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2017
Dias, AMGC, Roque ACA.  2017.  The future of protein scaffolds as affinity reagents for purification. Biotechnology and Bioengineering. 114:481–491., Number 3 Abstract

Affinity purification is one of the most powerful separation techniques extensively employed both at laboratory and production scales. While antibodies still represent the gold standard affinity reagents, others derived from non-immunoglobulin scaffolds emerged as interesting alternatives in particular for affinity purification. The lower costs of production, fast ligand development and high robustness are appealing advantages of non-immunoglobulin scaffolds. These have successfully been used in the affinity purification of relevant targets as antibodies, human serum albumin, transferrin and other biomarkers, as reviewed in this work. Furthermore, a critical assessment on the strengths, weaknesses, opportunities and threats related with the implementation of non-immunoglobulin scaffolds as ligands in affinity purification are discussed. This article is protected by copyright. All rights reserved.

Carvalho, HF, Barbosa A, Roque ACA, Iranzo O, Branco RJF.  2017.  Integration of Molecular Dynamics Based Predictions into the Optimization of de novo Protein Designs: Limitations and Benefits. Computation Protein Design. :181-201.
dos Santos, R, Carvalho AL, Roque ACA.  2017.  Renaissance of protein crystallization and precipitation in biopharmaceuticals purification. Biotechnology Advances. 35:–., Number 1: Elsevier Inc. AbstractWebsite

The current chromatographic approaches used in protein purification are not keeping pace with the increasing biopharmaceutical market demand. With the upstream improvements, the bottleneck shifted towards the downstream process. New approaches rely in Anything But Chromatography methodologies and revisiting former techniques with a bioprocess perspective. Protein crystallization and precipitation methods are already implemented in the downstream process of diverse therapeutic biological macromolecules, overcoming the current chromatographic bottlenecks. Promising work is being developed in order to implement crystallization and precipitation in the purification pipeline of high value therapeutic molecules. This review focuses in the role of these two methodologies in current industrial purification processes, and highlights their potential implementation in the purification pipeline of high value therapeutic molecules, overcoming chromatographic holdups.

2016
Palma, SICJ, Fernandes AR, Roque ACA.  2016.  An affinity triggered MRI nanoprobe for pH-dependent cell labeling. RSC Adv.. 6:113503–113512., Number 114: Royal Society of Chemistry AbstractWebsite

The pH-sensitive affinity pair composed by neutravidin and iminobiotin was used to develop a multilayered Magnetic Resonance Imaging (MRI) nanoprobe responsive to the acidic pH of tumor microenvironment. The multilayer system was assembled on meso-2,3-dimercaptosuccinic acid-coated iron oxide magnetic nanoparticles (MNP), which convey negative MRI contrast enhancement properties to the nanoprobe. The outer stealth PEG-layer is altered in acidic media due to the disruption of interactions between neutravidin–iminobiotin. As a consequence, the positively charged inner layer is exposed and enhances interactions with cells. The nanoprobe uptake by HCT116 cells cultured in vitro under acidic conditions had a 2-fold increase compared to the uptake at physiological pH. The uptake difference is particularly clear in T2-weighted MRI phantoms of cells incubated with the nanoprobes at both pH conditions. This work sets the proof-of-concept of a MNP-based MRI nanoprobe targeting acidic tumor microenvironment through the use of a specific bio-recognition interaction that is pH-sensitive. This tumor targeting strategy is potentially applicable to the generality of tumors since the typical hypoxic conditions and high glycolysis rate in cancer cells create an acidic environment common to the majority of cancer types.

Fernandes, CSM, dos Santos R, Ottengy S, Viecinski AC, Béhar G, Mouratou B, Pecorari F, Roque ACA.  2016.  Affitins for protein purification by affinity magnetic fishing. Journal of Chromatography A. 1457:50–58.: Elsevier B.V. AbstractWebsite

Currently most economical and technological bottlenecks in protein production are placed in the down-stream processes. With the aim of increasing the efficiency and reducing the associated costs, variousaffinity ligands have been developed. Affitins are small, yet robust and easy to produce, proteins derivedfrom the archaeal extremophilic “7 kDa DNA-binding” protein family. By means of combinatorial pro-tein engineering and ribosome display selection techniques, Affitins have shown to bind a diversity oftargets. In this work, two previously developed Affitins (anti-lysozyme and anti-IgG) were immobilizedonto magnetic particles to assess their potential for protein purification by magnetic fishing. The opti-mal lysozyme and human IgG binding conditions yielded 58 mg lysozyme/g support and 165 mg IgG/gsupport, respectively. The recovery of proteins was possible in high yield (≥95{%}) and with high purity,namely ≥95{%} and 81{%}, when recovering lysozyme from Escherichia coli supernatant and IgG from humanplasma, respectively. Static binding studies indicated affinity constants of 5.0 × 104M−1and 9.3 × 105M−1for the anti-lysozyme and anti-IgG magnetic supports. This work demonstrated that Affitins, which canbe virtually evolved for any protein of interest, can be coupled onto magnetic particles creating novelaffinity adsorbents for purification by magnetic fishing.

Batalha, IL, Zhou H, Lilley K, Lowe CR, Roque ACA.  2016.  Mimicking nature: Phosphopeptide enrichment using combinatorial libraries of affinity ligands. Journal of Chromatography A. 1457:76–87.: Elsevier B.V. AbstractWebsite

Phosphorylation is a reversible post-translational modification of proteins that controls a plethora of cellular processes and triggers specific physiological responses, for which there is a need to develop tools to characterize phosphorylated targets efficiently. Here, a combinatorial library of triazine-based synthetic ligands comprising 64 small molecules has been rationally designed, synthesized and screened for the enrichment of phosphorylated peptides. The lead candidate (coined A8A3), composed of histidine and phenylalanine mimetic components, showed high binding capacity and selectivity for binding mono- and multi-phosphorylated peptides at pH 3. Ligand A8A3 was coupled onto both cross-linked agarose and magnetic nanoparticles, presenting higher binding capacities (100-fold higher) when immobilized on the magnetic support. The magnetic adsorbent was further screened against a tryptic digest of two phosphorylated proteins ($\alpha$- and $\beta$-caseins) and one non-phosphorylated protein (bovine serum albumin, BSA). The MALDI-TOF mass spectra of the eluted peptides allowed the identification of nine phosphopeptides, comprising both mono- and multi-phosphorylated peptides.

Batalha, ÍL, Roque ACA.  2016.  Petasis-Ugi ligands: New affinity tools for the enrichment of phosphorylated peptides. Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences. 1031:86–93.: Elsevier B.V. AbstractWebsite

Affinity chromatography is a widespread technique for the enrichment and isolation of biologics, which relies on the selective and reversible interaction between affinity ligands and target molecules. Small synthetic affinity ligands are valuable alternatives due to their robustness, low cost and fast ligand development. This work reports, for the first time, the use of a sequential Petasis-Ugi multicomponent reaction to generate rationally designed solid-phase combinatorial libraries of small synthetic ligands, which can be screened for the selection of new affinity adsorbents towards biological targets. As a proof of concept, the Petasis-Ugi reaction was here employed in the discovery of affinity ligands suitable for phosphopeptide enrichment. A combinatorial library of 84 ligands was designed, synthesized on a chromatographic solid support and screened in situ for the specific binding of phosphopeptides binding human BRCA1C-terminal domains. The success of the reaction on the chromatographic matrix was confirmed by both inductively coupled plasma atomic emission spectroscopy and fluorescence microscopy. Three lead ligands were identified due to their superior performance in terms of binding capacity and selectivity towards the phosphorylated moiety on peptides, which showed the feasibility of the Petasis-Ugi reaction for affinity ligand development.

Batalha, ÍL, Roque ACA.  2016.  Phosphopeptide Enrichment Using Various Magnetic Nanocomposites: An Overview. Phospho-Proteomics. 1355(Methods in Molecular Biology):193–209. AbstractWebsite

Magnetic nanocomposites are hybrid structures consisting of an iron oxide (Fe3O4 /$\gamma$-Fe2O3 ) superparamagnetic core and a coating shell which presents affi nity for a specifi c target molecule. Within the scope of phosphopeptide enrichment, the magnetic core is usually fi rst functionalized with an intermediate layer of silica or carbon to improve dispersibility and increase specifi c area, and then with an outer layer of a phosphate-affi nity material. Fe3O4 -coating materials include metal oxides, rare earth metal-based compounds, immobilized-metal ions, polymers, and many others. This chapter provides a generic overview of the different materials that can be found in literature and their advantages and drawbacks.

Fernandes, CSM, Barbosa I, Castro R, Pina AS, Coroadinha AS, Barbas A, Roque ACA.  2016.  Retroviral particles are effectively purified on an affinity matrix containing peptides selected by phage-display. Biotechnology Journal. 11:1513–1524. Abstract

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Fernandes, CSM, Castro R, Coroadinha AS, Roque ACA.  2016.  Small synthetic ligands for the enrichment of viral particles pseudotyped with amphotropic murine leukemia virus envelope. Journal of Chromatography A. 1438:160–170.: Elsevier B.V. AbstractWebsite

Retroviral vectors gained popularity toward other viral vectors as they integrate their genome into hosts' genome, a characteristic required for the modification of stem cells. However, the production of viable particles for gene therapy is hampered by the low ratio of infectious to non-infectious viral particles after purification, low titers and limited number of competent viral receptors. We have developed de novo two fully synthetic triazine-based ligands that can selectively bind retroviral particles pseudotyped with amphotropic murine leukemia virus envelope (AMPHO4070A). A 78-membered library of triazine-based ligands was designed in silico and was virtually screened against the modeled structure of the AMPHO4070A protein. Ligands displaying the highest energy of binding were synthesized on cross-linked agarose and experimentally tested. Adsorbents containing ligands A5A10 and A10A11 showed selectivity toward viral particles containing the target protein (VLP-AMPHO), binding 19 ± 5 $μ$g/g support and 47 ± 13 $μ$g/g support, respectively. The elution conditions for both ligands were mild and with high recovery yields (80-100{%}), in comparison with common purification practices. These results were based on a lab-scale experimental setting with VLP integrity being confirmed through TEM. In particular, the elution buffer containing 12 mM imidazole allowed the recovery of intact amphotropic viral particles.

