ABSTRACT
Magnetic separation is a promising alternative to chromatography for enhancing the downstream processing (DSP) of monoclonal antibodies (mAbs). However, there is a lack of efficient magnetic particles for successful application. Aiming to fill this gap, we demonstrate the suitability of bare iron oxide nanoparticles (BION) with physical site-directed immobilization of an engineered Protein A affinity ligand (rSpA) as an innovative magnetic material. The rSpA ligand contains a short peptide tag that enables the direct and stable immobilization onto the uncoated BION surface without commonly required laborious particle activation. The resulting BION@rSpA have beneficial characteristics outperforming conventional Protein A-functionalized magnetic particles: a simple, fast, low-cost synthesis, a particle size in the nanometer range with a large effective specific surface area enabling large immunoglobulin G (IgG) binding capacity, and a high magnetophoretic velocity advantageous for fast processing. We further show rapid interactions of IgG with the easily accessible rSpA ligands. The binding of IgG to BION@rSpA is thereby highly selective and not impeded by impurity molecules in perfusion cell culture supernatant. Regarding the subsequent acidic IgG elution from BION@rSpA@IgG, we observed a hampering pH increase caused by the protonation of large iron oxide surfaces after concentrating the particles in 100 mM sodium acetate buffer. However, the pH can be stabilized by adding 50 mM glycine to the elution buffer, resulting in recoveries above 85% even at high particle concentrations. Our work shows that BION@rSpA enable efficient magnetic mAb separation and could help to overcome emerging bottlenecks in DSP.
Subject(s)
Immunoglobulin G , Magnetic Iron Oxide Nanoparticles , Materials Testing , Particle Size , Magnetic Iron Oxide Nanoparticles/chemistry , Ligands , Immunoglobulin G/chemistry , Immunoglobulin G/isolation & purification , Biocompatible Materials/chemistry , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/isolation & purification , Staphylococcal Protein A/chemistry , Staphylococcal Protein A/metabolism , Surface Properties , Ferric Compounds/chemistryABSTRACT
Membrane separations offer a compelling alternative to traditional chromatographic methods by overcoming mass transport limitations. We introduce an additional degree of freedom in modulating membrane chromatography by using metalized membranes in a potential-driven process. Investigating the impact of a gold coating on membrane characteristics, the sputtered gold layer enhances the surface conductivity with stable electrochemical behavior. However, this comes at the expense of reduced permeability, wettability, and static binding capacity (â¼ 474 µg g-1 of maleic acid). The designed device displayed a homogenous flow distribution, and the membrane electrodes exhibit predominantly capacitive behavior during potential application. Modulating the electrical potential during the adsorption and desorption phase strongly influenced the binding and elution behavior of anion-exchange membranes. Switching potentials between ±1.0 V vs. Ag/AgCl induces desorption, confirming the process principle. Elution efficiency reaches up to 58 % at -1.0 V vs. Ag/AgCl in the desorption phase without any alteration of the mobile phase. Increasing the potential perturbation ranging from +1.0 V to -1.0 V vs. Ag/AgCl resulted in reduced peak width and improved elution behavior, demonstrating the feasibility of electrochemically-modulated membrane chromatography. The developed process has great potential as a gentle and sustainable separation step in the biotechnological and chemical industry.
Subject(s)
Chromatography , Gold , Electrodes , Adsorption , Gold/chemistry , BiotechnologyABSTRACT
Magnetic nanoparticles are an attractive bioseparation tool due to their magnetic susceptibility and high adsorption capacity for different types of molecules. A major challenge for separation is to generate selectivity for a target molecule, or for a group of molecules in complex environments such as cell lysates. It is crucial to understand the factors that determine the targets' adsorption behavior in mixtures for triggering intended interactions and selectivity. Here we use a model system containing three molecules, each of them a common representative of the more abundant types of macromolecules in living systems: sodium oleate (SO), a fatty acid; bovine serum albumin (BSA), a protein; and dextran, a polysaccharide. Our results show that (a) the BSA adsorption capacity on the iron oxide material depends markedly on the pH, with the maximum capacity at the pI of the protein (0.39 g gMNP-1 ); (b) sodium oleate, a strongly negatively charged molecule, an organic anion, renders a maximum adsorption capacity of 0.40 g gMNP-1, even at pHs at which oleate as well as the nanoparticle surface are negatively charged; (c) the adsorbed masses of dextran, a neutral sugar, are lower than for the other two molecules, between 0.09 and 0.13 g gMNP-1, regardless of the system's pH. We observe an unexpected behavior in mixtures: SO completely prevents the adsorption of BSA, and dextran decreases the adsorption of the other competitors, SO and BSA, while adsorbing at the same capacities, unaffected by either the presence of the other two molecules or the pH. BSA does not decrease the oleate adsorption capacity. We demonstrate the essential role of pH in the adsorption of BSA (a protein) and SO (a fatty acid), as well as its impact in the structural organization of the oleate molecules in water. Moreover, we present exciting data on the adsorption of the molecules in competition, revealing the need to focus on interaction studies in more complex environments. This study attempts to open the scope of the current research of bio-nano interactions to not only proteins but also to mixtures, and generally to molecules with other physicochemical characteristics. Furthermore, we contribute to the understanding of multicomponent systems with the vision set in enhancing biomass exploitation and biofractionation processes.
