Publications by authors named "Ana M Azevedo"

57 Publications

Protein discrimination using erythrosin B-based GUMBOS in combination with UV-Vis spectroscopy and chemometrics.

Talanta 2021 Dec 23;240:123164. Epub 2021 Dec 23.

LAQV, REQUIMTE, Departamento de Ciências Químicas, Laboratório de Química Aplicada, Faculdade de Farmácia, Universidade do Porto, Rua Jorge Viterbo Ferreira 228, 4050-313, Porto, Portugal. Electronic address:

GUMBOS (Group of Uniform Materials Based on Organic Salts) have recently emerged as interesting materials for protein analysis due to their unique features and high tunability. In this regard, four novel erythrosin B (EB)-based GUMBOS were synthesized and their potential to discriminate among proteins with distinct properties (e.g., size, charge, and hydrophobicity) was assessed. These solid-phase materials were prepared using a single-step metathesis reaction between EB and various phosphonium and ammonium cations, namely tetrabutylphosphonium (P), tributylhexadecylphosphonium (P), tetrabutylammonium (N), and benzyldimethylhexadecylammonium (BDHA). Subsequently, the effect of pH (3.0, 4.5, and 6.0) and reaction time (5, 10, and 15 min) on the discriminatory power of synthesized GUMBOS was evaluated. Absorption spectra resulting from the interaction between EB-based GUMBOS and proteins were analyzed using partial least squares discriminant analysis (PLSDA). Unlike time, the pH value was determined to have influence over GUMBOS discrimination potential. Correct protein assignments varied from 86.5% to 100.0%, and the best discriminatory results were observed for [P][EB] and [N][EB] at pH 6.0. Additionally, these two GUMBOS allowed discrimination of protein mixtures containing different ratios of albumin and myoglobin, which appeared as individualized clusters in the PLSDA scores plots. Overall, this study showcases EB-based GUMBOS as simple synthetic targets to provide a label-free, cost-effective, rapid, and successful approach for discrimination of single proteins and their mixtures.
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http://dx.doi.org/10.1016/j.talanta.2021.123164DOI Listing
December 2021

Microchromatography integrated with impedance sensor for bioprocess optimization: Experimental and numerical study of column efficiency for evaluation of scalability.

J Chromatogr A 2022 Jan 8;1661:462678. Epub 2021 Nov 8.

IBB - Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal; Department of Bioengineering, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal. Electronic address:

In the last decade, there has been a growing interest in developing microfluidic systems as new scale-down models for accelerated and cost-effective biopharmaceutical process development. Nonetheless, the research in this field is still in its infancy and requires further investigation to simplify and accelerate the microfabrication process. In addition, integration of different label-free sensors into the microcolumn systems has utmost importance to minimize result discrepancies during the scale-up process. In this study, we developed a simple, low-cost integrated microcolumn (26 µl). Micromilling technology was employed to define the geometry and shape of microfluidic structures using poly(methylmethacrylate) (PMMA). The design of PMMA microstructure was transferred to polydimethylsiloxane (PDMS), and interdigitated planar microelectrodes (IDE) were integrated into the system. To evaluate the scalability of the developed microcolumn column, column performance was assessed and compared with a conventional 1-ml prepacked column. Computational Fluid Dynamics (CFD) studies were performed for both columns to understand the differences between theoretical and experimental results regarding retention time and peak broadening. Despite obtaining an acceptable asymmetric factor for the microcolumn (1.03 ± 0.02), the reduced plate height value was still higher than the recommended range with the value of 4.14 ± 0.18. Nevertheless, the consistency and significant improvement of microcolumn efficiency compared to previous studies provide the possibility of developing robust simulation tools for transferring acquired experimental data for larger-scale units.
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http://dx.doi.org/10.1016/j.chroma.2021.462678DOI Listing
January 2022

Microfluidics as a high-throughput solution for chromatographic process development - The complexity of multimodal chromatography used as a proof of concept.

J Chromatogr A 2021 Nov 8;1658:462618. Epub 2021 Oct 8.

iBB - Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal; Department of Bioengineering, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal. Electronic address:

High-throughput technologies are fundamental to expedite the implementation of novel purification platforms. The possibility of performing process development within short periods of time while saving consumables and biological material are prime features for any high-throughput screening device. In this work, a microfluidic device is evaluated as high-throughput solution for a complete study of chromatographic operation conditions on ten different multimodal resins. The potential of this class of purification solutions is generally hindered by its complexity. Taking this into consideration, the microfluidic platform was herein applied and assessed as a tool for high-throughput applications. The commercially available multimodal ligands were studied for the binding of three antibody-based biomolecules (polyclonal mixture of whole antibodies, Fab and Fc fragments) at different pH and salt conditions, in a total of 450 experiments. The results obtained with the microfluidic device were comparable to a standard 96-well filtering microplate high-throughput tool. Additionally, five of the ten multimodal ligands tested were packed into a bench-scale column to perform a final validation of the microfluidic results obtained. All the data acquired in this work using different screening protocols corroborate each other, showing that microfluidic chromatography is a valuable tool for the fast implementation of a new purification step, particularly, if the goal is to narrow the downstream possibilities by being a first point of decision.
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http://dx.doi.org/10.1016/j.chroma.2021.462618DOI Listing
November 2021

Manufacturing of bacteriophages for therapeutic applications.