Pina, AS, Carvalho S, Dias AMGC, Guilherme M, Pereira AS, Caraça LT, Coroadinha AS, Lowe CR, Roque ACA.  2016.  Tryptophan tags and de novo designed complementary affinity ligands for the expression and purification of recombinant proteins. Journal of Chromatography A. 1472:55–65. AbstractWebsite

A common strategy for the production and purification of recombinant proteins is to fuse a tag to the protein terminal residues and employ a “tag-specific” ligand for fusion protein capture and purification. In this work, we explored the effect of two tryptophan-based tags, NWNWNW and WFWFWF, on the expression and purification of Green Fluorescence Protein (GFP) used as a model fusion protein. The titers obtained with the expression of these fusion proteins in soluble form were 0.11 mg ml−1 and 0.48 mg ml−1 for WFWFWF and NWNWNW, respectively. A combinatorial library comprising 64 ligands based on the Ugi reaction was prepared and screened for binding GFP-tagged and non-tagged proteins. Complementary ligands A2C2 and A3C1 were selected for the effective capture of NWNWNW and WFWFWF tagged proteins, respectively, in soluble forms. These affinity pairs displayed 106 M−1 affinity constants and Qmax values of 19.11 ± 2.60 ug g−1 and 79.39 ug g−1 for the systems WFWFWF AND NWNWNW, respectively. GFP fused to the WFWFWF affinity tag was also produced as inclusion bodies, and a refolding-on column strategy was explored using the ligand A4C8, selected from the combinatorial library of ligands but in presence of denaturant agents.

Dias, AMGC, dos Santos R, Iranzo O, Roque ACA.  2016.  Affinity adsorbents for proline-rich peptide sequences: a new role for WW domains. RSC Adv.. 6:68979-68988.: The Royal Society of Chemistry AbstractWebsite

The WW domain derived from human Yes-associated protein (hYAP65_WW) recognizes proline-rich peptides. The structural and chemical robustness of WW domains makes them appealing candidates to target and capture these peptides in affinity purification processes. In this work{,} the chemical synthesis of the hYAP65_WW domain containing a terminal cysteine for oriented coupling onto the chromatographic matrix was successfully achieved by a fragment solution condensation reaction and by incorporation of pseudoproline dipeptide units. Both strategies yielded a hYAP65_WW protein with the characteristic WW domain folding. The purified hYAP65_WW domain was immobilized in a chromatographic matrix and tested for binding to a proline-rich peptide. The adsorbent bound 92 ng of peptide per mg of support and the elution was particularly efficient when employing a low pH or an increase in salt concentration. This work sets the ground for the application of WW domains as affinity reagents towards the capture and elution of peptides and proteins rich in proline sequences.

2015
Carvalho, HF, Roque ACA, Iranzo O, Branco RJF.  2015.  Comparison of the Internal Dynamics of Metalloproteases Provides New Insights on Their Function and Evolution, 2015/09/23. PLoS ONE. 10(9):e0138118-.: Public Library of Science AbstractWebsite

Metalloproteases have evolved in a vast number of biological systems, being one of the most diverse types of proteases and presenting a wide range of folds and catalytic metal ions. Given the increasing understanding of protein internal dynamics and its role in enzyme function, we are interested in assessing how the structural heterogeneity of metalloproteases translates into their dynamics. Therefore, the dynamical profile of the clan MA type protein thermolysin, derived from an Elastic Network Model of protein structure, was evaluated against those obtained from a set of experimental structures and molecular dynamics simulation trajectories. A close correspondence was obtained between modes derived from the coarse-grained model and the subspace of functionally-relevant motions observed experimentally, the later being shown to be encoded in the internal dynamics of the protein. This prompted the use of dynamics-based comparison methods that employ such coarse-grained models in a representative set of clan members, allowing for its quantitative description in terms of structural and dynamical variability. Although members show structural similarity, they nonetheless present distinct dynamical profiles, with no apparent correlation between structural and dynamical relatedness. However, previously unnoticed dynamical similarity was found between the relevant members Carboxypeptidase Pfu, Leishmanolysin, and Botulinum Neurotoxin Type A, despite sharing no structural similarity. Inspection of the respective alignments shows that dynamical similarity has a functional basis, namely the need for maintaining proper intermolecular interactions with the respective substrates. These results suggest that distinct selective pressure mechanisms act on metalloproteases at structural and dynamical levels through the course of their evolution. This work shows how new insights on metalloprotease function and evolution can be assessed with comparison schemes that incorporate information on protein dynamics. The integration of these newly developed tools, if applied to other protein families, can lead to more accurate and descriptive protein classification systems.

Fernandes, CSM, Gonçalves B, Sousa M, Martins DL, Barroso T, Pina AS, Peixoto C, Aguiar-Ricardo A, Roque ACA.  2015.  Biobased Monoliths for Adenovirus Purification. ACS Applied Materials & Interfaces. 7(12):6605-6612., Number 12 AbstractWebsite

Adenoviruses are important platforms for vaccine development and vectors for gene therapy, increasing the demand for high titers of purified viral preparations. Monoliths are macroporous supports regarded as ideal for the purification of macromolecular complexes, including viral particles. Although common monoliths are based on synthetic polymers as methacrylates, we explored the potential of biopolymers processed by clean technologies to produce monoliths for adenovirus purification. Such an approach enables the development of disposable and biodegradable matrices for bioprocessing. A total of 20 monoliths were produced from different biopolymers (chitosan, agarose, and dextran), employing two distinct temperatures during the freezing process (−20 °C and −80 °C). The morphological and physical properties of the structures were thoroughly characterized. The monoliths presenting higher robustness and permeability rates were further analyzed for the nonspecific binding of Adenovirus serotype 5 (Ad5) preparations. The matrices presenting lower nonspecific Ad5 binding were further functionalized with quaternary amine anion-exchange ligand glycidyltrimethylammonium chloride hydrochloride by two distinct methods, and their performance toward Ad5 purification was assessed. The monolith composed of chitosan and poly(vinyl) alcohol (50:50) prepared at −80 °C allowed 100% recovery of Ad5 particles bound to the support. This is the first report of the successful purification of adenovirus using monoliths obtained from biopolymers processed by clean technologies.

Palma, SI, Marciello M, Carvalho A, Veintemillas-Verdaguer S, Morales PM, Roque ACA.  2015.  Effects of phase transfer ligands on monodisperse iron oxide magnetic nanoparticles. Journal of Colloid & Interface Science. 437(1):147–155. AbstractWebsite

Oleic acid coated iron oxide nanoparticles synthesized by thermal decomposition in organic medium are highly monodisperse but at the same time are unsuitable for biological applications. Ligand-exchange reactions are useful to make their surface hydrophilic. However, these could alter some structural and magnetic properties of the modified particles. Here we present a comprehensive study and comparison of the effects of employing either citric acid (CA) or meso-2,3-dimercaptosuccinic acid (DMSA) ligand-exchange protocols for phase transfer of monodisperse hydrophobic iron oxide nanoparticles produced by thermal decomposition of Fe(acac)3 in benzyl ether. We show the excellent hydrodynamic size distribution and colloidal stability of the hydrophilic particles obtained by the two protocols and confirm that there is a certain degree of oxidation caused by the ligand-exchange. CA revealed to be more aggressive towards the iron oxide surface than DMSA and greatly reduced the saturation magnetization values and initial susceptibility of the resulting particles compared to the native ones. Besides being milder and more straightforward to perform, the DMSA ligand exchange protocol produces MNP chemically more versatile for further functionalization possibilities. This versatility is shown through the covalent linkage of gum Arabic onto MNP-DMSA using carboxyl and thiol based chemical routes and yielding particles with comparable properties.

Pina, AS, Dias AMGC, Ustok FI, Khoury GE, Fernandes CSM, Branco RJF, Lowe CR, Roque ACA.  2015.  Mild and cost-effective green fluorescent protein purification employing small synthetic ligands. Journal of Chromatography A. 1418:83-93. AbstractWebsite

Abstract The green fluorescent protein (GFP) is a useful indicator in a broad range of applications including cell biology, gene expression and biosensing. However, its full potential is hampered by the lack of a selective, mild and low-cost purification scheme. In order to address this demand, a novel adsorbent was developed as a generic platform for the purification of \{GFP\} or \{GFP\} fusion proteins, giving \{GFP\} a dual function as reporter and purification tag. After screening a solid-phase combinatorial library of small synthetic ligands based on the Ugi-reaction, the lead ligand (A4C7) selectively recovered \{GFP\} with 94% yield and 94% purity under mild conditions and directly from Escherichia coli extracts. Adsorbents containing the ligand \{A4C7\} maintained the selectivity to recover other proteins fused to GFP. The performance of \{A4C7\} adsorbents was compared with two commercially available methods (immunoprecipitation and hydrophobic interaction chromatography), confirming the new adsorbent as a low-cost viable alternative for \{GFP\} purification.

Palma, SI, Rodrigues CA, Carvalho A, Morales PM, Freitas F, Fernandes AR, Cabral JS, Roque ACA.  2015.  A value-added exopolysaccharide as a coating agent for MRI nanoprobes. Nanoscale. (7):14272-14283. AbstractWebsite

Fucopol, a fucose-containing exopolysaccharide (EPS) produced by the bacterium Enterobacter A47 DSM 23139 using glycerol as a carbon source, was employed as a new coating material for iron oxide magnetic nanoparticles (MNP). The coated particles were assessed as nanoprobes for cell labeling by Magnetic Resonance Imaging (MRI). The MNP were synthesized by a thermal decomposition method and transferred to aqueous medium by ligand-exchange reaction with meso-2,3-dimercaptosuccinic acid (DMSA). Covalent binding of EPS to DMSA-stabilized nanoparticles (MNP-DMSA) resulted in a hybrid magnetic-biopolymeric nanosystem (MNP-DMSA-EPS) with a hydrodynamic size of 170 nm, negative surface charge at physiological conditions and transverse to longitudinal relaxivities ratio, r2/r1, of 148. In vitro studies with two human cell lines (colorectal carcinoma - HCT116 - and neural stem/progenitor cells - ReNcell VM) showed that EPS promotes internalization of nanoparticles in both cell lines. In vitro MRI cell phantoms also showed superior performance of MNP-DMSA-EPS in ReNcell VM, for which iron dose-dependent MRI signal drop was obtained at relatively low iron concentrations (12 - 20 µg Fe/ml) and short incubation time. Furthermore, ReNcell VM multipotency was not affected by culture in the presence of MNP-DMSA or MNP-DMSA-EPS for 14 days. Our study suggests that Fucopol-coated MNP represent useful cell labeling nanoprobes for MRI.

