Evaluation of the impact of lipid fouling during the chromatographic purification of virus‐like particles from Saccharomyces cerevisiae

BACKGROUND: Production of recombinant virus‐like particles (VLPs) in yeast expression systems for use as vaccines requires cell disruption and detergent‐mediated steps to liberate the product. Typically, these release high levels of cellular components such as lipids that foul chromatography columns. This study compares the impact of applying lipid‐rich and lipid‐depleted feedstocks to hydrophobic interaction chromatography columns to quantify the loss of performance caused by the presence of host lipids over a total of 40 operational cycles. RESULTS: VLP binding capacity in the lipid‐rich feed was significantly lower than for the lipid‐depleted feed, with greater than 24% of the lipids remaining in the column after each cycle. Triacylglycerol was found to be the major contaminant. The effectiveness of subsequent caustic clean‐in‐place was limited, resulting in column hydrophobicity increasing over repeated loading cycles. This improved the effective VLP binding capacity and affinity, but also made product elution more difficult, and recovery decreased by more than 70% over the 40 cycles. CONCLUSION: Host cell lipids cause major fouling problems during VLP purification. Instead of screening for better CIP conditions, priority should be given to identifying suitable upstream lipid removal strategies in order to maintain column performance and so yield more economically viable processes. Copyright © 2009 Society of Chemical Industry     

Solubilisation study of water‐insoluble dye in cationic single/dimeric surfactant micelles: effect of headgroup,non‐polar tail,and spacer chain in aqueous and salt solution

The solubilisation of hydrophobic azo dye Orange OT in aqueous/salt solution in several cationic surfactant micelles was studied using UV‐vis spectroscopy. An attempt was made to correlate dye solubilising strength with adsorption/micellar characteristics. In our experiments we determined the change in solubilisation of hydrophobic dye when added to an aqueous solution of oppositely charged quaternary‐salt‐based cationic surfactants (conventional and gemini) and remarked on the probable location of the solubilised dye in the surfactant micelle. Results highlight the onset of dye solubilisation around the critical micelle concentration of each surfactant, which is influenced by the non‐polar tail, spacer, and polar headgroup, while no dye could be solubilised at concentrations below the critical micelle concentration. Orange OT solubilised almost linearly with increase in surfactant concentration at and above the critical micelle concentration. The change in colour intensity of the dye (darker below the critical micelle concentration, lighter at and above the critical micelle concentration) could be attributed to dye–surfactant interactions. Further dye solubilisation was observed in the presence of salt.     

Hydrophobic oxime ethers: a versatile class of pDNA and siRNA transfection lipids

The manipulation of the cationic lipid structures to increase polynucleotide binding and delivery properties, while also minimizing associated cytotoxicity, has been a principal strategy for developing next-generation transfection agents. The polar (DNA binding) and hydrophobic domains of transfection lipids have been extensively studied; however, the linking domain comprising the substructure used to tether the polar and hydrophobic domains has attracted considerably less attention as an optimization variable. Here, we examine the use of an oxime ether as the linking domain. Hydrophobic oxime ethers were readily assembled via click chemistry by oximation of hydrophobic aldehydes using an aminooxy salt. A facile ligation reaction delivered the desired compounds with hydrophobic domain asymmetry. Using the MCF-7 breast cancer, H1792 lung cancer and PAR C10 salivary epithelial cell lines, our findings show that lipoplexes derived from oxime ether lipids transfect in the presence of serum at higher levels than commonly used liposome formulations, based on both luciferase and green fluorescent protein (GFP) assays. Given the biological compatibility of oxime ethers and their ease of formation, this functional group should find significant application as a linking domain in future designs of transfection vectors.     