Subject(s)
Magnetite Nanoparticles , Oleic Acid , Oleic Acid/chemistry , Fatty Acids , Dextrans , Serum Albumin, Bovine/chemistryABSTRACT
The major interest in nanoparticles as an application platform for biotechnology arises from their high surface-to-volume ratio. Iron oxide nanoparticles (IONPs) are particularly appealing due to their superparamagnetic behavior, which enables bioseparation using external magnetic fields. In order to design advanced biomaterials, improve binding capacities and develop innovative processing solutions, a thorough understanding of the factors governing organic-inorganic binding in solution is critical but has not yet been achieved, given the wide variety of chemical and physical influences. This paper offers a critical review of experimental studies of the interactions between low cost IONPs (bare iron oxides, silica-coated or easily-functionalized surfaces) and the main groups of biomolecules: proteins, lipids, nucleic acids and carbohydrates. Special attention is devoted to the driving forces and interdependencies responsible of interactions at the solid-liquid interface, to the unique structural characteristics of each biomolecular class, and to environmental conditions influencing adsorption. Furthermore, studies focusing on mixtures, which are still rare, but absolutely necessary to understand the biocorona, are also included. This review concludes with a discussion of future work needed to fill the gaps in knowledge of bio-nano interactions, seeking to improve nanoparticles' targeting capabilities in complex systems, and to open the door for multipurpose recognition and bioseparation processes.
ABSTRACT
Carbon-based materials are the spearhead of research in multiple fields of nanotechnology. Moreover, their role as stationary phase in chromatography is gaining relevance. We investigate a material consisting of multiwall carbon nanotubes (CNTs) and superparamagnetic iron oxide nanoparticles towards its use as a mixed-mode chromatography material. The idea is to immobilize the ion exchange material iron oxide on CNTs as a stable matrix for chromatography processes without a significant pressure drop. Iron oxide nanoparticles are synthesized and used to decorate the CNTs via a co-precipitation route. They bind to the walls of oxidized CNTs, thereby enabling to magnetically separate the composite material. This hybrid material is investigated with transmission electron microscopy, magnetometry, X-ray diffraction, X-ray photoelectron and Raman spectroscopy. Moreover, we determine its specific surface area and its wetting behavior. We also demonstrate its applicability as chromatography material for amino acid retention, describing the adsorption and desorption of different amino acids in a complex porous system surrounded by aqueous media. Thus, this material can be used as chromatographic matrix and as a magnetic batch adsorbent material due to the iron oxide nanoparticles. Our work contributes to current research on composite materials. Such materials are necessary for developing novel industrial applications or improving the performance of established processes.
ABSTRACT
Owing to their high specific surface and low production cost, carbon materials are among the most important adsorption materials. Novel usages, for instance in pharmaceutical applications, challenge existing methods because charged and strongly polar substances need to be adsorbed. Here, we systematically investigate the highly complex adsorption equilibria of organic molecules having multiple protonation states as a function of pH. The adsorption behavior depends on intermolecular interactions within the solution (dissociation equilibria) and between adsorbed molecules on the carbon surface (electrostatic forces). For the model substances maleic acid and phenylalanine, we demonstrate that a custom-made genetic algorithm is able to extract up to nine parameters of a multispecies isotherm from experimental data covering a broad pH-range. The parameters, including adsorption affinities, interaction energies, and maximum loadings were also predicted by molecular dynamics simulations. Both approaches obtained a good qualitative and mostly also quantitative description of the adsorption behavior within a pH-range of 2-12. By combining the determined isotherms with mass balances, the final concentrations and pH-shifts of batch adsorption experiments can be predicted. The developed modeling tools can be easily adapted to other types of pH-dependent, multispecies adsorbates and therefore will help to optimize adsorption-based processes in different fields.