Biotechnol Adv 2021 Jul-Aug;49:107758. Epub 2021 Apr 22.

iBB - Institute for Bioengineering and Biosciences, Department of Bioengineering, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1, 1049-001 Lisbon, Portugal. Electronic address:

Bacteriophages, or simply phages, are the most abundant biological entities on Earth. One of the most interesting characteristics of these viruses, which infect and use bacteria as their host organisms, is their high level of specificity. Since their discovery, phages became a tool for the comprehension of basic molecular biology and originated applications in a variety of areas such as agriculture, biotechnology, food safety, veterinary, pollution remediation and wastewater treatment. In particular, phages offer a solution to one of the major problems in public health nowadays, i.e. the emergence of multidrug-resistant bacteria. In these situations, the use of virulent phages as therapeutic agents offers an alternative to the classic, antibiotic-based strategies. The development of phage therapies should be accompanied by the improvement of phage biomanufacturing processes, both at laboratory and industrial scales. In this review, we first present some historical and general aspects related with the discovery, usage and biology of phages and provide a brief overview of the most relevant phage therapy applications. Then, we showcase current processes used for the production and purification of phages and future alternatives in development. On the production side, key factors such as the bacterial physiological state, the conditions of phage infection and the operation parameters are described alongside with the different operation modes, from batch to semi-continuous and continuous. Traditional purification methods used in the initial phage isolation steps are then described followed by the presentation of current state-of-the-art purification approaches. Continuous purification of phages is finally presented as a future biomanufacturing trend.
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http://dx.doi.org/10.1016/j.biotechadv.2021.107758DOI Listing
June 2021

Development of an automated yeast-based spectrophotometric method for toxicity screening: Application to ionic liquids, GUMBOS, and deep eutectic solvents.

Chemosphere 2021 Aug 18;277:130227. Epub 2021 Mar 18.

LAQV, REQUIMTE, Departamento de Ciências Químicas, Faculdade de Farmácia, Universidade do Porto, Porto, Portugal. Electronic address:

Saccharomyces cerevisiae has been used as a eukaryotic model organism for studying the toxic effects of various compounds. In this context, an automated spectrophotometric method based on the enzymatic reduction of methylene blue dye to a colorless product by living yeast cells was implemented in a sequential injection analysis system. Loss of yeast viability/impaired metabolic activity was monitored by an increase in optical density at 664 nm. To prove the usefulness of this approach, the toxicity of ILs (ionic liquids), GUMBOS (group of uniform materials based on organic salts), and DESs (deep eutectic solvents) was examined. Differences obtained between IC values confirmed the impact of structural elements on each compounds' toxicity. While DESs appeared to be less toxic than ILs, GUMBOS were found to be among the most toxic compounds to yeast cells and thus can be viewed as promising antimicrobial candidates. The automated methodology showed satisfactory repeatability and reproducibility (RSD < 9%), which is in good agreement with Green Chemistry principles. In fact, the method required consumption of only 40 μL of reagents and produced less than 2 mL of effluents per cycle. Thus, the developed assay can be used as an alternative tool for toxicity screening.
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http://dx.doi.org/10.1016/j.chemosphere.2021.130227DOI Listing
August 2021

mRNA vaccines manufacturing: Challenges and bottlenecks.

Vaccine 2021 04 24;39(16):2190-2200. Epub 2021 Mar 24.

Department of Biochemical Engineering, University College London, Bernard Katz Building, Gordon Street, London WC1H 0AH, United Kingdom. Electronic address:

Vaccines are one of the most important tools in public health and play an important role in infectious diseases control. Owing to its precision, safe profile and flexible manufacturing, mRNA vaccines are reaching the stoplight as a new alternative to conventional vaccines. In fact, mRNA vaccines were the technology of choice for many companies to combat the Covid-19 pandemic, and it was the first technology to be approved in both United States and in Europe Union as a prophylactic treatment. Additionally, mRNA vaccines are being studied in the clinic to treat a number of diseases including cancer, HIV, influenza and even genetic disorders. The increased demand for mRNA vaccines requires a technology platform and cost-effective manufacturing process with a well-defined product characterisation. Large scale production of mRNA vaccines consists in a 1 or 2-step in vitro reaction followed by a purification platform with multiple steps that can include Dnase digestion, precipitation, chromatography or tangential flow filtration. In this review we describe the current state-of-art of mRNA vaccines, focusing on the challenges and bottlenecks of manufacturing that need to be addressed to turn this new vaccination technology into an effective, fast and cost-effective response to emerging health crises.
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http://dx.doi.org/10.1016/j.vaccine.2021.03.038DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7987532PMC
April 2021

GUMBOS and nanoGUMBOS in chemical and biological analysis: A review.

Anal Chim Acta 2020 Oct 9;1133:180-198. Epub 2020 Jul 9.

LAQV, REQUIMTE, Departamento de Ciências Químicas, Laboratório de Química Aplicada, Faculdade de Farmácia, Universidade do Porto, Rua Jorge Viterbo Ferreira, 228, 4050-313, Porto, Portugal. Electronic address:

GUMBOS (group of uniform materials based on organic salts) is a novel class of materials that exhibits similar features to those of ionic liquids, but have melting points between 25 and 250 °C. GUMBOS can be easily converted into nanomaterials (nanoGUMBOS), with advantages of working at nanoscale. Due to the huge number of possible cation-anion combinations, these materials can be multifunctional and designed for a specific task. This review highlights the possibility of fine-tuning GUMBOS physical and chemical properties in view of changing their ionic counterparts. Their outstanding potential for analytical applications is shown through recent developments in areas such as sensing, and solid-phase extraction. Available methods for synthesis of nanoGUMBOS, and their different outcomes in shapes and optical properties are described, with pros and cons being outlined. Finally, an analysis is made of opportunities and challenges faced by this class of organic ionic materials.
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http://dx.doi.org/10.1016/j.aca.2020.06.028DOI Listing
October 2020

Purification of Plasmid DNA by Multimodal Chromatography.