Palma, SICJ, Carvalho A, Silva J, Martins P, Marciello M, Fernandes AR, del Puerto Morales M, Roque ACA.  2015.  Covalent coupling of gum arabic onto superparamagnetic iron oxide nanoparticles for MRI cell labeling: physicochemical and in vitro characterization. Contrast Media & Molecular Imaging. 10:320–328., Number 4 AbstractWebsite

Gum arabic (GA) is a hydrophilic composite polysaccharide derived from exudates of Acacia senegal and Acacia seyal trees. It is biocompatible, possesses emulsifying and stabilizing properties and has been explored as coating agent of nanomaterials for biomedical applications, namely magnetic nanoparticles (MNPs). Previous studies focused on the adsorption of GA onto MNPs produced by co-precipitation methods. In this work, MNPs produced by a thermal decomposition method, known to produce uniform particles with better crystalline properties, were used for the covalent coupling of GA through its free amine groups, which increases the stability of the coating layer. The MNPs were produced by thermal decomposition of Fe(acac)3 in organic solvent and, after ligand-exchange with meso-2,3-dimercaptosuccinic acid (DMSA), GA coating was achieved by the establishment of a covalent bond between DMSA and GA moieties. Clusters of several magnetic cores entrapped in a shell of GA were obtained, with good colloidal stability and promising magnetic relaxation properties (r2/r1 ratio of 350). HCT116 colorectal carcinoma cell line was used for in vitro cytotoxicity evaluation and cell-labeling efficiency studies. We show that, upon administration at the respective IC50, GA coating enhances MNP cellular uptake by 19 times compared to particles bearing only DMSA moieties. Accordingly, in vitro MR images of cells incubated with increasing concentrations of GA-coated MNP present dose-dependent contrast enhancement. The obtained results suggest that the GA magnetic nanosystem could be used as a MRI contrast agent for cell-labeling applications. Copyright © 2015 John Wiley & Sons, Ltd.

Dias, AMGC, Iranzo O, Roque ACA.  2015.  An in silico and chemical approach towards small protein production and application in phosphoproteomics. RSC Adv.. 5:19743-19751.: The Royal Society of Chemistry AbstractWebsite

The human Pin1 WW domain (hPin1_WW) is a 38 residue protein which specifically recognizes ligands rich in proline and phosphorylated in Ser and Thr residues. This work presents a protocol for the improved chemical synthesis and modification of this protein through automated microwave assisted synthesis combined with the incorporation of pseudoproline units in the protein sequence. After purification{,} the protein was characterized by Mass Spectrometry and Circular Dichroism spectroscopy with results comparable to the same WW domain chemically synthesized by other strategies or biologically expressed. The protein was further immobilized on a matrix and tested for the selective binding and mild elution of phosphorylated sequences at Ser{,} Thr and Tyr residues. These results suggest that hPin1_WW is a useful protein scaffold for the purification of phosphorylated species in pTyr and pSer{,} which can be easily produced and modified by chemical methods.

Alves, BM, Borlido L, Rosa SASL, Silva MFF, Aires-Barros MR, Roque ACA, Azevedo AM.  2015.  Purification of human antibodies from animal cell cultures using gum arabic coated magnetic particles. Journal of Chemical Technology & Biotechnology. 90:838–846., Number 5: John Wiley & Sons, Ltd AbstractWebsite

BACKGROUND The emergence of monoclonal antibodies (mAbs) as new biopharmaceutical products requires the development of new purification methods that are not only effective but are able to reduce production costs. To address the problematic recovery of mAbs, gum arabic (GA) coated magnetic particles (MPs) were used for the purification of human antibodies from animal cells supernatants. RESULTS MPs were synthesized via co-precipitation and exhibited a spherical-like physical aspect, with an average hydrodynamic diameter of 473 nm and a zeta potential of –26 mV. The adsorption and elution of IgG on these adsorbents was thoroughly studied. Adsorption of human IgG was enhanced at pH 6, for which a qmax of 244 mg IgG g−1 MPs and Kd of 25 mg L−1 were obtained. Increasing salt concentrations at a basic pH (1 mol L−1 NaCl at pH 11) were found to improve the elution of bound IgG. The MPs were challenged with an artificial protein mixture containing human IgG, albumin, insulin and apo-transferrin. An overall yield of 84% was achieved, retrieving 92% of bound IgG. CONCLUSIONS MPs were successfully used for the capture of monoclonal antibodies from two distinct mammalian cell cultures, a Chinese hamster ovary (CHO) and a hybridoma cell culture supernatants. The elution yields were high, ranging between 84% and 94%, with overall yields ranging from 72% to 88%. Final purities of 85% were reached for hybridoma cell supernatants. © 2014 Society of Chemical Industry

2014
Pina, AS, Batalha IL, Roque ACA.  2014.  Affinity Tags in Protein Purification and Peptide Enrichment: An Overview. Protein Downstream Processing: Design, Development and Application of High and Low-Resolution Methods. (Labrou, Nikolaos, Ed.).:147-168.: Springer Abstract

The reversible interaction between an affinity ligand and a complementary receptor has been widely explored in purification systems for several biomolecules. The development of tailored affinity ligands highly specific towards particular target biomolecules is one of the options in affinity purification systems. However, both genetic and chemical modifications on proteins and peptides widen the application of affinity ligand-tag receptor pairs towards universal capture and purification strategies. In particular, this chapter will focus on two case studies highly relevant for biotechnology and biomedical areas, namely, the affinity tags and receptors employed on the production of recombinant fusion proteins and the chemical modification of phosphate groups on proteins and peptides and the subsequent specific capture and enrichment, a mandatory step before further proteomic analysis.

Dhadge, VL, Hussain A, Azevedo AM, Aires-Barros MR, Roque ACA.  2014.  Boronic acid-modified magnetic materials for antibody purification. J. R. Soc. Interface. 11(91):20130875. AbstractWebsite

Aminophenyl boronic acids can form reversible covalent ester interactions with cis-diol-containing molecules, serving as a selective tool for binding glycoproteins as antibody molecules that possess oligosaccharides in both the Fv and Fc regions. In this study, amino phenyl boronic acid (APBA) magnetic particles (MPs) were applied for the magnetic separation of antibody molecules. Iron oxide MPs were firstly coated with dextran to avoid non-specific binding and then with 3-glycidyloxypropyl trimethoxysilane to allow further covalent coupling of APBA (APBA_MP). When contacted with pure protein solutions of human IgG (hIgG) and bovine serum albumin (BSA), APBA_MP bound 170 ± 10 mg hIgG g−1 MP and eluted 160 ± 5 mg hIgG g−1 MP, while binding only 15 ± 5 mg BSA g−1 MP. The affinity constant for the interaction between hIgG and APBA_MP was estimated as 4.9 × 105 M−1 (Ka) with a theoretical maximum capacity of 492 mg hIgG adsorbed g−1 MP (Qmax), whereas control particles bound a negligible amount of hIgG and presented an estimated theoretical maximum capacity of 3.1 mg hIgG adsorbed g−1 MP (Qmax). APBA_MPs were also tested for antibody purification directly from CHO cell supernatants. The particles were able to bind 98% of IgG loaded and to recover 95% of pure IgG (purity greater than 98%) at extremely mild conditions.

Pina, AS, Lowe CR, Roque ACA.  2014.  Challenges and opportunities in the purification of recombinant tagged proteins. Biotechnology Advances. 32(2):366-381. AbstractWebsite

The purification of recombinant proteins by affinity chromatography is one of the most efficient strategies due to the high recovery yields and purity achieved. However, this is dependent on the availability of specific affinity adsorbents for each particular target protein. The diversity of proteins to be purified augments the complexity and number of specific affinity adsorbents needed, and therefore generic platforms for the purification of recombinant proteins are appealing strategies. This justifies why genetically encoded affinity tags became so popular for recombinant protein purification, as these systems only require specific ligands for the capture of the fusion protein through a pre-defined affinity tag tail. There is a wide range of available affinity pairs “tag-ligand” combining biological or structural affinity ligands with the respective binding tags. This review gives a general overview of the well-established “tag-ligand” systems available for fusion protein purification and also explores current unconventional strategies under development.

Pina, AS, Batalha ÍL, Fernandes CSM, Aoki MA, Roque ACA.  2014.  Exploring the potential of magnetic antimicrobial agents for water disinfection. Water Research. 66:160–168. AbstractWebsite

Industrial and urban activities yield large amounts of contaminated groundwater, which present a major health issue worldwide. Infectious diseases are the most common health risk associated with drinking-water and wastewater remediation is a major concern of our modern society. The field of wastewater treatment is being revolutionized by new nano-scale water disinfection devices which outperform most currently available technologies. In particular, iron oxide magnetic nanoparticles (MNPs) have been widely used in environmental applications due to their unique physical–chemical properties. In this work, poly(ethylene) glycol (PEG)-coated MNPs have been functionalized with (RW)3, an antimicrobial peptide, to yield a novel magnetic-responsive support with antimicrobial activity against Escherichia coli K-12 DSM498 and Bacillus subtilis 168. The magnetic-responsive antimicrobial device showed to be able to successfully disinfect the surrounding solution. Using a rapid high-throughput screening platform, the minimal inhibitory concentration (MIC) was determined to be 500 μM for both strains with a visible bactericidal effect.

