In situ magnetic separation of antibody fragments from Escherichia coli in complex media.

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

Dipicolinic acid as a novel spore-inspired excipient for antibody formulation

Ionic excipients are commonly used in aqueous therapeutic monoclonal antibody (mAb) formulations. Novel excipients are of industrial interest, with a recent focus on Arg salt forms and their application as viscosity reducing and stabilizing additives. Here, we report that the calcium salt of dipicolinic acid (DPA, pyridine-2,6-dicarboxylic acid), uniquely present in nature in the core of certain bacterial spores, reduces the viscosity of a mAb formulated at 150mg/mL, below that achieved by Arg hydrochloride at the same concentration (10mM). DPA also reduced the reversible phase separation of the same formulation, which characteristically occurs for this mAb upon cooling to 4°C. Differential scanning calorimetry and differential scanning fluorimetry did not reveal a conformation destabilisation of the mAb in the presence of 10mM DPA, or by the related quinolinic acid (QA, pyridine-2,3-dicarboxylic acid). However, fluorescence spectrophotometry did reveal localised (aromatic) conformational changes to the mAb attributed to DPA, dependent on the salt form. While precise mechanisms of action remain to be identified, our preliminary data suggest that these DPA salts are worthy of further investigation as novel ionic excipient for biologics formulation.MedImmun

Sobrecarga de ferro transfusional em portadores de anemia falciforme: comparação entre ressonância magnética e ferritina sérica

OBJETIVO: Identificar variáveis preditoras de sobrecarga de ferro em portadores de anemia falciforme e correlacionar indicadores bioquímicos e imaginológicos. MATERIAiS E MÉTODOS: Foi realizado estudo transversal envolvendo 32 portadores de anemia falciforme, que foram submetidos a dosagem sérica de ferro, ferritina e a ressonância magnética do fígado. Foram realizadas cinco sequências gradiente-eco e uma spin-eco. A intensidade de sinal foi obtida em cada sequência pelas médias das regiões de interesse no fígado e musculatura paravertebral para obter a razão da intensidade de sinal (RIS) fígado/músculo. A partir da RIS foi obtida a concentração hepática estimada de ferro (CHEF) pela fórmula: e[5,808 - (0,877 × T2*) - (1,518 × PI)], onde T2* é a RIS na sequência com TE de 13 ms e PI é a RIS da sequência com ponderação intermediária. Os pacientes foram agrupados segundo o regime de transfusão de hemácias (regulares mensais versus esporádicas). RESULTADOS: Os grupos transfusionais foram comparados pelas variáveis clínico-laboratoriais, sendo significativas as diferenças entre RIS, CHEF e ferritina sérica: o grupo que recebeu transfusões regulares apresentou sobrecarga de ferro hepático mais intensa. CONCLUSÃO: A ressonância magnética foi ferramenta eficiente para avaliação de sobrecarga hepática de ferro em portadores de anemia falciforme

Platforms for enrichment of phosphorylated proteins and peptides in proteomics

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. © 2011 Elsevier Ltd

Gum arabic coated magnetic nanoparticles with affinity ligands specific for antibodies

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 1μm, but the introduction of the triazine ligands leads to a decrease on MNPs size. The non-functionalized MNP_GA bound 28mg IgG/g, two times less than bare MNP (60mg IgG/g). MNP_GA modified with ligand 22/8 bound 133mg 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 Qmáx of 344mg IgG/g support and Ka of 1.5×10,5 M. The studies on different elution conditions indicated that although the 0.05M 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 0.05M glycine-NaOH (pH 11) buffer. ©2010 John Wiley & Sons, Ltd

The interaction of polymer-coated magnetic nanoparticles with seawater

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 48 h, 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

Mimicking nature: Phosphopeptide enrichment using combinatorial libraries of affinity ligands

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 (α- and β-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

β-Hairpins as peptidomimetics of human phosphoprotein-binding domains

Phosphoprotein-binding domains interact with cognate phosphorylated targets ruling several biological processes. The impairment of such interactions is often associated with disease development, namely cancer. The breast cancer susceptibility gene 1 (BRCA1) C-terminal (BRCT) domain is involved in the control of complex signaling networks of the DNA damage response. The capture and identification of BRCT-binding proteins and peptides may be used for the development of new diagnostic tools for diseases with abnormal phosphorylation profiles. Here we show that designed cyclic β-hairpin structures can be used as peptidomimetics of the BRCT domain, with high selectivity in binding to a target phosphorylated peptide. The amino acid residues and spatial constraints involved in the interaction between a phosphorylated peptide (GK14-P) and the BRCT domain were identified and crafted onto a 14-mer β-hairpin template in silico. Several cyclic peptides models were designed and their binding towards the target peptide and other phosphorylated peptides evaluated through virtual screening. Selected cyclic peptides were then synthesized, purified and characterized. The high affinity and selectivity of the lead cyclic peptide towards the target phosphopeptide was confirmed, and the possibility to capture it using affinity chromatography demonstrated. This work paves the way for the development of cyclic β-hairpin peptidomimetics as a novel class of affinity reagents for the highly selective identification and capture of target molecules

Biocompatible and bioactive gum Arabic coated iron oxide magnetic nanoparticles

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 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 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. © 2009 Elsevier B.V. All rights reserved

In vitro studies with mammalian cell lines and gum arabic-coated magnetic nanoparticles

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. © 2010 John Wiley & Sons, Ltd