 in all the preparations) to the kinetics of plasmid liberation as initial approach to model the phenomena.

Peroxidase from jackfruit: Purification, characterization and thermal inactivation

Peroxidase (POD) from jackfruit bulb was purified using ammonium sulfate precipitation, hydrophobic interaction and gel filtration columns. The POD was a dimer with a molecular weight of 104kDa. The Km and Vmax values for guaiacol, gallic acid and o‑phenylenediamine (OPD) were estimated. OPD was the most suitable substrate. The enzyme showed its maximum activity at pH5.5 and 55–60°C. The activation energy (Ea) of heat inactivation was estimated to be 206.40kJ/mol. The enthalpy, free energy and entropy values for the thermal inactivation were also determined. The POD activity was enhanced by K+, Zn2+, Ba2+, citric acid, malic acid, benzoic acid and EDTA·Na2, but inhibited by Cu2+, Ca2+, glutathione, cysteine and ascorbic acid. Chemical modification indicated a histidine residue was located in the enzyme active site. The POD activity in fruit extracts significantly decreased when heated at 80°C and 90°C. The ferric-reducing antioxidant power, ABTS radical scavenging activity and total phenolics decreased with increasing heating temperature and time.

Performance and preparation of cation‐exchange chromatographic stationary phases without negatively charged groups for protein separation

AbstractWe synthesized a novel polymer‐based stationary phase modified by N‐methyl‐d‐glucamine. Although there is a tertiary amine group on the ligand of the special stationary phase, the performance of protein separation is cation exchange chromatography, instead of anion exchange chromatography. The similar retention behavior was also observed in ion exchange chromatography mode of two similar stationary phases and a commercial hydrophobic interaction chromatography column, which have no negatively charged groups. It could be caused by the hydrogen‐bonding interaction between chloride ions and hydroxyl groups.

Green Fluorescent Protein Purification as a Didactic Tool During Practical Classes For Undergraduates Students of UFAM

INTRODUCTION: The Green Fluorescent Protein (GFP), originated from the jellyfish Aequorea victoria has broadly applicability for cellular and molecular biology research. Its spectral characteristics make it practical to be detect by UV-A (black light) lamp during the purification procedure. Moreover, this approach implementation during a practical class allows the exploring of fluorescence features. OBJETIVES: the purpose of this investigation was to teach the concepts and principles of protein purification during a practical class using recombinant GFP protein. MATERIAL E METHODS: Transformed E. coli JM110 expressing GFP were resuspended in buffer solution (Tris-HCl 20 mM pH 8.0, 150 mM NaCl, 5 mM EDTA, 20% (NH4)2SO4) following the sonication step. The lysate was submitted to the purification through hydrophobic interaction chromatography column (HIC). After analysis of chromatogram, some collected fractions were quantified by Bradford assay and evaluated by SDS-PAGE. Besides that, the GFP presences were measured at an excitation wavelength of 488 nm on a spectrofluorimeter. RESULTS AND DISCUSSION: Before the experiments, the students were encouraged to explore the biochemistry characteristics of GFP, assessing protein data banks and published articles. These guided questions conducted to discussion of the purification strategy choosen. The GFP purification enabled the visual observation of chromatography principles necessary for the theory assimilation. During the chromatography running, we used a UV-A lamp which allowed a greatly exploration of concepts beyond this technique such as the sample injection, the GFP column retention, and the elution step. The chromatogram obtaneid were analysed and correlated to the collected fractions. Our next step was the efficiency analysis generated by the GFP measurement, total protein quantification and the analytical method SDS-PAGE. CONCLUSION: Collectively, we observed in this class the clear development of chromatography concepts by adopting the GFP protein as a target for purification protocol.

Mechanistic modeling of hydrophobic interaction chromatography for monoclonal antibody purification: process optimization in the quality by design paradigm

AbstractBACKGROUNDA mechanistic model has been developed for hydrophobic interaction chromatography (HIC) based purification of monoclonal antibodies (mAbs). The model has been used to gain thorough process understanding as desired by the quality by design paradigm. The model is based on the general rate model coupled with an exponentially modified Langmuir adsorption model. The latter has been modeled by using the solvophobic theory for correlating protein adsorption equilibrium on the hydrophobic media with the mobile phase salt concentration.RESULTSThe proposed model satisfactorily predicts the elution profile of the monoclonal antibody product under consideration and its aggregates on a HIC column (Phenyl Sepharose 6 Fast FlowTM low sub) operated in linear gradient mode. The model has been used to elucidate the underlying mechanism behind separation of monomer and aggregate species. A comparison of the relative time scales for the various mass transfer events for mAb and aggregate species indicates that the intra‐particle (pore) diffusion transport of these molecules to their respective binding sites within the porous HIC resin is the rate limiting step whereas convection, film diffusion, and binding kinetics are relatively faster (time scales are 10−1 to 10−3 order of magnitude times the time scale of intra‐particle pore diffusion).CONCLUSIONSThe proposed methodology for modeling can be successfully implemented to achieve efficient aggregate removal by HIC using minimal experimentation compared with the traditional experimentally extensive approaches. © 2017 Society of Chemical Industry

A step-wise approach to define binding mechanisms of surrogate viral particles to multi-modal anion exchange resin in a single solute system.