Dhadge, VL, Morgado PI, Freitas F, Reis MA, Azevedo AM, Aires-Barros R, Roque ACA.  2014.  An extracellular polymer at the interface of magnetic bioseparations. Journal of the Royal Society Interface. 11(100):20140743. AbstractWebsite

FucoPol, a fucose-containing extracellular polysaccharide (EPS) produced by bacterium Enterobacter A47 using glycerol as the carbon source, was employed as a coating material for magnetic particles (MPs), which were subsequently functionalized with an artificial ligand for the capture of antibodies. The performance of the modified MPs (MP–EPS-22/8) for antibody purification was investigated using direct magnetic separation alone or combined with an aqueous two-phase system (ATPS) composed of polyethylene glycol (PEG) and dextran. In direct magnetic capturing, and using pure protein solutions of human immunoglobulin G (hIgG) and bovine serum albumin (BSA), MP–EPS-22/8 bound 120 mg hIgG g−1 MPs, whereas with BSA only 10 ± 2 mg BSA g−1 MPs was achieved. The hybrid process combining both the ATPS and magnetic capturing leads to a good performance for partitioning of hIgG in the desired phase as well as recovery by the magnetic separator. The MPs were able to bind 145 mg of hIgG g−1 of particles which is quite high when compared with direct magnetic separation. The theoretical maximum capacity was calculated to be 410 ± 15 mg hIgG adsorbed g−1 MPs with a binding affinity constant of 4.3 × 104 M−1. In multiple extraction steps, the MPs bound 92% of loaded hIgG with a final purity level of 98.5%. The MPs could easily be regenerated, recycled and re-used for five cycles with only minor loss of capacity. FucoPol coating allowed both electrostatic and hydrophobic interactions with the antibody contributing to enhance the specificity for the targeted products.

Barroso, T, Casimiro T, Ferraria A, Mattioli F, Aguiar-Ricardo A, Roque ACA.  2014.  Hybrid monoliths for magnetically-driven protein separations. Adv. Funct. Mater.. 24(28):4528–4541. AbstractWebsite

Monoliths represent powerful platforms for isolation of large molecules with high added value. This work presents a hybrid approach for antibody (Ab) capture and release. Using mostly natural polymers and clean processes, it is possible to create macroporous monoliths with well-defined porous networks, tuneable mechanical properties, and easy functionalization with a biomimetic ligand specific for Ab. Magnetic nanoparticles (MNPs) are embedded on the monolith network to confer a controlled magnetic response that facilitates and accelerates Ab recovery in the elution step. The hybrid monolithic systems prepared with agarose or chitosan/poly(vinyl alcohol) (PVA) blends exhibit promising binding capacities of Abs directly from cell-culture extracts (120 ± 10 mg Ab g−1 support) and controlled Ab magnetically-assisted elution yielding 95 ± 2% recovery. Moreover, a selective capture of mAbs directly from cell culture extracts is achieved yielding a final mAb preparation with 96% of purity.

Kadar, E, Batalha ÍL, Fisher A, Roque ACA.  2014.  The interaction of polymer-coated magnetic nanoparticles with seawater. Science of The Total Environment. 487:771-777. AbstractWebsite

Laboratory studies were conducted to evaluate the interaction between bare and polymer-coated magnetic nanoparticles (MNPs) with various environmentally relevant carrying solutions including natural oceanic seawater with and without addition of algal exopolymeric substances (EPS). The MNPs were coated with three different stabilising agents, namely gum Arabic (GA-MNP), dextran (D-MNP) and carboxymethyl-dextran (CMD-MNP). The colloidal stability of the suspensions was evaluated over 48 h and we demonstrated that: (i) hydrodynamic diameters increased over time regardless of carrying solution for all MNPs except the GA-coated ones; however, the relative changes were carrying solution- and coat-dependent; (ii) polydispersity indexes of the freshly suspended MNPs are below 0.5 for all coated MNPs, unlike the much higher values obtained for the uncoated MNPs; (iii) freshly prepared MNP suspensions (both coated and uncoated) in Milli-Q (MQ) water show high colloidal stability as indicated by zeta-potential values below -30 mV, which however decrease in absolute value within 48 h for all MNPs regardless of carrying solution; (iv) EPS seems to "stabilise" the GA-coated and the CMD-coated MNPs, but not the uncoated or the D-coated MNPs, which form larger aggregates within 48 h; (v) despite this aggregation, iron (Fe)-leaching from MNPs is sustained over 48h, but remained within the range of 3-9% of the total iron-content of the initially added MNPs regardless of suspension media and capping agent. The environmental implications of our findings and biotechnological applicability of MNPs are discussed.

Dhadge, VL, Rosa S, Azevedo A, Aires-Barros R, Roque ACA.  2014.  Magnetic Aqueous Two Phase Fishing: An Hybrid Process Technology for Antibody Purification. J. Chromatogr. A. 1339:59-64. AbstractWebsite

The potential to combine aqueous two-phase extraction (ATPE) with magnetic separation was here investigated with the aim of developing a selective non-chromatographic method for the purification of antibodies from cell culture supernatants. Aqueous two-phase systems (ATPS) composed of polyethylene glycol (PEG) and dextran were supplemented with several surface modified magnetic particles (MPs) at distinct salt concentrations. The partition of pure human IgG in the upper and lower phases as well as the amount adsorbed at the MPs surface was investigated, indicating that MPs coated with dextran and gum Arabic established the lowest amount of non-specific interactions. The binding capacity of gum arabic coated particles modified with aminophenyl boronic acid (GA-APBA-MP) was were found to be excellent in combination with the ATPS system, yielding high yields of antibody recovery (92%) and purity (98%) from cell culture supernatants. The presence of MPs in the ATPS was found to speed up phase separation (from 40 to 25 min), to consume a lower amount of MPs (half of the amount needed in magnetic fishing) and to increase the yield and purity of a mAb purified from a cell culture supernatant, when compared with ATPE or magnetic fishing processes alone.

Barroso, T, Branco RJF, Aguiar‐Ricardo A, Roque ACA.  2014.  Structural evaluation of an alternative Protein A biomimetic ligand for antibody purification. Journal of Computer-Aided Molecular Design. 28(1):25-34. AbstractWebsite

Affinity chromatography is one of the most common techniques employed at the industrial-scale for antibody purification. In particular, the purification of human immunoglobulin G (hIgG) has gained relevance with the immobilization of its natural binding counterpart—Staphylococcus aureus Protein A (SpA) or with the recent development of biomimetic affinity ligands, namely triazine-based ligands. These ligands have been developed in order to overcome economic and leaching issues associated to SpA. The most recent triazine-based ligand—TPN-BM, came up as an analogue of 2-(3-amino-phenol)-6-(4-amino-1-naphthol)-4-chloro-sym-triazine ligand also known as ligand 22/8 with improved physico-chemical properties and a greener synthetic route. This work intends to evaluate the potential of TPN-BM as an alternative affinity ligand towards antibody recognition and binding, namely IgG, at an atomic level, since it has already been tested, after immobilization onto chitosan-based monoliths and demonstrated interesting affinity behaviour for this purpose. Herein, combining automated molecular docking and molecular dynamics simulations it was predicted that TPN-BM has high propensity to bind IgG through the same binding site found in the crystallographic structure of SpA_IgG complex, as well as theoretically predicted for ligand 22/8_IgG complex. Furthermore, it was found that TPN-BM established preferential interactions with aromatic residues at the Fab domain (Trp 50, Tyr 53, Tyr 98 and Trp 100), while in the Fc domain the main interactions are based on hydrogen bonds with pH sensitive residues at operational regime for binding and elution like histidines (His 460, His 464, His 466). Moreover, the pH dependence of TPN-BM_IgG complex formation was more evident for the Fc domain, where at pH 3 the protonation state and consequently the charge alteration of histidine residues located at the IgG binding site induced ligand detachment which explains the optimal elution condition at this pH observed experimentally.

Pina, AS, Guilherme M, Pereira AS, Fernandes CSM, Branco RJF, Lowe CR, Roque ACA.  2014.  A tailor made affinity pair “tag-receptor” for the purification of fusion proteins. ChemBioChem. 15(10):1423-35. AbstractWebsite

A novel affinity “tag–receptor” pair was developed as a generic platform for the purification of fusion proteins. The hexapeptide RKRKRK was selected as the affinity tag and fused to green fluorescent protein (GFP). The DNA fragments were designed, cloned in Pet-21c expression vector and expressed in E. coli host as soluble protein. A solid-phase combinatorial library based on the Ugi reaction was synthesized: 64 affinity ligands displaying complementary functionalities towards the designed tag. The library was screened by affinity chromatography in a 96-well format for binding to the RKRKRK-tagged GFP protein. Lead ligand A7C1 was selected for the purification of RKRKRK fusion proteins. The affinity pair RKRKRK-tagged GFP with A7C1 emerged as a promising solution (Ka of 2.45×105 M−1). The specificity of the ligand towards the tag was observed experimentally and theoretically through automated docking and molecular dynamics simulations.

Fernandes, CSM, Pina AS, Dias AMGC, Branco RJF, Roque ACA.  2014.  A theoretical and experimental approach toward the development of affinity adsorbents for GFP and GFP-fusion proteins purification. Journal of Biotechnology. 186:13-20. AbstractWebsite

The green fluorescent protein (GFP) is widely employed to report on a variety of molecular phenomena, but its selective recovery is hampered by the lack of a low-cost and robust purification alternative. This work reports an integrated approach combining rational design and experimental validation toward the optimization of a small fully-synthetic ligand for GFP purification. A total of 56 affinity ligands based on a first-generation lead structure were rationally designed through molecular modeling protocols. The library of ligands was further synthesized by solid-phase combinatorial methods based on the Ugi reaction and screened against Escherichia coli extracts containing GFP. Ligands A4C2, A5C5 and A5C6 emerged as the new lead structures based on the high estimated theoretical affinity constants and the high GFP binding percentages and enrichment factors. The elution of GFP from these adsorbents was further characterized, where the best compromise between mild elution conditions, yield and purity was found for ligands A5C5 and A5C6. These were tested for purifying a model GFP-fusion protein, where ligand A5C5 yielded higher protein recovery and purity. The molecular interactions between the lead ligands and GFP were further assessed by molecular dynamics simulations, showing a wide range of potential hydrophobic and hydrogen-bond interactions.