Physicochemical investigation and spectral properties of Sunset Yellow dye in cetyltrimethylammonium bromide micellar solution under different pH conditions

To mimic binding to biological membranes, the extent of interaction of the efficient food dye Sunset Yellow with a cationic surfactant, cetyltrimethylammonium bromide, was probed by quantifying its absorption and fluorescence emission spectra as a function of premicellar to post‐micellar surfactant concentration. The characteristics of the partition behaviour and spectral‐luminescent properties of Sunset Yellow–surfactant have been evaluated qualitatively as well as quantitatively for various cetyltrimethylammonium bromide concentrations. With the help of mathematical models, the water–micelle partition coefficient, the Sunset Yellow–cetyltrimethylammonium bromide binding constant, the binding capacity, and the degree of counterion binding were calculated at 25 °C under various pH conditions. The solubilisation of Sunset Yellow is also discussed from the thermodynamics viewpoint by considering the standard free energy change of dye solubilisation and the Sunset Yellow–cetyltrimethylammonium bromide binding energy. The high values of all these interactions indicate that the dye, being organic in nature, is partitioned largely from the aqueous environment to the palisade layer of the micelle. The solubilisation of the dye is enhanced by switching the pH to suppress the self‐aggregation of dye molecules. Combined with electrostatic forces, the hydrophobic interactions also play a promising role in the solubilisation of Sunset Yellow in the outer core of the micellar interior.     

Study of Antioxidative Properties of Some Mono Amino‐Acid‐Type and Dipeptide‐Type Surfactants

Surfactants of tryptophan, tyrosine, and histidine were successfully synthesized. They were then modified to obtain dipeptide‐type surfactants with better solubility in both polar and nonpolar solvents, giving more emphasis to the development of water‐soluble amino‐acid‐type surfactants. All developed materials were designed to have functionality as antioxidants (AOX). The investigation of free radical scavenging capacity at pre‐micellar concentrations revealed greater Trolox equivalent (TE, relative scavenging capacity to Trolox) results for Trp‐type surfactants followed by Tyr‐type and His‐type. As expected, the synthesized surfactants exhibited comparable AOX properties to their parent amino acids. The AOX behavior of these surfactants was also checked in micellar concentrations, and the trend was found to be analogous: Trp‐type > Tyr‐type > His‐type surfactants. However, all of them were acting as better AOX in the micellar state than in the monomeric state. Investigating the synergistic effect found no interaction between the “amino acid–surfactant” pairs or the “surfactant–surfactant” pairs with varying ratios. They showed total AOX activity with respect to that of each material. This work opens up the door to the application of some new oil and water‐soluble amino‐acid‐type surfactants having auspicious AOX activity.     

Study of the Preparation of Flavone Imprinted Silica Microspheres and Their Molecular Recognition Function

The aim of this study is to prepare flavone imprinted silica with good sphericity. Previous studies have demonstrated that imprinted silica can be synthesized using a simple sol‐gel method and a grinding‐sieving process. Despite the simplicity of the synthetic method, the appearance of the final product obtained is often very irregular and nonuniform. This product has many utilization issues and problems, e.g., residues and low operability. In this study, one type of novel spherical flavone imprinted silica was synthesized. A sol‐gel method and molecular imprinting technique were employed. By using luteolin as a template molecule, aminopropyltriethoxysilane (APTS) as the functional monomer, tetraethoxysilane (TEOS) as the cross‐linking agent, molecularly imprinted silica microspheres were prepared via a two‐stage reaction. The preparation conditions were monitored by the appearance of the products. SEM images show that under optimum conditions, the diameters of these microspheres are ca. 20–30 μm. The amino group adsorption peaks can be seen from the IR spectra. Results from elemental analysis indicate that 3.206 μmol of amino groups were successfully introduced into 1 g of product. This microspheric adsorbent has a high adsorption capacity to the template molecule, high adsorption selectivity and reproducibility. The adsorption capacity amounted to 32.987 mg/g and the separation factor was up to 84.3, while the adsorption capacity hardly decreased after 15 cycles.     

Complexation of Surfactant/β‐Cyclodextrin to Inhibit Surfactant Adsorption onto Sand,Kaolin, and Shale for Applications in Enhanced Oil Recovery Processes. Part II: Dynamic Adsorption Analysis