Methods Mol Biol 2021 ;2197:193-205

Department of Bioengineering, Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal.

Multimodal (MM) chromatography can be described as a chromatographic method that uses more than one mode of interaction between the target molecule and the ligand to achieve a particular separation. Owing to its advantages over traditional chromatography, such as higher selectivity and capacity, its application for the purification of biomolecules with therapeutic interest has been widely studied. The potential of MM chromatography for the purification of plasmid DNA has been demonstrated. In this chapter, a downstream process for the purification of supercoiled plasmid DNA using MM chromatography with two different ligands-Capto™ adhere and PPA HyperCell™-is described. In both the cases, the purification process yields a high purity and highly homogeneous sc plasmid product.
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http://dx.doi.org/10.1007/978-1-0716-0872-2_10DOI Listing
March 2021

Primary Purification of Plasmid DNA Using Differential Isopropanol Precipitation.

Methods Mol Biol 2021 ;2197:151-165

Department of Bioengineering, Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal.

A method for the intermediate recovery of plasmid DNA (pDNA) from alkaline lysates is described that comprises differential isopropanol precipitation steps. In a first low-cut precipitation, a smaller amount of isopropanol (20% v/v) is used so that only high molecular weight RNA precipitates. After solid liquid separation, a high-cut precipitation is performed by bringing isopropanol concentration to 70% v/v to precipitate pDNA. Tests made with lysates show that the differential precipitation increases purity threefold compared to the conventional one-step precipitation at 70% v/v without affecting pDNA recovery (>80%).
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http://dx.doi.org/10.1007/978-1-0716-0872-2_8DOI Listing
March 2021

Effect of Two Different Pose Estimation Approaches on Lower Extremity Biomechanics in Professional Dancers.

Annu Int Conf IEEE Eng Med Biol Soc 2019 Jul;2019:2947-2950

Different algorithms can be used to estimate the pose of musculoskeletal models in biomechanical studies. Visual 3D uses segment optimization whereas OpenSim uses global optimization. Thus, our purpose was to study whether the two approaches would influence the estimation of lower extremity biomechanical parameters. Marker trajectories and ground reaction forces of 6 professional dancers were collected during a single-leg forward jump-landing. The same data set was processed using both approaches. Our findings suggested that the sagittal knee and ankle angles and moments were highly comparable between the two approaches. The ankle sagittal angle and moment showed the lowest offset. On the other hand, the choice of a kinematic model was likely to affect the hip, more evident in the frontal and transverse planes. This may be due to different factors such as the pelvis and femur positions or larger amount of soft tissue in the thigh.
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http://dx.doi.org/10.1109/EMBC.2019.8857683DOI Listing
July 2019

Oxford foot model kinematics in landings: A comparison between professional dancers and non-dancers.

J Sci Med Sport 2020 Apr 31;23(4):347-352. Epub 2019 Oct 31.

George Mason University, Sports Medicine Assessment, Research & Testing (SMART) Laboratory, USA; Department of Bioengineering, George Mason University. Electronic address:

Objectives: Dancers frequently perform jump-landing activities, with the foot-ankle complex playing an essential role to attenuate the landing forces. However, scarce research has been conducted in professional dancers multi-segmented foot in landings. The aim of this study was to compare the multi-segmented foot kinematics between professional dancers and non-dancers, during forward and lateral single-leg jump-landings.

Design: Descriptive group comparison.

Methods: Marker trajectories and synchronized ground reaction forces of 15 professional dancers and 15 non-dancers were collected using motion capture and a force plate, during multidirectional single-leg jump-landings. Sagittal and frontal hindfoot-tibia, forefoot-hindfoot, and hallux-forefoot kinematics of the multi-segmented foot model were computed at initial contact, peak vertical ground reaction force and peak knee flexion. Repeated measures ANOVAs were conducted (p < 0.05).

Results: Professional dancers landed with higher hindfoot-tibia and forefoot-hindfoot plantarflexion angles at initial contact (p < 0.001), and hindfoot-tibia dorsiflexion angles at peak vertical ground reaction force and peak knee flexion (p < 0.001) than non-dancers. Also, dancers exhibited higher sagittal hindfoot-tibia and forefoot-hindfoot excursions than non-dancers (p < 0.001). No statistically significant differences were found in the frontal plane.

Conclusions: The multi-segmented foot allows a comprehensive kinematic analysis of the different foot joints. In jump-landings, professional dancers higher hindfoot-tibia, and forefoot-hindfoot plantarflexion at initial contact, compared to non-dancers, contributed to a subsequent higher foot joints excursion. This pattern is commonly linked to a better shock absorption mechanism in landings.
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http://dx.doi.org/10.1016/j.jsams.2019.10.018DOI Listing
April 2020

Foot modeling affects ankle sagittal plane kinematics during jump-landing.

J Biomech 2019 Nov 12;96:109337. Epub 2019 Sep 12.