2013
Barroso, T, Hussain A, Roque ACA, Aguiar‐Ricardo A.  2013.  Functional monolithic platforms: Chromatographic tools for antibody purification. Biotechnology journal. 8(6):671–681. AbstractWebsite

Polymer monoliths are an efficient platform for antibody purification. The use of monoclonal antibodies (mAbs) and engineered antibody structures as therapeutics has increased exponentially over the past few decades. Several approaches use polymer monoliths to purify large quantities of antibody with defined clinical and performance requirements. Functional monolithic supports have attracted a great deal of attention as they offer practical advantages for antibody purification, such as more rapid analysis, smaller sample volume requirements and the opportunity for a greater target molecule enrichment. This review focuses on the development of synthetic and natural polymer-based monoliths for antibody purification. The materials and methods employed in monolith production are discussed, highlighting the properties of each system. We also review the structural characterization techniques available using monolithic systems and their performance under different chromatographic approaches to antibody capture and release. Finally, a summary of monolithic platforms developed for antibody separation is presented, as well as expected trends in research to solve current and future challenges in this field. This review comprises a comprehensive analysis of proposed solutions highlighting the remarkable potential of monolithic platforms.

Barroso, T, Lourenço A, Araújo M, Bonifácio VDB, Roque ACA, Aguiar-Ricardo A.  2013.  A green approach toward antibody purification: a sustainable biomimetic ligand for direct immobilization on (bio)polymeric supports. Journal of Molecular Recognition. 26(12):662-671.
Cerff, M, Scholz A, Franzreb M, Batalha IL, Roque ACA, Posten C.  2013.  In situ magnetic separation of antibody fragments from Escherichia coli in complex media. BMC biotechnology. 13(1):44. AbstractWebsite

Background
In situ magnetic separation (ISMS) has emerged as a powerful tool to overcome process constraints such as product degradation or inhibition of target production. In the present work, an integrated ISMS process was established for the production of his-tagged single chain fragment variable (scFv) D1.3 antibodies (?D1.3?) produced by E. coli in complex media. This study investigates the impact of ISMS on the overall product yield as well as its biocompatibility with the bioprocess when metal-chelate and triazine-functionalized magnetic beads were used.

Results
Both particle systems are well suited for separation of D1.3 during cultivation. While the triazine beads did not negatively impact the bioprocess, the application of metal-chelate particles caused leakage of divalent copper ions in the medium. After the ISMS step, elevated copper concentrations above 120?mg/L in the medium negatively influenced D1.3 production. Due to the stable nature of the model protein scFv D1.3 in the biosuspension, the application of ISMS could not increase the overall D1.3 yield as was shown by simulation and experiments.

Conclusions
We could demonstrate that triazine-functionalized beads are a suitable low-cost alternative to selectively adsorb D1.3 fragments, and measured maximum loads of 0.08?g D1.3 per g of beads. Although copper-loaded metal-chelate beads did adsorb his-tagged D1.3 well during cultivation, this particle system must be optimized by minimizing metal leakage from the beads in order to avoid negative inhibitory effects on growth of the microorganisms and target production. Hereby, other types of metal chelate complexes should be tested to demonstrate biocompatibility. Such optimized particle systems can be regarded as ISMS platform technology, especially for the production of antibodies and their fragments with low stability in the medium. The proposed model can be applied to design future ISMS experiments in order to maximize the overall product yield while the amount of particles being used is minimized as well as the number of required ISMS steps.

Borlido, L, Azevedo AM, Roque ACA, Aires-Barros MR.  2013.  Magnetic separations in biotechnology. Biotechnology Advances. 31(8):1374-1385. AbstractWebsite

Magnetic separations are probably one of the most versatile separation processes in biotechnology as they are able to purify cells, viruses, proteins and nucleic acids directly from crude samples. The fast and gentle process in combination with its easy scale-up and automation provide unique advantages over other separation techniques. In the midst of this process are the magnetic adsorbents tailored for the envisioned target and whose complex synthesis spans over multiple fields of science. In this context, this article reviews both the synthesis and tailoring of magnetic adsorbents for bioseparations as well as their ultimate application.

Borlido, L, Moura L, Azevedo AM, Roque ACA, Aires‐Barros MR, Farinha JPS.  2013.  Stimuli‐Responsive magnetic nanoparticles for monoclonal antibody purification. Biotechnology Journal. 8(6):709–717. AbstractWebsite

Monoclonal antibodies (mAbs) are important therapeutic proteins. One of the challenges facing large-scale production of monoclonal antibodies is the capacity bottleneck in downstream processing, which can be circumvented by using magnetic stimuli-responsive polymer nanoparticles. In this work, stimuli-responsive magnetic particles composed of a magnetic poly(methyl methacrylate) core with a poly(N-isopropylacrylamide-co-acrylic acid) (P(NIPAM-co-AA)) shell cross-linked with N, N'-methylenebisacrylamide were prepared by miniemulsion polymerization. The particles were shown to have an average hydrodynamic diameter of 317 nm at 18°C, which decreased to 277 nm at 41°C due to the collapse of the thermo-responsive shell. The particles were superparamagnetic in behavior and exhibited a saturation magnetization of 12.6 emu/g. Subsequently, we evaluated the potential of these negatively charged stimuli-responsive magnetic particles in the purification of a monoclonal antibody from a diafiltered CHO cell culture supernatant by cation exchange. The adsorption of antibodies onto P(NIPAM-co-AA)-coated nanoparticles was highly selective and allowed for the recovery of approximately 94% of the mAb. Different elution strategies were employed providing highly pure mAb fractions with host cell protein (HCP) removal greater than 98%. By exploring the stimuli-responsive properties of the particles, shorter magnetic separation times were possible without significant differences in product yield and purity.

2012
Santana, SDF, Pina AS, Roque ACA.  2012.   Immobilization of enterokinase on magnetic supports for the cleavage of fusion proteins. Journal of Biotechnology. 161:378–382. AbstractWebsite

Magnetic nanobiocatalysts for tag cleavage on fusion proteins have been prepared by immobilizing
enterokinase (EK) onto iron oxide magnetic nanoparticles coated with biopolymers. Two different
chemistries have been explored for the covalent coupling of EK, namely carbodiimide (EDC coupling)
and maleimide activation (Sulfo coupling). Upon immobilization, EK initial activity lowered but EDC coupling lead to higher activity retention. Regarding the stability ofthe nanobiocatalysts,thesewere recycled
up to ten times with the greater activity losses observed in the first two cycles. The immobilized EK also
proved to cleave a control fusion protein and to greatly simplify the separation of the enzyme from the
reaction mixture.

Ataíde, F, Azevedo C, Clemente JJ, Cunha AE, Freitas F, Reis MAM, Roque ACA, Oliveira R.  2012.  Analysis of oxygen transport enhancement by functionalized magnetic nanoparticles (FMP) in bioprocesses. New Biotechnology. 29S:S75.Website
Cardoso, MM, Peça IN, Roque ACA.  2012.  Antibody-Conjugated Nanoparticles for Therapeutic Applications. Current Medicinal Chemistry. 19(19):3103-3127. AbstractWebsite

A great challenge to clinical development is the delivery of chemotherapeutic agents, known to cause severe toxic effects, directly to diseased sites which increase the therapeutic index whilst minimizing off-target side effects. Antibody-conjugated nanoparticles offer great opportunities to overcome these limitations in therapeutics. They combine the advantages given by the nanoparticles with the ability to bind to their target with high affinity and improve cell penetration given by the antibodies. This specialized vehicle, that can encapsulate several chemotherapeutic agents, can be engineered to possess the desirable properties, allowing overcoming the successive physiological conditions and to cross biological barriers and reach a specific tissue or cell. Moreover, antibody-conjugated nanoparticles have shown the ability to be internalized through receptor-mediated endocytosis and accumulate in cells without being recognized by the P-glycoprotein, one of the main mediators of multi-drug resistance, resulting in an increase in the intracellular concentration of drugs. Also, progress in antibody engineering has allowed the manipulation of the basic antibody structure for raising and tailoring specificity and functionality. This review explores recent developments on active drug targeting by nanoparticles functionalized with monoclonal antibodies (polymeric micelles, liposomes and polymeric nanoparticles) and summarizes the opportunities of these targeting strategies in the therapy of serious diseases (cancer, inflammatory diseases, infectious diseases, and thrombosis).

Barroso, T, Roque ACA, Aguiar-Ricardo A.  2012.  Bioinspired and Sustainable Chitosan Based Monoliths for Antibody Capture and Release. RSC ADV. 2(30):11285-11294. AbstractWebsite

Chitosan-based monoliths activated by plasma technology induced the coupling of a robust biomimetic ligand, previously reported as an artificial Protein A, with high yields while minimizing the environmental impact of the procedure. Due to the high porosity, good mechanical and tunable physicochemical properties of the affinity chitosan-based monoliths, it is possible to achieve high binding capacities (150 ± 10 mg antibody per gram support), and to recover 90 ± 5% of the bound protein with 98% purity directly from cell-culture extracts. Therefore, the chitosan-based monoliths prepared by clean processes exhibit a remarkable performance for the one-step capture and recovery of pure antibodies or other biological molecules with biopharmaceutical relevance.