Surfactant adsorption onto solid surfaces is problematic in some industrial processes, such as in surfactant flooding for enhanced oil recovery. In this work, it was hypothesized that the use of a surfactant delivery system could prevent surfactant adsorption onto solid surfaces. Therefore, the encapsulation of sodium dodecyl sulfate (SDS) into the hydrophobic core of β‐cyclodextrin (β‐CD) to generate a surfactant delivery system (SDS/β‐CD) was evaluated in this work. This complexation was characterized using optical and scanning electron microscopy (SEM), and Fourier transform infrared spectroscopy (FT‐IR). Dynamic adsorption evaluation was applied to determine the effectiveness of the complexation in inhibiting surfactant adsorption onto a variety of solid adsorbents including sand, and mixtures of sand–kaolin and sand–shale. Surfactant adsorption was also evaluated applying the quartz crystal microbalance technology (QCM‐D). The formation and morphology of the complexation was confirmed by optical microscopy, SEM, and FT‐IR. Dynamic adsorption tests demonstrated the effectiveness of the surfactant delivery approach in preventing the adsorption of surfactant (up to 74 % adsorption reduction). The QCM‐D technology confirmed these observations. Several mechanisms were proposed to explain the inhibition of surfactant adsorption including steric hindrance, self‐association of inclusion complexes, hydrophilicity increase, and disruption of hemimicelles formation.     

Cross‐linked Poly(arylene ether ketone) Proton Exchange Membranes with High Ion Exchange Capacity for Fuel Cells

Sulfonated poly(arylene ether ketone) (SPAEK) possessing the pendant carboxylic acid groups was synthesized. The carboxylic acid groups of SPAEK were reacted with a cross‐linking reagent to prepare a cross‐linked membrane with a high ion exchange capacity (IEC), a high oxidative stability, and an excellent mechanical strength. The cross‐linking hindered the mobility of the polymer chains and thus strongly affected the water uptake and the methanol permeability of the membranes. Also, as the cross‐linker used in this study bore sulfonic acid groups, cross‐linking did not lead to a noticeable loss of the proton conductivity. The cross‐linked SPAEK membrane with 20% cross‐linking density, CSPAEK‐20% membrane, exhibited a high proton conductivity of 0.045 S cm^–1 associated with a high IEC value of 1.78 mmol g^–1 but a low methanol permeability of 4.3 × 10^–7 cm² s^–1. The CSPAEK‐20% membrane also showed excellent cell performance and oxidation resistance.     

(Meth)acrylic Cross‐Linked Polymer Microparticles: Synthesis by Dispersion Polymerization and Particle Characterization

(Meth)acrylic cross‐linked polymer microparticles have been synthesized by dispersion polymerization in organic media. They were produced by radical copolymerization of mono(meth)acrylate monomers with a certain concentration of a diacrylate as cross‐linker, in a mixture of two organic solvents, heptane and propan‐2‐ol. The reactive surfactant (“surfmer”) was a low solubility parameter acrylate monomer, based on an aliphatic ester group (generally C₁₈) or an isobornyl cycle, which gave auto‐dispersing character to the microparticles. By using glycidyl methacrylate in the monomer mixture, oxirane groups were introduced in the particles. The influence of the major synthesis parameters such as solvent composition, monomer composition and concentration, and initiator concentration has been investigated regarding the size and the molar mass of the cross‐linked polymer microparticles. The study was completed with the influence of the nature and the concentration of the surfmer, the concentration of cross‐linking agent and the composition of monomers feed. Stable cross‐linked microparticles ranging from z‐average radius of gyration, R_z = 20 nm to R_z > 60 nm were obtained by varying the synthesis conditions. The smallest microparticles were prepared with a blend of heptane and propan‐2‐ol in the 50/50 ratio by weight. Increasing the surfmer concentration or reducing the monomer concentration in the reaction mixture usually led to smaller microparticles. The longer the aliphatic chain of the surfmer, the smaller the microparticles. Minimum sizes were obtained for cross‐linking agent concentrations between 5 and 7.5 mol‐%, depending on monomers composition. For higher concentrations, macrogelation may occur during the synthesis.     

Evaluation of polymer hydrophobic recovery behavior following H2O plasma processing

Inductively coupled radio frequency (rf) H₂O vapor plasma was used to modify a range of polymers to better elucidate the dependence of hydrophobic recovery on polymer composition and structure. Freshly modified and aged samples were examined using scanning electron microscopy, (SEM), X‐ray photoelectron spectroscopy, (XPS), and water contact angle (wCA) goniometry. Initially, wettability was increased on each polymer surface due to the implantation of polar oxide groups, an effect exacerbated by increased surface roughness with plasma treatment. Polypropylene and polystyrene exhibit nearly complete hydrophobic recovery as polar groups are subsumed as samples age. High‐density polyethylene and polycarbonate exhibit minimal hydrophobic recovery, owing to plasma‐induced cross‐linking and intrinsic thermal stability, respectively. Overall, the hydrophobic performance following H₂O plasma modification is similar to other oxidizing plasmas, suggesting that recovery behavior is intrinsic to the polymer. © 2015 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2015 , 132, 41978.     