ABSTRACT
Nanoparticles are acquiring an ever increasing role in analytical technologies for enhanced applications such as signalling of hazardous dyes. One challenge for the synthesis of hybrid nanomaterials is to control their shape, size and properties. The colloidal and interfacial properties of initial nanoparticles are decisive for the formation, growth and characteristics of nanohybrids. Our objective is to combine the advantages of iron oxide nanoparticles for magnetic separation with nanoscale gold for a surface enhanced Raman scattering (SERS) effect which could be used e.g. for improved detection of dye molecules. We synthesized iron oxide nanoparticles (â¼10 nm) with a high saturation magnetization of around 80 Am2 kg-1 and coupled nanoscale gold to these particles. The focus was set in testing multiple approaches to combine these two materials with the goal of understanding and discussing the effect of the colloidal stability of iron oxide nanoparticles on the properties of the hybrid material. Stability is a seldom addressed issue; however, it plays a critical role for guaranteeing a homogeneous distribution of the gold on the iron oxide surface. We characterized the produced materials with UV/Vis spectroscopy, dynamic light scattering, and transmission electron microscopy, and their capability to enhance Raman signals is investigated. The seed-mediated growth method of oleate and PEG-stabilized magnetic particles yielded the best enhancement of Raman scattering for identification of the dye Rhodamin 6G. This approach can be used to couple gold nanoparticles to other surfaces and microfluidic devices. The presented method might pave the way to further applications in diagnostics or also in environmental approaches and beyond.
ABSTRACT
Monoclonal antibodies are key molecules in medicine and pharmaceuticals. A potentially crucial drawback for faster advances in research here is their high price due to the extremely expensive antibody purification process, particularly the affinity capture step. Affinity chromatography materials have to demonstrate the high binding capacity and recovery efficiency as well as superior chemical and mechanical stability. Low-cost materials and robust, faster processes would reduce costs and enhance industrial immunoglobulin purification. Therefore, exploring the use of alternative materials is necessary. In this context, we conduct the first comparison of the performance of magnetic nanoparticles with commercially available chromatography resins and magnetic microparticles with regard to immobilizing Protein G ligands and recovering immunoglobulin G (IgG). Simultaneously, we demonstrate the suitability of bare as well as silica-coated and epoxy-functionalized magnetite nanoparticles for this purpose. All materials applied have a similar specific surface area but differ in the nature of their matrix and surface accessibility. The nanoparticles are present as micrometer agglomerates in solution. The highest Protein G density can be observed on the nanoparticles. IgG adsorbs as a multilayer on all materials investigated. However, the recovery of IgG after washing indicates a remaining monolayer, which points to the specificity of the IgG binding to the immobilized Protein G. One important finding is the impact of the ligand-binding stoichiometry (Protein G surface coverage) on IgG recovery, reusability, and the ability to withstand long-term sanitization. Differences in the materials' performances are attributed to mass transfer limitations and steric hindrance. These results demonstrate that nanoparticles represent a promising material for the economical and efficient immobilization of proteins and the affinity purification of antibodies, promoting innovation in downstream processing.
Subject(s)
Epoxy Resins/chemistry , Immunoglobulin G/chemistry , Magnetite Nanoparticles/chemistry , Particle Size , Porosity , Surface PropertiesABSTRACT
Downstream processing needs more innovative ideas to advance and overcome current bioprocessing challenges. Chromatography is by far the most prevalent technique used by a conservative industrial sector. Chromatography has many advantages but also often represents the most expensive step in a pharmaceutical production process. Therefore, alternative methods as well as further processing strategies are urgently needed. One promising candidate for new developments on a large scale is magnetic separation, which enables the fast and direct capture of target molecules in fermentation broths. There has been a small revolution in this area in the last 10-20 years and a few papers dealing with the use of magnetic separation in bioprocessing examples beyond the analytical scale have been published. Since each target material is purified with a different magnetic separation approach, the comparison of processes is not trivial but would help to understand and improve magnetic separation and thus making it attractive for the technical scale. To address this issue, we report on the latest achievements in magnetic separation technology and offer an overview of the progress of the capture and separation of biomolecules derived from biotechnology and food technology. Magnetic separation has great potential for high-throughput downstream processing in applied life sciences. At the same time, two major challenges need to be overcome: (1) the development of a platform for suitable and flexible separation devices and (2) additional investigations of advantageous processing conditions, especially during recovery. Concentration and purification factors need to be improved to pave the way for the broader use of magnetic applications. The innovative combination of magnetic gradients and multipurpose separations will set new magnetic-based trends for large scale downstream processing.