George Mason University, Sports Medicine Assessment, Research & Testing (SMART) Laboratory, Manassas, VA, USA. Electronic address:

The foot-ankle complex is a key-element to mitigate impact forces during jump-landing activities. Biomechanical studies commonly model the foot as a single-segment, which can provide different ankle kinematics compared to a multi-segmented model. Also, it can neglect intersegmental kinematics of the foot-ankle joints, such as the hindfoot-tibia, forefoot-hindfoot, and hallux-forefoot joints, that are used during jump-landing activities. The purpose of this short communication was to compare ankle kinematics between a three- and single-segmented foot models, during forward and lateral single-leg jump-landings. Marker trajectories and synchronized ground reaction forces of 30 participants were collected using motion capture and a force plate, during multidirectional single-leg jump-landings. Ankle kinematics were computed using a three- (hindfoot-tibia) and a single-segmented (ankle) foot models, at initial contact (IC), peak vertical ground reaction force (PvGRF) and peak knee flexion (PKF). Repeated measures ANOVAs were conducted (p < 0.05). The findings of this study showed that during lateral and forward jump-landing directions, the three-segmented foot model exhibited lower hindfoot-tibia dorsiflexion angles (PvGRF and PKF, p < 0.001) and excursions (sagittal: p < 0.001; frontal: p < 0.05) during the weightbearing acceptance phase than the single-segmented model. Overall, the two foot models provided distinctive sagittal ankle kinematics, with lower magnitudes in the hindfoot-tibia of the three-segmented foot. Furthermore, the three-segmented foot model may provide additional and representative kinematic data of the ankle and foot joints, to better comprehend its function, particularly in populations whose foot-ankle complex plays an important role (e.g., dancers).
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http://dx.doi.org/10.1016/j.jbiomech.2019.109337DOI Listing
November 2019

Microfluidic platform for rapid screening of bacterial cell lysis.

J Chromatogr A 2020 Jan 10;1610:460539. Epub 2019 Sep 10.

IBB - Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal; Department of Bioengineering, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal. Electronic address:

Over the past decade significant progress has been found in the upstream production processes, shifting the main bottlenecks in current manufacturing platforms for biopharmaceuticals towards the downstream processing. Challenges in the purification process include reducing the production costs, developing robust and efficient purification processes as well as integrating both upstream and downstream processes. Microfluidic technologies have recently emerged as effective tools for expediting bioprocess design in a cost-effective manner, since a large number of variables can be evaluated in a small time frame, using reduced volumes and manpower. Their modularity also allows to integrate different unit operations into a single chip, and consequently to evaluate the effect of each stage on the overall process efficiency. This paper describes the development of a diffusion-based microfluidic device for the rapid screening of continuous chemical lysis conditions. The release of a recombinant green fluorescent protein (GFP) expressed in Escherichia coli (E. coli) was used as model system due to the simple evaluation of cell growth and product concentration by fluorescence. The concept can be further applied to any biopharmaceutical production platform. The microfluidic device was successfully used to test the lytic effect of both enzymatic and chemical lysis solutions, with lysis efficiency of about 60% and close to 100%, respectively, achieved. The microfluidic technology also demonstrated the ability to detect potential process issues, such as the increased viscosity related with the rapid release of genomic material, that can arise for specific lysis conditions and hinder the performance of a bioprocess. Finally, given the continuous operation of the lysis chip, the microfluidic technology has the potential to be integrated with other microfluidic modules in order to model a fully continuous biomanufacturing process on a chip.
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http://dx.doi.org/10.1016/j.chroma.2019.460539DOI Listing
January 2020

Biochemical Engineering Science-Sustainable Processes and Economies.

Biotechnol J 2019 08;14(8):e1900276

Instituto Superior Técnico, Universidade de Lisboa, Lisbon, 1049-001, Portugal.

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http://dx.doi.org/10.1002/biot.201900276DOI Listing
August 2019

Mobile-Phase Modulators as Salt Tolerance Enhancers in Phenylboronate Chromatography: Thermodynamic Evaluation of the Mechanisms Underlying the Adsorption of Monoclonal Antibodies.

Biotechnol J 2019 Oct 13;14(10):e1800586. Epub 2019 Aug 13.

CICS-UBI-Health Sciences Research Centre, University of Beira Interior, Avenida Infante D. Henrique, 6200-506, Covilhã, Portugal.

Phenylboronate chromatography has been employed for bioseparation applications though details concerning the mechanisms of interaction between the ligand and macromolecules remain widely unknown. Here, the phenomena underlying the adsorption of an anti-human interleukin-8 (anti-IL8) monoclonal antibody (mAb) onto an m-aminophenylboronic acid (m-APBA) ligand in the presence of different mobile-phase modulators (NaF/MgCl /(NH ) SO ) and under different pH values (7.5/8.5/9.0) is investigated. Flow microcalorimetry (FMC) is applied to measure instantaneous heat energy transfer, providing insights about the role of specific and nonspecific interactions involved in the adsorptive process. Results show that the adsorption of anti-IL8 mAb to m-APBA is enthalpically driven, corroborating the presence of the reversible esterification reaction between boronic acid or boronates and cis-diol-containing molecules. Nevertheless, for all mobile-phase modulators studied, changes in thermogram profiles are observed as well as reductions in the net heat of adsorption when increasing the pH. Overall, FMC and parallel chromatographic experiments data suggest that ligand salt tolerance could be enhanced using mobile-phase modulators, with all salts studied promoting the specific cis-diol interactions and reducing nonspecific interactions. The last feature is more noticeable at pH values above ligand's pK , mainly due to the ability of NaF and (NH ) SO to diminish electrostatic interactions when compared to the commonly used NaCl.
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http://dx.doi.org/10.1002/biot.201800586DOI Listing
October 2019

Optimizing the Performance of Chromatographic Separations Using Microfluidics: Multiplexed and Quantitative Screening of Ligands and Target Molecules.