Optimization of Continuous Synthesis of Cross‐Linked Chitosan Nanoparticles Using Microreactors

A process for continuous synthesis of cross‐linked chitosan‐sodium tripolyphosphate (CS‐TPP) nanoparticles is optimized using microreactors for its comparison with a batch stirred reactor. The effect of various parameters including residence time, concentration of CS, pH of the CS solutions, and stabilizing surfactant concentration was modeled by population balance equations (PBEs) to determine size, growth, and nucleation rates of the CS‐TPP nanoparticles. The smallest particle size was obtained at lower residence time, lower concentration of CS, pH 5, and using a surfactant concentration above its critical micellar concentration. The particles obtained from the microreactors are agglomerated but are smaller in size as compared to those obtained from the batch reactor. The system was also optimized for the minimum particle size applying the estimated growth rate and the PBEs.     

Synthesis of thermoresponsive polymeric micelles of PNIPAAm‐b‐OMMA as a drug carrier for loading and controlled release of prednisolone

We synthesized a drug delivery system of poly(N‐isopropylacrylamide)‐b‐oligo(methyl methacrylate) (PNIPAAm‐b‐OMMA) via polycondensation of two homopolymers in 1,4‐dioxane. The products are characterized by FT‐IR and ¹H‐NMR spectra and TEM. The PNIPAAm‐b‐OMMA copolymer micelles in aqueous solution present the same lower critical solution temperature (LCST) as the unmodified PNIPAAm, owing to the formation of a core–shell micellar structure that the hydrophilic shell shields the hydrophobic inner OMMA core from interacting with water. The micelle carriers exhibit two heterogeneous microdomains: a hydrophobic inner core capable of highly solubilizing hydrophobic prednisolone molecules, plus a hydrated outer shell that stabilizes this micellar structure below its LCST. Moreover, the micelle carriers show reversible thermoresponsive aggregation/dispersion in response to temperature cycles through the LCST. By using the antiinflammation drug prednisolone as model drug, it is found that the PNIPAAm‐b‐OMMA drug carrier could prolong the release time and control the release amount by changing the temperature. Accordingly, this copolymer micelle may provide as an effective drug carrier for drug control and release. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2009     

Modifying the rheological behavior of associative triblock copolymers in aqueous media through surfactant additives

The rheological behavior of aqueous micellar solutions of a triblock copolymer, bearing two small hydrophobic end-blocks of polybutadiene and a large hydrophilic middle block of poly(sodium methacrylate), was studied in the presence of a cationic (dodecyl trimethyl ammonium bromide) or an anionic (sodium dodecyl sulfate) surfactant. Depending on the concentration and the charges of the interacting (with the water soluble middle block) surfactants, the rheological behavior of the triblock copolymer micelles (which resemble compact spheres, based on scattering studies) can be altered dramatically. The surfactant additives can either solidify a liquid-like system (low triblock concentration, dominated by loops) or alternatively liquefy a gel (high triblock concentration, dominated by bridges). Apparently, the hydrophobic tails of the surfactants prefer to join the hydrophobic polybutadiene cores of the triblocks, whereas by increasing the surfactant concentration the core functionality can be changed. In addition interactions between the oppositely charged hydrophilic surfactant heads and middle blocks can yield complexes producing new hydrophobic domains. These findings suggest possibilities for controlling complex fluid rheology.     