Sandu, ICA, Schäfer S, Magrini D, Bracci S, Roque ACA.  2012.  Cross-Section and Staining-Based Techniques for Investigating Organic Materials in Painted and Polychrome Works of Art: A Review.. Microscopy and Microanalysis. 18(4):860-875. AbstractWebsite

The article presents a review of the use of cross-section and staining techniques for investigating natural organic materials (mainly proteinaceous and oil-based binders/varnishes) in painted and polychrome artworks, considering the requirements of conservation practice and routine diagnostics. The reviewed literature calls attention to the importance of using cross sections to prepare samples for optical microscopy and to different properties of embedding resins; the most appropriate instrumental conditions for optical microscopy; and the advantages and disadvantages of the most common staining techniques. A few case studies were selected to illustrate the use of autofluorescence (intrinsic fluorescence) and induced fluorescence (using specific staining tests and fluorophore-labeled antibodies) for mapping and identifying organic paint materials in cross sections. New directions of research in cross-section analyses and fluorescence-based techniques for the identification and mapping of artistic materials are presented. The complementary use of different stains on the same cross section, further exploration of intrinsic and induced fluorescence of aged versus fresh materials, and applicability of cross-section observation and staining as complementary methods for assessing the effectiveness of restoration treatments, such as cleaning and consolidation, are discussed in the last section of the article.

Santana, SDF, Dhadge VL, Roque ACA.  2012.  Dextran-Coated Magnetic Supports Modified with a Biomimetic Ligand for IgG Purification. ACS Applied Materials and Interfaces. 4(11):5907–5914. AbstractWebsite

extran-coated iron oxide magnetic particles modified with ligand 22/8, a protein A mimetic ligand, were prepared and assessed for IgG purification. Dextran was chosen as the agent to modify the surface of magnetic particles by presenting a negligible level of nonspecific adsorption. For the functionalization of the particles with the affinity ligand toward antibodies, three methods have been explored. The optimum coupling method yielded a theoretical maximum capacity for human IgG calculated as 568 ± 33 mg/g and a binding affinity constant of 7.7 × 104 M–1. Regeneration, recycle and reuse of particles was also highly successful for five cycles with minor loss of capacity. Moreover, this support presented specificity and effectiveness for IgG adsorption and elution at pH 11 directly from crude extracts with a final purity of 95% in the eluted fraction.

Borlido, L, Azevedo AM, Sousa AG, Oliveira PH, Roque ACA, Aires-Barros MR.  2012.  Fishing human monoclonal antibodies from a CHO cell supernatant with boronic acid magnetic particles. Journal of Chromatography B. 903:163-170. AbstractWebsite

In this work we have evaluated the potential of boronic acid functionalized magnetic particles for the one-step capture of a human monoclonal antibody (mAb) from a Chinese hamster ovary (CHO) cell culture supernatant. For comparison, Protein A coated magnetic particles were also used. The most important factor influencing the overall process yield and product purity in boronic acid particles was found to be the binding pH. Basic pH values promoted higher purities while resulting in decreased yields due to the competing effects of molecules such as glucose and lactate present in the cell culture supernatant. After optimization, the particles were successfully used in a multi-cycle purification process of the mAb from the CHO feedstock. Boronic acid particles were able to achieve an average overall yield of 86% with 88% removal of CHO host cell proteins (HCP) when the binding was performed at pH 7.4, while at pH 8.5 these values were 58% and 97%, respectively. In both cases, genomic DNA removal was in excess of 97%. Comparatively, Protein A particles recorded an average overall yield of 80% and an HCP removal greater than 99%. The adsorption of the mAb to the boronic acid particles was shown to be mediated by strong affinity interactions. Overall, boronic acid based purification processes can offer a cost-effective alternative to Protein A as the direct capturing step from the mammalian cell culture.

Sandu, ICA, Roque ACA, Matteini P, Schäfer S, Agati G, Correia CR, Viana JFFP.  2012.  Fluorescence recognition of proteinaceous binders in works of art by a novel integrated system of investigation. Microscopy Research and Technique. 75(3):316-24. AbstractWebsite

Fluorescence microscopy and microspectrofluorometry are important tools in the characterization and identification of proteins, offering a great range of applications in conservation science. Because of their high selectivity and sensitivity, the combination of these techniques can be exploited for improved recognition and quantification of proteinaceous binders in paintings and polychromed works of art. The present article explores an analytical protocol integrating fluorescence microscopy and fluorometry for both identification and mapping of proteinaceous binders (in particular egg and glues) in paint samples. The study has been carried out on historically accurate reconstructions simulating the structure and composition of tempera and oil paints containing these binders. To assess the spatial distribution of specific proteins within the paint layers, cross-sections from the reconstructions were analyzed by fluorescence imaging after staining with an exogenous fluorophore. Reference fluorescence spectra for each layer were acquired by a multichannel spectral analyzer and compared after Gaussian deconvolution. The results obtained demonstrated the effectiveness of the integrated protocol, highlighting the potential for the use of fluorescent staining coupled with microspectrofluorometry as a routine diagnostic tool in conservation science. The current work creates a set of fully characterized reference samples for further comparison with those from actual works of art.

Batalha, IL, Lowe CR, Roque ACA.  2012.  Platforms for enrichment of phosphorylated proteins and peptides in proteomics. Trends in Biotechnology. 30(2):100-110. AbstractWebsite

Protein phosphorylation is a complex and highly dynamic process involved in numerous biological events. Abnormal phosphorylation is one of the underlying mechanisms for the development of cancer and metabolic disorders. The identification and absolute quantification of specific phospho-signatures can help elucidate protein functions in signaling pathways and facilitate the development of new and personalized diagnostic and therapeutic tools. This review presents a variety of strategies currently utilized for the enrichment of phosphorylated proteins and peptides before mass spectrometry analysis during proteomic studies. The investigation of specific affinity reagents, allied to the integration of different enrichment processes, is triggering the development of more selective, rapid and cost-effective high-throughput automated platforms.

Branco, RJF, Dias AMGC, Roque ACA.  2012.  Understanding the molecular recognition between antibody fragments and protein A biomimetic ligand. Journal of Chromatography A. 1244:106-115. AbstractWebsite

Affinity chromatography with protein A from Staphylococcus aureus (SpA) is the most widespread and
accepted methodology for antibody capture during the downstream process of antibody manufacturing.
A triazine based ligand (ligand 22/8) was previously developed as an inexpensive and robust alternative
to SpA chromatography (Li et al. [12] and Teng et al. [11]). Despite the experimental success, there is no
structural information on the binding modes of ligand 22/8 to antibodies, namely to Immunoglobulin G
(IgG) molecules and fragments. In this work, we addressed this issue by a molecular docking approach
allied to molecular dynamics simulations. Theoretical results confirmed the preference of the synthetic
ligand to bind IgG through the binding site found in the crystallographic structure of the natural complex
between SpA and the Fc fragment of IgG. Our studies also suggested other unknown “hot-spots” for
specific binding of the affinity ligand at the hinge between VH and CH1 domains of Fab fragment. The best
docking poses were further analysed by molecular dynamics studies at three different protonation states
(pH 3, 7 and 11). The main interactions between ligand 22/8 and the IgG fragments found at pH 7 were
weaker at pH 3 and pH 11 and in these conditions the ligand start losing tight contact with the binding
site, corroborating the experimental evidence for protein elution from the chromatographic adsorbents
at these pH conditions.

2011
Borlido, L, Azevedo AM, Roque ACA, Aires-Barros MR.  2011.  Potential of boronic acid functionalized magnetic particles in the adsorption of human antibodies under mammalian cell culture conditions. Journal of Chromatography A. 1218(43):7821-7827. AbstractWebsite

In this work, we systematically evaluated the potential of using boronic acid functionalized magnetic particles in the capturing of human immunoglobulin G under typical mammalian cell culture conditions. For comparison, Protein A coated magnetic particles were also used. The binding pH was found to significantly influence the adsorption isotherms of boronic acid particles with the higher capacities (0.216 g IgG/g support) being observed at pH 7.4. Comparatively, this value was 0.109 g IgG/g support, for Protein A particles under the same conditions. Both particles revealed very fast adsorption kinetics with more than 70% of the maximum binding capacity being achieved in a few seconds. The effect of glucose and lactate, which are known to interact with boronic acid, was evaluated. For glucose, the binding capacity was significantly influenced by the pH and decreased as pH increased. At pH 9.5, a 70% lower binding capacity was observed for glucose concentrations as low as 0.5 g/l. The effect of lactate was less pronounced and almost pH independent reaching at most 20% decrease in binding capacity. Nevertheless, the effect of both molecules was always lower at pH 7.4. The optimization of the elution conditions enabled complete recovery of bound IgG from boronic acid particles using 50mM Tris-HCl, 200 mM sorbitol, 200 mM NaCl at pH 8.5.

Dias, AMGC, Hussain A, Marcos AS, Roque ACA.  2011.  A biotechnological perspective on the application of iron oxide magnetic colloids modified with polysaccharides. Biotechnology Advances. 29:142–155., Number 1 AbstractWebsite

Iron oxide magnetic nanoparticles {(MNPs)} alone are suitable for a broad spectrum of applications, but the low stability and heterogeneous size distribution in aqueous medium represent major setbacks. These setbacks can however be reduced or diminished through the coating of {MNPs} with various polymers, especially biopolymers such as polysaccharides. Polysaccharides are biocompatible, non-toxic and renewable; in addition, they possess chemical groups that permit further functionalization of the {MNPs.} Multifunctional entities can be created through decoration with specific molecules e.g. proteins, peptides, drugs, antibodies, biomimetic ligands, transfection agents, cells, and other ligands. This development opens a whole range of applications for iron oxide nanoparticles. In this review the properties of magnetic structures composed of {MNPs} and several polysaccharides {(Agarose}, Alginate, Carrageenan, Chitosan, Dextran, Heparin, Gum Arabic, Pullulan and Starch) will be discussed, in view of their recent and future biomedical and biotechnological applications.