Biotechnol J 2019 Oct 23;14(10):e1800593. Epub 2019 Jul 23.

IBB - Institute for Bioengineering and Biosciences Instituto Superior Técnico, Universidade de Lisboa, Avenida Rovisco Pais 1, 1049-001, Lisbon, Portugal.

The optimization of chromatography ligands for the purification of biopharmaceuticals is highly demanded to meet the needs of the pharmaceutical industry. In the case of monoclonal antibodies (mAbs), synthetic ligands comprising multiple types of interactions (multimodal) provide process and economic advantages compared to protein-based affinity ligands. However, optimizing the operation window of these ligands requires the development of effective high-throughput screening platforms. Here, a novel microfluidics-based methodology to perform rapid and multiplexed screening of various multimodal ligands relative to their ability to bind different target molecules is demonstrated. The microfluidic structure comprises three individual chambers (≈8 nL each) packed with different types of chromatography beads in series with the feed flow. An artificial mixture composed of immunoglobulin G (IgG) and bovine serum albumin, labeled with different thiol-reactive neutral fluorescent dyes, is used as a model to quantitatively optimize the performance (yield and purity) of the separation. This approach can potentially be used as a predictive analytical tool in the context of mAb purification, allowing low consumption of molecules and providing results in <3 min. Furthermore, this versatile approach can potentially be extended not only with respect to the number of different resins and target molecules, but also for parallel analysis of multiple conditions.
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http://dx.doi.org/10.1002/biot.201800593DOI Listing
October 2019

Minimizing the Influence of Fluorescent Tags on IgG Partition in PEG-Salt Aqueous Two-Phase Systems for Rapid Screening Applications.

Biotechnol J 2019 Aug 17;14(8):e1800640. Epub 2019 May 17.

IBB-Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001, Lisbon, Portugal.

Aqueous two-phase extraction (ATPE) has been showing significant potential in the biopharmaceutical industry, allowing the selective separation of high-value proteins directly from unclarified cell culture supernatants. In this context, effective high-throughput screening tools are critical to perform a rapid empirical optimization of operating conditions. In particular, microfluidic ATPE screening devices, coupled with fluorescence microscopy to continuously monitor the partition of fluorophore-labeled proteins, have been recently demonstrated to provide short diffusion distances and rapid partition, using minimal reagent volumes. Nevertheless, the currently overlooked influence of the labeling procedure on partition must be carefully evaluated to validate the extrapolation of results to the unlabeled molecule. Here, three fluorophores with different global charge and reactivity selected to label immunoglobulin G (IgG) at degrees of labeling (DoL) ranging from 0.5 to 7.6. Labeling with BODIPY FL maleimide (DoL = 0.5), combined with tris(2-carboxyethyl) phosphine (TCEP) to generate free thiol groups, is the most promising strategy to minimize the influence of the fluorophore on partition. In particular, the partition coefficient (K ) measured in polyethylene glycol (PEG) 3350-phosphate systems with and without the addition of NaCl using microtubes (batch) or microfluidic devices (continuous) is comparable to those quantified for the native protein.
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http://dx.doi.org/10.1002/biot.201800640DOI Listing
August 2019

Professional Dancers Distinct Biomechanical Pattern during Multidirectional Landings.

Med Sci Sports Exerc 2019 03;51(3):539-547

Sports Medicine Assessment, Research & Testing (SMART) Laboratory, George Mason University, Manassas, VA.

Purpose: This study aimed to compare lower extremity (LE) biomechanics between professional dancers (PD) and nondancers (ND) during multidirectional single-leg landings.

Methods: Fifteen PD (27 ± 7 yr, 1.69 ± 0.1 m, 57.8 ± 9.3 kg) and 15 ND (25 ± 5 yr, 1.69 ± 0.1 m, 66 ± 10.2 kg) performed single-leg jumps in three directions: 1) lateral, 2) diagonal, and 3) forward. Dominant LE biomechanical data were collected using a motion capture system. Data were processed in Visual3D. LE kinematic (hip, knee, and ankle joint angles in sagittal and frontal planes, and range of motion [ROM]) and kinetics (hip and knee internal joint moments and vertical ground reaction force) variables were analyzed at initial contact (IC), peak vertical ground reaction force (PvGRF), and peak knee flexion (PKF). Repeated-measures ANOVA was conducted (P < 0.05).

Results: At IC, statistically significant interactions were found for ankle frontal and hip sagittal angles (P < 0.05). The main effects for groups and jump directions were attained (P < 0.05). PD at IC had lower hip and knee flexion and higher ankle plantarflexion than ND. PD had significantly higher knee (PD, 41 ± 6.1; ND, 33.8 ± 8.4) and ankle (PD, 53.7 ± 3.4; ND, 38.9 ± 8.9) ROM than ND. At IC, the lateral jump had higher hip abduction moment, hip abduction, and ankle inversion and lower hip flexion and ankle plantarflexion than the forward and diagonal jumps. The lateral jump (15.5 ± 7.7) had higher hip excursion than the forward jump (12.7 ± 5.4).