Effect of cross‐linking time on the thermal and mechanical properties and pervaporation performance of poly(vinyl alcohol) membrane cross‐linked with fumaric acid used for dehydration of isopropanol

Cross‐linked poly (vinyl alcohol) membranes were prepared using fumaric acid as the cross‐linking agent and were used for the pervaporation separation of water/isopropanol mixtures. Cross‐linking process was carried out at 150°C at three different times of 10, 30, and 60 min. The membranes were characterized by different known methods of FT‐IR, TGA, XRD as well as tensile test. The effects of cross‐linking time on the thermal and mechanical properties of the membranes and also their pervaporation performance were investigated. Formation of more ester groups by increasing the cross‐linking time was confirmed by the FT‐IR results. TGA analyses showed that thermal stability of the membranes is improved by prolonging the duration of cross‐linking process. This was due to the formation of more compact structure in the membranes. The XRD results revealed that the crystalline regions of the membranes were relatively diminished with an increase in the cross‐linking time. No specific trend was observed for the variation of tensile strength at break with the cross‐linking time. The PVA membrane cross‐linked for 60 min showed high selectivity of 1492 for water permeation for the feed mixture containing 10 wt % water. The temperature dependency of the permeation flux was investigated using Arrhenius relationship, and the activation energy values were calculated for total permeation (E_p), water (E_pw), and IPA (E_pIPA) fluxes. Lower value of E_pw in comparison with E_pIPA supported excellent dehydration performance of the cross‐linked membranes. Despite large increase in activation energy of water with prolonged cross‐linking time, the selectivity was improved. © 2012 Wiley Periodicals, Inc. J Appl Polym Sci, 2013     

Chemo‐enzymatic synthesis and evaluation of novel structured phenolic lipids as potential lipophilic antioxidants

Chemo‐enzymatic synthesis of structured triacylglycerol bearing ferulic acid as a phenolic acid at sn‐1/3 position is described in the present work. Four compounds of varying chain lengths, namely 3‐(4‐hydroxy‐3‐methoxy‐phenyl)‐acrylic acid‐2,3‐bis‐hexanoyloxy‐propyl ester, 3‐(4‐hydroxy‐3‐methoxy‐phenyl)‐acrylic acid‐2,3‐bis‐octanoyloxy‐propyl ester, 3‐(4‐hydroxy‐3‐methoxy‐phenyl)‐acrylic acid‐2,3‐bis‐dodecanoyloxy‐propyl ester, and 3‐(4‐hydroxy‐3‐methoxy‐phenyl)‐acrylic acid‐2,3‐bis‐9‐octadecenoyloxy‐propyl ester were synthesized, and their structures were confirmed by IR, NMR, and MS. Antioxidant activity of the structured phenolic lipids were evaluated using three different in vitro antioxidant assays such as 2, 2‐diphenyl‐1‐picrylhydrazyl free radical (DPPH^•) scavenging, antioxidant potency in lipid matrix using rancimat, and by the rate of inhibition of autoxidation of linoleic acid in micelles. Ferulic acid and dodecylgallate were used as reference antioxidant compounds. DPPH^• assay did not show any improvement in the antioxidant activity of ferulic acid with lipophilic modification. However, the antioxidant potency of the structured phenolic lipids measured by rancimat method as well as by the rate of inhibition of autoxidation of linoleic acid in micelle showed improvement in antioxidant activity compared to ferulic acid. This is probably due to better solubility of the synthesized phenolic lipids in a hydrophobic medium and appropriate anchorage in Tween 20 micelle. The observed activities of the structured phenolic lipids are comparable to dodecyl gallate in rancimat assay, but superior to dodecyl gallate in Tween 20 micellar system.     

Preparation and characterization of hyaluronan/chitosan scaffold cross‐ linked by 1‐ethyl‐3‐(3‐dimethylaminopropyl) carbodiimide

A novel biocompatible scaffold was prepared by cross‐linking hyaluronan (HA) and chitosan (CS). The carboxyl groups of HA were activated by 1‐ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide (EDC) and then cross‐linked with amino groups of CS by forming amide bonds. The HA/CS scaffold thus prepared was characterized using Fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM) and differential scanning calorimetry. FTIR spectra showed that the absorbance of the amide (1550 cm^?1) and carbonyl (1633 cm^?1) bond in the cross‐linked scaffold was stronger than that in HA or CS. SEM micrographs showed that the cross‐linked scaffold produced at low EDC concentration had an intertwisted ribbon‐like microstructure, while the product prepared at higher EDC concentration had a porous structure. The concentration of EDC in the reaction system greatly affected the structure and properties of the HA/CS scaffold. The prepared scaffold could strongly resist degradation by hyaluronidase, free radicals in vitro and stress. Copyright © 2007 Society of Chemical Industry     