2010
Batalha, IL, Hussain A, Roque ACA.  2010.  Gum Arabic coated magnetic nanoparticles with affinity ligands specific for antibodies, oct. Journal of Molecular Recognition. 23:462–471., Number 5 AbstractWebsite

A novel magnetic support based on gum Arabic {(GA)} coated iron oxide magnetic nanoparticles {(MNP)} has been endowed with affinity properties towards immunoglobulin G {(IgG)} molecules. The success of the in situ triazine ligand synthesis was confirmed by fluorescence assays. Two synthetic ligands previously developed for binding to {IgG}, named as ligand 22/8 (artificial Protein A) and ligand 8/7 (artificial Protein L) were immobilized on to {MNPs} coated with {GA} {(MNP\_GA).} The dimension of the particles core was not affected by the surface functionalization with {GA} and triazine ligands. The hydrodynamic diameters of the magnetic supports indicate that the coupling of {GA} leads to the formation of larger agglomerates of particles with about 1 microm, but the introduction of the triazine ligands leads to a decrease on {MNPs} size. The non-functionalized {MNP\_GA} bound 28 mg {IgG/g}, two times less than bare {MNP} (60 mg {IgG/g).} {MNP\_GA} modified with ligand 22/8 bound 133 mg {IgG/g} support, twice higher than the value obtained for ligand 8/7 magnetic adsorbents (65 mg/g). Supports modified with ligand 22/8 were selected to study the adsorption and the elution of {IgG.} The adsorption of human {IgG} on this support followed a Langmuir behavior with a Q(máx) of 344 mg {IgG/g} support and K(a) of 1.5 x 10(5) M. The studies on different elution conditions indicated that although the 0.05 M citrate buffer {(pH} 3) presented good recovery yields (elution 64% of bound protein), there was occurrence of iron leaching at this acidic {pH.} Therefore, a potential alternative would be to elute bound protein with a 0.05 M {glycine-NaOH} {(pH} 11) buffer.

Barroso, T, Temtem M, Hussain A, Aguiar-Ricardo A, Roque ACA.  2010.  Preparation and characterization of a cellulose affinity membrane for human immunoglobulin G (IgG) purification, feb. Journal of Membrane Science. 348:224–230., Number 1-2 AbstractWebsite

This paper reports the design, preparation and characterization of cellulose affinity membranes for antibody purification using a new methodology. Cellulose membranes were prepared from polymer-ionic liquid solutions, namely 1-butyl-3-methylimidazolium chloride {([BMIM][Cl])}, by the water induced phase inversion process. After functionalization with a synthetic ligand 2-(3-aminophenol)-6-(4-amino-1-naphthol)-4-chloro-s-triazine (ligand 22/8), these were evaluated as affinity supports for human immunoglobulin G {(IgG).} Membranes were characterized in terms of morphology {(SEM)}, porosity (mercury porosimetry), hydrophilicity (contact angle measurement), transport properties (permeability) and mechanical performance {(DMA).} Membranes prepared with varying cellulose contents (5 and 10&\#xa0;wt.% cellulose in ionic liquid solutions) lead to films with different properties. The 10&\#xa0;wt.% cellulose membrane presented enhanced morphological and mechanical properties, however, the morphology of this membrane was significantly altered after ligand coupling. Adsorption isotherms for human {IgG} onto 10&\#xa0;wt.% matrix activated with ligand 22/8 were obtained. Preliminary results showed that the bovine serum albumin {(BSA)}, a model impurity, did not adsorb onto the membrane while up to 6&\#xa0;mg {IgG/g} was bound and 2&\#xa0;mg {IgG/g} recovered.

Bicho, A, Peça IN, Roque ACA, Cardoso MM.  2010.  Anti-CD8 conjugated nanoparticles to target mammalian cells expressing CD8. International Journal of Pharmaceutics. 399:80–86., Number 1-2 AbstractWebsite

This work aimed at the development of targeted drug delivery systems using nanoparticles fused with antibodies. The antibody anti-human {CD8} was coupled onto {PLGA} nanoparticles, and the ability of these particles to specifically target cells expressing {CD8} was studied. The obtained particles were found to be of spherical shape exhibiting a size between 350 and 600 nm. In vitro experiments with different cellular cultures {(TE671}, {CHO} and {HEK293)} using unmodified nanoparticles containing rhodamine have shown that particles were present on their surface within 48 h of incubation. In vitro tests using {anti-CD8} conjugated nanoparticles in {CHO} cell cultures indicated that all transfected cells which express {CD8} show these particles on their surface within 1h of incubation. These results demonstrated that, in a shorter time, the produced particles can target cells expressing {CD8} on their surface which offers the ability to reduce drug side effects. The antibody-coupled nanoparticles represent a promising approach to improve the efficacy of active targeting for lymphoblastic leukaemia therapy.

Pina, AS, Lowe CR, Roque ACA.  2010.  Comparison of Fluorescence Labelling Techniques for the Selection of Affinity Ligands from Solid-Phase Combinatorial Libraries. Separation Science and Technology. 45:2187–2193., Number 15 Abstract

This study reports the comparison of fluorimetric techniques (fluorescence microscopy and spectrofluorimetry on a 96-well format) for the on-bead screening of combinatorial libraries of affinity ligands for chromatographic separations. Two solid-phase libraries of synthetic ligands based on distinct scaffolds were synthesized by combinatorial chemistry. The libraries comprising ligands representing different hydrophobic/hydrophilic properties and sizes were tested for binding to randomly selected biomolecules (labelled with a fluorophore). Fluorescence microscopy was revealed to be a reliable and reproducible technique for the detection of lead ligands which strongly bound the target biomolecule. Results obtained by fluorescence intensity measurements in a 96-well format were less consistent, mainly due to challenges related with the accurate dispensing of the solid support.

Ferreira, IMPLV, Pinho O, Monteiro D, Faria S, Cruz S, Perreira A, Roque ACA, Tavares P.  2010.  Short communication: effect of kefir grains on proteolysis of major milk proteins. Journal of Dairy Science. 93:27–31., Number 1 AbstractWebsite

The effect of kefir grains on the proteolysis of major milk proteins in milk kefir and in a culture of kefir grains in pasteurized cheese whey was followed by reverse {phase-HPLC} analysis. The reduction of kappa-, alpha-, and beta-caseins {(CN)}, alpha-lactalbumin {(alpha-LA)}, and beta-lactoglobulin {(beta-LG)} contents during 48 and 90 h of incubation of pasteurized milk {(100mL)} and respective cheese whey with kefir grains (6 and 12 g) at 20 degrees C was monitored. Significant proteolysis of {alpha-LA} and kappa-, alpha-, and beta-caseins was observed. The effect of kefir amount (6 and 12 {g/100mL)} was significant for {alpha-LA} and alpha- and {beta-CN.} {alpha-Lactalbumin} and {beta-CN} were more easily hydrolyzed than {alpha-CN.} No significant reduction was observed with respect to {beta-LG} concentration for 6 and 12 g of kefir in {100mL} of milk over 48 h, indicating that no significant proteolysis was carried out. Similar results were observed when the experiment was conducted over 90 h. Regarding the cheese whey kefir samples, similar behavior was observed for the proteolysis of {alpha-LA} and {beta-LG:} {alpha-LA} was hydrolyzed between 60 and 90% after 12h (for 6 and 12 g of kefir) and no significant {beta-LG} proteolysis occurred. The proteolytic activity of lactic acid bacteria and yeasts in kefir community was evaluated. Kefir milk prepared under normal conditions contained peptides from proteolysis of {alpha-LA} and kappa-, alpha-, and beta-caseins. Hydrolysis is dependent on the kefir:milk ratio and incubation time. {beta-Lactoglobulin} is not hydrolyzed even when higher hydrolysis time is used. Kefir grains are not appropriate as adjunct cultures to increase {beta-LG} digestibility in whey-based or whey-containing foods.

Bicho, A, Roque ACA, Cardoso AS, Domingos P, Batalha ÍL.  2010.  In vitro studies with mammalian cell lines and gum arabic‐coated magnetic nanoparticles. Journal of Molecular Recognition. 23:536–542., Number 6 AbstractWebsite

Iron oxide magnetic nanoparticles {(MNPs)} were synthesized by the chemical co-precipitation method and coated with gum arabic {(GA)} by physical adsorption and covalent attachment. Cultures of mammalian cell lines {(HEK293}, {CHO} and {TE671)} were grown in the presence of uncoated and {GA-coated} {MNPs.} Cellular growth was followed by optical microscopy in order to assess the proportion of cells with particles, alterations in cellular density and the presence of debris. The in vitro assays demonstrated that cells from different origins are affected differently by the presence of the nanoparticles. Also, the methods followed for {GA} coating of {MNPs} endow distinct surface characteristics that probably underlie the observed differences when in contact with the cells. In general, the nanoparticles to which the {GA} was adsorbed had a smaller ability to attach to the cells' surface and to compromise the viability of the cultures. Copyright © 2010 John Wiley & Sons, Ltd.

2009
Pina, AS, Roque ACA.  2009.  Studies on the molecular recognition between bioactive peptides and angiotensin-converting enzyme, apr. Journal of Molecular Recognition. 22:162–168., Number 2 AbstractWebsite

High blood pressure or hypertension is a condition affecting many individuals and represents a controllable risk factor for cardiovascular diseases such as coronary heart disease and stroke. A non-pharmacological approach to manage these includes the application of food components with antihypertensive activity. Milk protein-derived peptides have been exploited as natural hypotensive agents, namely the peptides {Val-Pro-Pro} {(VPP)} and {Ile-Pro-Pro} {(IPP)}, already commercialized in functional foods as a potential alternative to synthetic drugs. These bioactive peptides inhibit in vitro and in vivo the Angiotensin I-converting enzyme {(ACE)}, a protein with an important role in blood pressure regulation. In this work, we attempted to elucidate the possible mode of interaction between the peptides and {ACE}, including mechanisms of binding to the cofactor Zn2+, and further contrast this with the known mode of inhibition exerted by synthetic drugs {(Captopril}, Enalaprilat and Lisinopril). The bioactive peptide {Ala-Leu-Pro-Met-His-Ile-Arg} {(ALPMHIR)}, also known to inhibit the enzyme {ACE} but with a lower efficiency than {VPP} and {IPP}, was utilized in the docking studies for comparison. It was observed that the best docking poses obtained for {VPP} and {IPP} were located at the {ACE} catalytic site with very high resemblance to the drugs mode of interaction, including the coordination with Zn2+. As for {ALPMHIR}, the best docking poses were located in the narrow {ACE} channel outside the catalytic site, representing higher affinity energies and fewer resemblances with the interaction established by drugs.