Conclusion: The higher extended posture of PD at IC promoted an efficient use of the knee and ankle ROM to dissipate the landing forces. Regardless of the group, jump directions also solicited different biomechanical responses, particularly between lateral and forward directions. These strategies should be considered for implementation in prevention programs, as it can foster adequate LE neuromuscular control.
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http://dx.doi.org/10.1249/MSS.0000000000001817DOI Listing
March 2019

Thermodynamics of the adsorption of monoclonal antibodies in phenylboronate chromatography: Affinity versus multimodal interactions.

J Chromatogr A 2018 Sep 17;1569:118-127. Epub 2018 Jul 17.

iBB- Institute for Bioengineering and Biosciences, Department of Bioengineering, Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal. Electronic address:

The aim of this work was to investigate the complex phenomena underlying the adsorption of an anti-human IL-8 (anti-IL8) monoclonal antibody (mAb) to m-aminophenylboronate (m-APBA) by Flow Microcalorimetry (FMC) and to understand the role of non-specific interactions in the adsorption process. FMC was exploited as a dynamic on-line method to measure instantaneous heat energy transfers in order to understand the surface phenomena underlying mAb's adsorption towards the synthetic ligand m-APBA under different pH (7.5, 8.5, 9.0, 9.5 and 10.0) and salt concentrations (0 and 150 mM NaCl). Results showed that the adsorption of anti-IL8 mAb to m-APBA is enthalpically driven (ΔH<0), as expected for the predominant reversible esterification reaction between boronates and cis-diols-containing molecules. For all the pH conditions studied, thermograms presented a first exothermic peak, characteristic of the reversible esterification reaction between mAb (pI≥9.3) and m-APBA (pKa = 8.8), except at pH 9.0 in the presence of 150 mM NaCl, for which the thermogram presented a first endothermic peak. The heat of adsorption (ΔH) obtained at conditions where cis-diol interactions were predominant was approximately -243 ± 38 kJ/mol against -82 ± 14 kJ/mol (p-value < 0.05) obtained at pH 9.0 with 150 mM NaCl. The observed shift in the thermogram profile at pH 9.0, 150 mM NaCl, and the consequent decrease of 60-70% in ΔH were indicative of the promotion of electrostatic interactions between the protein and the ligand. Overall, and whereas the binding of the PBA ligand to mAb molecules has been described for decades as being affinity-based, our study demonstrates the multimodal behaviour of this interaction and contributes towards the understanding of the adsorption thermodynamics.
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http://dx.doi.org/10.1016/j.chroma.2018.07.050DOI Listing
September 2018

Multiplexed microfluidic fluorescence immunoassay with photodiode array signal acquisition for sub-minute and point-of-need detection of mycotoxins.

Lab Chip 2018 05;18(11):1569-1580

Instituto de Engenharia de Sistemas e Computadores - Microsistemas e Nanotecnologias (INESC MN) and IN - Institute of Nanoscience and Nanotechnology, Lisbon, Portugal.

Portable, rapid, cost effective and simple analytical tools are in increasing demand to facilitate the routine monitoring of target chemical/biological compounds at the point-of-need. Such devices are highly relevant within the context of food safety, particularly concerning the screening of highly toxic and strictly regulated mycotoxins. To achieve ultrarapid detection of mycotoxins, namely aflatoxin B1, ochratoxin A and deoxynivalenol, at the point-of-need, a novel multiplexed bead-based microfluidic competitive immunosensor, coupled with an array of a-Si:H thin-film photodiodes for integrated fluorescence signal acquisition, is reported. Simultaneously measuring the initial binding rate for each analyte of the sample under analysis against an internal reference, this device provided limits of detection below 1 ng mL-1 for all mycotoxins in a single-step assay and within 1 minute after mixing the sample under analysis with a fluorescent conjugate. The compatibility of the device with the analysis of mycotoxins spiked in corn samples was further demonstrated after performing a sample preparation procedure based on aqueous two-phase extraction. The short times of analysis and sensitivities in the low ng mL-1 range make these devices potentially competitive with the lateral flow devices that are currently the standard for this application. Furthermore, this device architecture and concept is amenable of being expanded to other analytes in food safety, biomedical and other applications.
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http://dx.doi.org/10.1039/c8lc00259bDOI Listing
May 2018

Enhancement of lateral flow assay performance by electromagnetic relocation of reporter particles.

PLoS One 2018 8;13(1):e0186782. Epub 2018 Jan 8.

Department of Chemical and Biomolecular Engineering, University of Houston, Houston, Texas, United States of America.

Lateral flow assays (LFAs) are a widely-used point-of care diagnostic format, but suffer from limited analytical sensitivity, especially when read by eye. It has recently been reported that LFA performance can be improved by using magnetic reporter particles and an external magnetic field applied at the test line. The mechanism of sensitivity/performance enhancement was suggested to be concentration/retardation of reporter particles at the test line. Here we demonstrate an additional mechanism of particle relocation where reporter particles from the lower depths of the translucent LFA strip relocate to more-visible locations nearer to the top surface, producing a more visible signal. With a magnetic field we observed an improvement in sensitivity of human chorionic gonadotropin (hCG) detection from 1.25 ng/mL to 0.31 ng/mL. We also observed an increase of the color intensity per particle in test lines when the magnetic field was present.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0186782PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5757911PMC
January 2018

M13 bacteriophage purification using poly(ionic liquids) as alternative separation matrices.