Multi-modal anion exchange resins combine properties of both anion exchange and hydrophobic interaction chromatography for commercial protein polishing and may provide some viral clearance as well. From a regulatory viral clearance claim standpoint, it is unclear if multi-modal resins are truly orthogonal to either single-mode anion exchange or hydrophobic interaction columns. To answer this, a strategy of solute surface assays and High Throughput Screening of resin in concert with a scale-down model of large scale chromatography purification was employed to determine the predominant binding mechanisms of a panel of bacteriophage (i.e., PR772, PP7, and ϕX174) to multi-modal and single mode resins under various buffer conditions. The buffer conditions were restricted to buffer environments suggested by the manufacturer for the multi-modal resin. Each phage was examined for estimated net charge expression and relative hydrophobicity using chromatographic based methods. Overall, PP7 and PR772 bound to the multimodal resin via both anionic and hydrophobic moieties, while ϕX174 bound predominantly by the anionic moiety. Biotechnol. Bioeng. 2017;114: 1487-1494. © 2017 Wiley Periodicals, Inc.

Protein separation in carousel multicolumn setup. Performance analysis and experimental validation

To overcome limitations of periodic separations of proteins in batch chromatographic columns Carousel Multi-Column Setup (CMS) has been recently suggested and theoretically analyzed in a previous study (R. Bochenek, W. Marek, W. Piątkowski, D. Antos, J. Chromatogr. A, 1301 (2013) 60–72). In this system, feed and mobile phase streams are subsequently delivered through parallel columns to mimic their countercurrent movement with respect to the fluid flow. All fluxes in the system are synchronized to ensure continuous feed delivery, which however causes reduction in the size of the operating window compared to batchwise-operating systems.In this study to improve the performance of CMS, additional process variables have been considered, such as the flow rate gradient and feed concentration. Though altering both variables allowed improving the separation selectivity and extending the operating window, the feed concentration appeared to be the most influential parameter affecting the process performance. Moreover, a procedure for practical realization of protein separations in CMS has been developed, including hints about the process design, configuration of columns and detectors, and use of pumps. As the case study, the separation of a ternary mixture of proteins, i.e., cytochrome C, lysozyme and immunoglobulin G, on hydrophobic interaction columns was used. A target product was a protein with intermediate adsorption strength that was isolated out of a more and less strongly adsorbed compound.

Lipoteichoic Acid of Enterococcus faecalis Inhibits the Differentiation of Macrophages into Osteoclasts

IntroductionEnterococcus faecalis is associated with persistent endodontic infection and refractory apical periodontitis. Recently, we have shown that heat-killed E. faecalis attenuates osteoclast differentiation. Because lipoteichoic acid (LTA) is a major virulence factor of gram-positive bacteria, we investigated the effect of LTA from E. faecalis (EfLTA) on osteoclast differentiation. MethodsEfLTA was purified through organic solvent extraction, hydrophobic interaction column chromatography, and ion exchange column chromatography. Bone marrow cells from C57BL/6 or Toll-like receptor 2–deficient mice were incubated with macrophage colony-stimulating factor (M-CSF) for 5 days to generate macrophages (bone marrow–derived macrophages [BMMs]). The cells were differentiated into osteoclasts with M-CSF and receptor activator of NF-κB ligand (RANKL) in the presence or absence of EfLTA. The degree of osteoclast differentiation was determined by tartrate-resistant acid phosphatase staining. The expression of NFATc1 and c-Fos transcription factors was determined by Western blotting. A phagocytosis assay was performed by measuring the uptake of carboxyfluorescein diacetate succinimidyl ester–stained E. faecalis. An enzyme-linked immunosorbent assay was used to determine the amount of cytokines and chemokines. ResultsWhen BMMs were treated with EfLTA, osteoclast differentiation was attenuated. EfLTA inhibited the RANKL-induced expression of NFATc1 and c-Fos. EfLTA inhibition of osteoclast differentiation was not observed in TLR2-deficient BMMs. In addition, EfLTA sustained the phagocytic capacity of BMMs even after the differentiation into osteoclasts, whereas it induced the expression of inflammatory cytokines and chemokines. ConclusionsEfLTA inhibits the differentiation of macrophages into osteoclasts and thereby maintains the phagocytic and inflammatory capacities of macrophages, potentially contributing to refractory apical periodontitis.

On-line determination by small angle X-ray scattering of the shape of hen egg white lysozyme immediately following elution from a hydrophobic interaction chromatography column.

This study documents the use of an integrated approach, involving on-line hydrophobic interaction chromatography interfaced with Small Angle X-ray Scattering (HIC-SAXS) measurements, to monitor the conformational status of proteins immediately upon elution from a chromatographic column operated at different temperatures. Moreover, this approach provides an additional avenue to interrogate the changes in protein shape that may occur across the eluted chromatographic peak. To this end, radii of gyration were extrapolated from the Guinier approximation with the HIC-SAXS data, whilst pair distribution functions and bead model simulations were generated by using the indirect transform program GNOM and ab initio reconstruction with GASBOR to provide further insight into protein conformational changes that occur during hydrophobic interaction chromatography.