Modulation of food texture using controlled heteroaggregation of lipid droplets: Principles and applications

This article reviews the development and potential application of novel food‐grade materials based on heteroaggregation of oppositely charged particles. These particles typically consist of a hydrophobic lipid core and an electrically charged emulsifier shell. Heteroaggregation leads to the formation of emulsion‐based products that are highly viscous or gel‐like at much lower droplet concentrations than in nonaggregated systems. Heteroaggregation may therefore be useful for developing food products with novel textural characteristics or for creating reduced‐calorie versions of full fat foods. We give an overview of the principles of heteroaggregation, the relationship between emulsion microstructure and texture, and possible commercial applications of this method. © 2013 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 130: 3833–3841, 2013     

Kinetic study on cross‐linking and blowing behavior of EVA/EPDM/CPE high elasticity material

Blend of ethylene‐vinyl acetate copolymer (EVA), ethylene‐propylene‐diene terpolymer rubber (EPDM), and chlorinated polyethylene (CPE) filled with talc was cross‐linked with dicumyl peroxide and blown simultaneously with azodicarbonamide for preparation of high elasticity material by injecting, cross‐linking, and blowing process. It had excellent mechanical properties and elasticity. It was found that mechanical properties were a function of cross‐linking and blowing temperature and time and formula. A good cross‐linking and blowing temperature was in the range of 440–450 K and the optimum temperature and time were 448 K and 360 s, respectively. Kinetics of both cross‐linking and blowing reactions were investigated by closed nonrotor torque rheometer. The results showed that both cross‐linking and blowing reactions were first order. The activation energy of the cross‐linking reaction was higher than that of the blowing reaction. With increasing of EDPM content, the activation energy of the cross‐linking reaction reduced whereas it increased for the blowing reaction. The gas cell morphology was characterized by scanning electron microscopy (SEM). The results of SEM and mechanical property analyses showed that the morphology and mechanical properties were improved by incorporating CPE. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci, 2007     

Self-Assembled Structures of Catanionic Associations: How to Optimize Vesicle Formation?

Like other amphiphilic compounds, bolaforms do not always possess surfactant properties; it depends on the spacer chain length and the nature of the polar head group (both sufficiently hydrophobic or hydrophilic, respectively, to intensify the amphiphilic properties). In this regard, unsymmetrical bolaamphiphiles bearing a sugar polar head group and a carboxylic acid function at the opposite ends of a hydrophobic binding spacer were synthesized. These biocompatible sugar-derived bolaforms were associated with basic fatty amines, by an acid–base reaction, to obtain catanionic mixtures. Associations with 1,7-lactobionamidoheptanoic acid and decylamine or octylamine spontaneously form stable 200–600 nm vesicles. This new type of association may find an application in drug delivery since catanionic vesicles can transport active substances inside the hydrophilic core, as well as hydrophobic drugs within the bilayer.     

Synthesis,Characterization, and Evaluation of Gel‐Type Poly(4‐Vinylpyridine) Resins for Plutonium Sorption

Abstract

Three different cross‐linked (4, 8, and 12%) gel‐type strong‐base poly(4‐vinylpyridine) resins (PVP) have been synthesized and characterized by elemental analysis, IR, exchange capacity, and moisture content. The uptake of plutonium and uranium was measured as a function of nitric acid concentration using all the three PVP resins. Plutonium sorption and elution kinetics experiments were also performed on all three PVP resins and compared with the benchmark, a gel‐type quarternary ammonium type anion‐exchange resin. The plutonium sorption rate decreases with the increase in cross‐linkage of the resin. All the three PVP resins exhibit better elution kinetics compared to the benchmark. The results on kinetic experiments performed on all three‐gel‐type resins indicated 8% gel‐type PVP resin with 50–100 mesh as a better candidate for plutonium processing or purification. Radiation degradation studies were carried out on the 8% PVP resin by gamma irradiation up to 200 MRad. The irradiated resins were characterized by IR, TGA, and SEM.

The exchange capacity, moisture content, and plutonium uptake were also evaluated for the irradiated PVP resins in comparison with the benchmark. The results indicated a better radiation stability for PVP resin over the benchmark.