J Chromatogr A 2018 Jan 6;1532:246-250. Epub 2017 Dec 6.

iBB-Institute for Bioengineering and Biosciences, Department of Bioengineering, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal.

M13 is a filamentous, non-lytic bacteriophage that infects Escherichia coli via the F pilus. Currently, phage M13 is widely used in phage display technology and bio-nanotechnology, and is considered a possible antibacterial therapeutic agent, among other applications. Conventional phage purification involves 5-7 operational steps, with high operational costs and significant product loss (approximately 60%). In this work, we propose a scalable purification process for M13 bacteriophage using a novel stationary phase based on a polymeric ionic liquid (PIL) with a positively charged backbone structure. Poly (1-vinyl-3-ethyl imidazolium bis(trifluoromethylsulfonyl) imide) - poly(VEIM-TFSI) predominantly acted as an anion exchanger under binding-elution mode. This revealed to be a rapid and simple method for the recovery of phage M13 with an overall separation yield of over 70% after a single downstream step. To the best of our knowledge, PILs have never been used as separation matrices for biological products and the results obtained, together with the large number of cations and anions available to prepare PILs, illustrate well the large potential of the proposed methodology.
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http://dx.doi.org/10.1016/j.chroma.2017.12.005DOI Listing
January 2018

Colorimetric detection of D-dimer in a paper-based immunodetection device.

Anal Biochem 2017 12 15;538:5-12. Epub 2017 Sep 15.

iBB- Institute for Bioengineering and Biosciences, Department of Bioengineering, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001 Lisboa, Portugal. Electronic address:

A microfluidic paper-based analytical device (μPADs) immunoassay for detection of the blood native biomarker D-dimer is reported. The μPAD is created by wax printing on a single piece of chromatographic paper and combined with an anti-D-dimer capture antibody and conjugates of anti-D-dimer antibody with 40 nm gold nanoparticles. The presence of D-dimer in buffer/simulated plasma samples is successfully reported for concentrations as low as 15 ng D-dimer/mL. Linearity between signal intensity and D-dimer concentration is observed up to 100 ng/mL. Using an appropriate dilution, the test could be used to yield positive results only for those samples with a D-dimer concentration above the clinically relevant threshold range of 250-500 ng/mL. Finally, the merits and pitfalls of using μPADs as compared to lateral flow devices in immunoassays are discussed.
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http://dx.doi.org/10.1016/j.ab.2017.09.009DOI Listing
December 2017

Monoclonal Antibodies Production Platforms: An Opportunity Study of a Non-Protein-A Chromatographic Platform Based on Process Economics.

Biotechnol J 2017 Dec 6;12(12). Epub 2017 Oct 6.

iBB - Institute for Bioengineering and Biosciences, Department of Bioengineering, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, No. 1, Lisbon 1049-001, Portugal.

Monoclonal antibodies currently dominate the biopharmaceutical market with growing sales having reached 80 billion USD in 2016. As most top-selling mAbs are approaching the end of their patent life, biopharmaceutical companies compete fiercely in the biosimilars market. These two factors present a strong motivation for alternative process strategies and process optimization. In this work a novel purification strategy for monoclonal antibodies comprising phenylboronic acid multimodal chromatography for capture followed by polishing by ion-exchange monolithic chromatography and packed bed hydrophobic interaction chromatography is presented and compared to the traditional protein-A-based process. Although the capital investment is similar for both processes, the operation cost is 20% lower for the novel strategy. This study shows that the new process is worthwhile investing in and could present a viable alternative to the platform process used by most industrial players.
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http://dx.doi.org/10.1002/biot.201700260DOI Listing
December 2017

A multiplexed microfluidic toolbox for the rapid optimization of affinity-driven partition in aqueous two phase systems.

J Chromatogr A 2017 Sep 5;1515:252-259. Epub 2017 Aug 5.

Department of Bioengineering, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal; IBB-Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal. Electronic address:

Antibodies and other protein products such as interferons and cytokines are biopharmaceuticals of critical importance which, in order to be safely administered, have to be thoroughly purified in a cost effective and efficient manner. The use of aqueous two-phase extraction (ATPE) is a viable option for this purification, but these systems are difficult to model and optimization procedures require lengthy and expensive screening processes. Here, a methodology for the rapid screening of antibody extraction conditions using a microfluidic channel-based toolbox is presented. A first microfluidic structure allows a simple negative-pressure driven rapid screening of up to 8 extraction conditions simultaneously, using less than 20μL of each phase-forming solution per experiment, while a second microfluidic structure allows the integration of multi-step extraction protocols based on the results obtained with the first device. In this paper, this microfluidic toolbox was used to demonstrate the potential of LYTAG fusion proteins used as affinity tags to optimize the partitioning of antibodies in ATPE processes, where a maximum partition coefficient (K) of 9.2 in a PEG 3350/phosphate system was obtained for the antibody extraction in the presence of the LYTAG-Z dual ligand. This represents an increase of approx. 3.7 fold when compared with the same conditions without the affinity molecule (K=2.5). Overall, this miniaturized and versatile approach allowed the rapid optimization of molecule partition followed by a proof-of-concept demonstration of an integrated back extraction procedure, both of which are critical procedures towards obtaining high purity biopharmaceuticals using ATPE.
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http://dx.doi.org/10.1016/j.chroma.2017.07.094DOI Listing
September 2017

A simple method for point-of-need extraction, concentration and rapid multi-mycotoxin immunodetection in feeds using aqueous two-phase systems.