Roque, ACA, Bispo S, Pinheiro ARN, Antunes JMA, Gonçalves D, Ferreira HA.  2009.  Antibody immobilization on magnetic particles. Journal of Molecular Recognition. 22:77–82., Number 2 AbstractWebsite

Magnetic particles {(MNPs)} offer attractive possibilities in biotechnology. {MNPs} can get close to a target biological entity, as their controllable sizes range from a few nanometres up to tens of nanometres, and their surface can be modified to add affinity and specificity towards desired molecules. Additionally, they can be manipulated by an external magnetic field gradient. In this work, the study of ferric oxide {(Fe3O4)} {MNPs} with different coating agents was conducted, particularly in terms of strategies for antibody attachment at the surfaces (covalent and physical adsorption) and the effects of blocking buffer composition and incubation times on the specific and non-specific interactions observed. The considered biological model system consisted of a coating antibody (goat {IgG)}, bovine serum albumin {(BSA)} as blocking agent, and a complementary antibody labelled with {FITC} (anti-goat {IgG).} The detection of antibody binding was followed by fluorescence microscopy and the intensity of the signals quantified. The ratio between the mean grey values of negative and positive controls, as well as the maximum intensity attainable in positive controls, were considered in the evaluation of the assays efficiency. The covalent immobilization of the coating antibody was more successful as opposed to protein adsorption. For covalent immobilization, silica-coated {MNPs}, a 5% (w/v) concentration of {BSA} in the blocking buffer and incubation times of 1 h produced the best results in terms of assay sensitivity. However, when conducting the assay for incubation periods of 10 min, the fluorescence signal was reduced by 44% but the assay specificity was maintained.

Hussain, A, Pina AS, Roque ACA.  2009.  Bio-recognition and detection using liquid crystals. Biosensors and Bioelectronics. 25:1–8., Number 1 AbstractWebsite

Liquid crystals {(LCs)} are used extensively by the electronics industry as display devices. Advances in the understanding of the liquid crystalline phase and the chemistry therein lead to the development of {LC} exhibiting faster switching speed with greater twist angle. This in turn lead to the emergence of liquid crystal displays, rendering dial-and-needle based displays (such as those used in various meters) and cathode ray tubes obsolete. In this article, we review the history of {LC} and their emergence as an invaluable material for display devices and the more recent discovery of their use as sensing elements in biosensors. This new application of {LC} as tools in the development of fast and simple biosensors is envisaged to gain more importance in the foreseeable future.

Roque, ACA, Bicho A, Batalha IL, Cardoso AS, Hussain A.  2009.  Biocompatible and bioactive gum Arabic coated iron oxide magnetic nanoparticles. Journal of Biotechnology. 144:313–320., Number 4 AbstractWebsite

The surface modification of iron oxide magnetic nanoparticles {(MNPs)} with gum Arabic {(GA)} via adsorption and covalent coupling was studied. The adsorption of {GA} was assessed during {MNP} chemical synthesis by the co-precipitation method {(MNP\_GA)}, and after {MNP} synthesis on both bare magnetite and {MNP\_GA.} The covalent immobilization of {GA} at the surface of aldehyde-activated {(MNP\_GAAPTES)} or aminated {MNPs} {(MNP\_GAEDC)} was achieved through free terminal amino and carboxylate groups from {GA.} The presence of {GA} at the surface of the {MNPs} was confirmed by {FTIR} and by the quantification of {GA} by the bicinchoninic acid test. Results indicated that the maximum of {GA} coating was obtained for the covalent coupling of {GA} through its free carboxylate groups {(MNP\_GAEDC)}, yielding a maximum of 1.8&\#xa0;g of {GA} bound/g of dried particles. The hydrodynamic diameter of {MNPs} modified with {GA} after synthesis resulted in the lowest values, in opposition to the {MNPs} co-precipitated with {GA} which presented the tendency to form larger aggregates of up to 1&\#xa0;μm. The zeta potentials indicate the existence of negatively charged surfaces before and after {GA} coating. The potential of the {GA} coated {MNPs} for further biomolecule attachment was assessed through anchorage of a model antibody to aldehyde-functionalized {MNP\_GA} and its subsequent detection with an {FITC} labeled anti-antibody.

Pina, AS, Hussain A, Roque ACA.  2009.  An historical overview of drug discovery. Ligand-Macromolecule Interactions in Drug Discovery. (Roque, A. C. A., Ed.).:3-12., USA: Humana Press Inc. Abstract

Drug Discovery in modern times straddles three main periods. The first notable period can be traced to the nineteenth century where the basis of drug discovery relied on the serendipity of the medicinal chemists. The second period commenced around the early twentieth century when new drug structures were found, which contributed for a new era of antibiotics discovery. Based on these known structures, and with the development of powerful new techniques such as molecular modelling, combinatorial chemistry, and automated high-throughput screening, rapid advances occurred in drug discovery towards the end of the century. The period also was revolutionized by the emergence of recombinant DNA technology, where it became possible to develop potential drugs target candidates. With all the expansion of new technologies and the onset of the "Omics" revolution in the twenty-first century, the third period has kick-started with an increase in biopharmaceutical drugs approved by FDA/EMEA for therapeutic use.

Roque, ACA.  2009.  Ligand-Macromolecule Interactions in Drug Discovery. , U.S.A.: Methods in Molecular Biology, Humana Press Inc.Website
2008
Roque, ACA, Wilson OC.  2008.  Adsorption of gum Arabic on bioceramic nanoparticles. Materials Science & Engineering C.- Biomimetic and Supramolecular Systems. 28:443–447., Number 3 Abstract

n/a

2007
Roque, ACA, Lowe CR.  2007.  Affinity chromatography: History, Perspectives, Limitations and Prospects. Affinity Chromatography: Methods and Protocols. (M. Zachariou, Ed.).:1-23., U.S.A.: Humana Press Inc. Abstract

Biomolecule separation and purification has until very recently steadfastly remained one of the more empirical aspects of modern biotechnology. Affinity chromatography, one of several types of adsorption chromatography, is particularly suited for the efficient isolation of biomolecules. This technique relies on the adsorbent bed material that has biological affinity for the substance to be isolated. This review is intended to place affinity chromatography in historical perspective and describe the current status, limitations and future prospects for the technique in modern biotechnology.

Roque, ACA, Silva CSO, Taipa ÂM.  2007.  Affinity-based methodologies and ligands for antibody purification: Advances and perspectives. Journal of Chromatography A. 1160:44–55., Number 1-2 AbstractWebsite

Many successful, recent therapies for life-threatening diseases such as cancer and rheumatoid arthritis are based on the recognition between native or genetically engineered antibodies and cell-surface receptors. Although naturally produced by the immune system, the need for antibodies with unique specificities and designed for single application, has encouraged the search for novel antibody purification strategies. The availability of these products to the end-consumer is strictly related to manufacture costs, particularly those attributed to downstream processing. Over the last decades, academia and industry have developed different types of interactions and separation techniques for antibody purification, affinity-based strategies being the most common and efficient methodologies. The affinity ligands utilized range from biological to synthetic designed molecules with enhanced resistance and stability. Despite the successes achieved, the purification “paradigm” still moves interests and efforts in the continuous demand for improved separation performances. This review will focus on recent advances and perspectives in antibody purification by affinity interactions using different techniques, with particular emphasis on affinity chromatography.

Ferreira, IMPLVO, Eça R, Pinho O, Tavares P, Pereira A, Roque AC.  2007.  Development and Validation of an HPLC/UV Method for Quantification of Bioactive Peptides in Fermented Milks. Journal of Liquid Chromatography & Related Technologies. 30:2139–2147., Number 14 Abstract

The simultaneous separation and quantification of two casein peptides {(IPP}, {VPP)} presenting potent inhibitory activity of angiotensin-converting-enzyme {(ACE)} and casein in fermented milks was developed. Gradient elution was carried out at a flow-rate of 1 {mL/min}, using a mixture of two solvents. Solvent A was 0.1% {TFA} in water and solvent B was acetonitrile-water-trifluoracetic acid 95:5:0.1. The effluent was monitored by {UV} detector at 214 nm. Calibration curves were constructed in the interval of 0.01-1.0 {mg/mL} for {VPP}, 0.005-1.0 {mg/mL} for {IPP}, and 0.05-3.0 {mg/mL} for casein. R2 invariably exceeded 0.999. The detection limits were 0.004 for {VPP}, 0.002 {mg/mL} for {IPP}, and 0.02 {mg/mL} for casein. Repeatability of the method was evaluated by six consecutive injections of two standard solutions containing {VPP}, {IPP}, and casein. The {RSD} values for concentration were all below 5.08%. Recovery studies were carried out to determine the accuracy of the method. Recoveries ranged between 88 and 98.2%. The methodology was applied, not only, for the monitorization of {VPP}, {IPP}, and casein in commercial fermented milks labeled as presenting antihypertensive properties, but also, in milk with different degrees of fermentation by L. Helveticus, and in other commercial functional fermented milks, such as, those presenting cholesterol lowering properties.

Roque, ACA, Lowe CR.  2007.  Rationally designed ligands for use in Affinity Chromatography: An artificial Protein L. Affinity Chromatography: Methods and Protocols. (M. Zachariou, Ed.).:93-110., U.S.A.: Humana Press Inc. Abstract

Synthetic affinity ligands can circumvent the drawbacks of natural immunoglobulin (Ig)-binding proteins by imparting resistance to chemical and biochemical degradation and to in situ sterilization, as well as ease and low cost of production. Protein L (PpL), isolated from Peptostreptococcus magnus strains, interacts with the Fab (antigen-binding fragment) portion of Igs, specifically with kappa light chains, and represents an almost universal ligand for the purification of antibodies. The concepts of rational design and solid-phase combinatorial chemistry were used for the discovery of a synthetic PpL mimic affinity ligand. The procedure presented in this chapter represents a general approach with the potential to be applied to different systems and target proteins.