J Chromatogr A 2017 Aug 5;1511:15-24. Epub 2017 Jul 5.

IBB - Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal; Department of Bioengineering, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal. Electronic address:

The rapid detection of mycotoxins in feed samples is becoming an increasingly relevant challenge for the food production sector, in order to effectively enforce current regulations and assure food and feed safety. To achieve rapid mycotoxin detection, several biosensing strategies have been published, many reaching assay times of the order of a few minutes. However, the vast majority of these rely on sample preparation based on volatile organic solvents, often comprising complex multi-step procedures and devoid of clean-up and/or concentration effects. Here, a novel sample preparation methodology based on a green, non-toxic and inexpensive polyethylene glycol-sodium citrate aqueous two-phase system is reported, providing single-step extraction and concentration of three target mycotoxins within 20min: aflatoxin B1 (AFB1), ochratoxin A (OTA) and deoxynivalenol (DON). With point-of-need applications in mind, the extraction procedure was optimized and validated using a rapid multi-toxin microfluidic competitive immunoassay. The assay was successfully tested with spiked complex solid matrices including corn, soy, chickpea and sunflower-based feeds and limits of detection of 4.6ngg±15.8%, 24.1ngg±8.1% and 129.7ngg±53.1% (±CV) were obtained in corn for AFB1, OTA and DON, respectively. These sensitivities are fit-for-purpose at the required regulatory and recommended limits for animal feed, providing an effective and safe semi-quantitative mycotoxin analysis that can be performed in the field.
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http://dx.doi.org/10.1016/j.chroma.2017.07.004DOI Listing
August 2017

Environmental Impact of Ionic Liquids: Recent Advances in (Eco)toxicology and (Bio)degradability.

ChemSusChem 2017 06 19;10(11):2321-2347. Epub 2017 May 19.

LAQV, Requimte, Departamento de Ciências Químicas, Laboratório de Química Aplicada, Faculdade de Farmácia, Universidade do Porto, Rua Jorge Viterbo Ferreira 228, 4050-313, Porto, Portugal.

This Review aims to integrate the most recent and pertinent data available on the (bio)degradability and toxicity of ionic liquids for global and critical analysis and on the conscious use of these compounds on a large scale thereafter. The integrated data will enable focus on the recognition of toxicophores and on the way the community has been dealing with them, with the aim to obtain greener and safer ionic liquids. Also, an update of the most recent biotic and abiotic methods developed to overcome some of these challenging issues will be presented. The review structure aims to present a potential sequence of events that can occur upon discharging ionic liquids into the environment and the potential long-term consequences.
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http://dx.doi.org/10.1002/cssc.201700261DOI Listing
June 2017

Assessment of ionic liquids' toxicity through the inhibition of acylase I activity on a microflow system.

Chemosphere 2017 Apr 16;173:351-358. Epub 2017 Jan 16.

LAQV, REQUIMTE, Departamento de Ciências Químicas, Faculdade de Farmácia, Universidade do Porto, Rua Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal. Electronic address:

Acylase I (ACY I) plays a role in the detoxication and bioactivation of xenobiotics as well in other physiological functions. In this context, an automated ACY I assay for the evaluation of ionic liquids' (ILs) toxicity was developed. The assay was implemented in a sequential injection analysis (SIA) system and was applied to eight commercially available ILs. The SIA methodology was based on the deacetylation of N-acetyl-l-methionine with production of l-methionine, which was determined using fluorescamine. ACY I inhibition in the presence of ILs was monitored by the decrease of fluorescence intensity. The obtained results confirmed the influence of ILs' structural elements on its toxicity and revealed that pyridinium and phosphonium cations, longer alkyl side chains and tetrafluoroborate anion displayed higher toxic effect on enzyme activity. The developed methodology proved to be robust and exhibited good repeatability (RSD < 1.3%, n = 10), leading also to a reduction of reagents consumption and effluents production. Thus, it is expected that the proposed assay can be used as a novel tool for ILs' toxicity screening.
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http://dx.doi.org/10.1016/j.chemosphere.2016.12.138DOI Listing
April 2017

Predicting protein partition coefficients in aqueous two phase system.

J Chromatogr A 2016 Oct 1;1470:50-58. Epub 2016 Oct 1.

iBB-Institute for Bioengineering and Biosciences, Instituto Superior Técnico, University of Lisbon, Av. Rovisco Pais, 1049-001 Lisbon, Portugal.

The present work aims to achieve an additional insight into the protein partitioning behavior in aqueous two phase systems (ATPSs), together with a study on the viability of a semi-empirical model based on continuum electrostatics to predict the protein partition characteristics. The partitioning behaviors of 14 globular proteins, with different properties, were explored in three polymer/polymer ATPSs. By the Collander equation, a linear correlation between protein partitioning coefficients in all systems was observed. Using the semi-empirical model it was possible to predict the partitioning behavior of proteins. The electrostatic energy depends on the protein size and ATPSs characteristics and varies in agreement with the difference in phase dielectric constants. Linear correlation of nonpolar energy, and the solvent accessible surface area was observed. Polymer structure and concentration have a significant influence on model viability. A good qualitative prediction of preferred phase for studied proteins was observed.
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http://dx.doi.org/10.1016/j.chroma.2016.09.072DOI Listing
October 2016
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