ABSTRACT
Magnetic separation is a promising alternative to conventional methods in downstream processing. This can facilitate easier handling, fewer processing steps, and more sustainable processes. Target materials can be extracted directly from crude cell lysates in a single step by magnetic nanoadsorbents with high-gradient magnetic fishing (HGMF). Additionally, the use of hazardous consumables for reducing downstream processing steps can be avoided. Here, we present proof of principle of one-step magnetic fishing from crude Escherichia coli cell lysate of a green fluorescent protein (GFP) with an attached hexahistidine (His6)-tag, which is used as the model target molecule. The focus of this investigation is the upscale to a liter scale magnetic fishing process in which a purity of 91% GFP can be achieved in a single purification step from cleared cell lysate. The binding through the His6-tag can be demonstrated, since no significant binding of nontagged GFP toward bare iron oxide nanoparticles (BIONs) can be observed. Nonfunctionalized BIONs with primary particle diameters of around 12 nm, as used in the process, can be produced with a simple and low-cost coprecipitation synthesis. Thus, HGMF with BIONs might pave the way for a new and greener era of downstream processing.
ABSTRACT
Superparamagnetic nanoparticles have recently gained much attention due to their broad range of applicability including medical in vivo technologies, sensors, and as supports for catalysts. As magnetic affinity materials, they can be utilized for the development of new purification strategies for pharmaceuticals and other target molecules from crude lysates. Here, a short peptide tag based on a glutamate sequence is introduced and the adsorption of pure protein as well as protein from crude cell lysate at different conditions is demonstrated. Fused to a model protein this tag can be used to recognize and purify this protein from a fermentation broth by bare iron oxide nanoparticles (BIONs). Binding of up to 0.2 g protein per g nanoparticles can be achieved and recovered easily by switching to a citrate buffered system. For a deeper understanding of the separation process, the aggregation and agglomeration of the nanoparticle protein systems were monitored for binding and elution steps. Furthermore, an upscaling of the process to the liter scale and the separation of a green fluorescent protein (GFP) containing the affinity tag to purities of 70% from Escherichia coli fermentation broth was possible in a one step process by means of high gradient magnetic separation (HGMS).
Subject(s)
Ferric Compounds/chemistry , Magnetite Nanoparticles/chemistry , Oligopeptides/chemistry , Peptides/chemistry , Proteins/chemistry , Adsorption , Chromatography, Affinity/methods , Escherichia coli/metabolism , Green Fluorescent Proteins/chemistry , Magnetics/methodsABSTRACT
Potential-controlled tensiometry is a voltage-induced method which enables measuring the contact angle between a powder bed and a liquid medium through the capillary rise method. This analytical tool provides a fine-grained technique for understanding wetting behavior of powders as well as solid surfaces in connection with the application of an electrical potential. In this work, the powder bed was brought into contact with an aluminum rod connected to a portable lightweight DAC-module (digital to analog converter) powered by a lithium-polymer battery (LiPo). The presented analytical device can be charged up to ±1000 mV. Both the power source and the DAC-module are lightweight in order to be conveniently attached to a force tensiometer without incorporating complex wiring. In this setup, we tested multiwall carbon nanotubes (MWCNT) and glassy carbon particles. An influence of the potential on the wetting behavior of glassy carbon particles is observed which demonstrates the working principle of the device. Surprisingly, no significant effect of the potential on the wetting behavior of MWCNT is indicated in the range studied. This technique can be a valuable tool to analyze the effect of changing surface properties applying electrical gradients on materials.
ABSTRACT
Microalgae continue to gain in importance as a bioresource, while their harvesting remains a major challenge at the moment. This study presents findings on microalgae separation using low-cost, easy-to-process bare iron oxide nanoparticles with the additional contribution of the upscaling demonstration of this simple, adhesion-based process. The high affinity of the cell wall for the inorganic surface enables harvesting efficiencies greater than 95% for Scenedesmus ovalternus and Chlorella vulgaris. Successful separation is possible in a broad range of environmental conditions and primarily depends on the nanoparticle-to-microalgae mass ratio, whereas the effect of pH and ionic strength are less significant when the mass ratio is chosen properly. The weakening of ionic concentration profiles at the interphase due to the successive addition of deionized water leads the microalgae to detach from the nanoparticles. The process works efficiently at the liter scale, enabling complete separation of the microalgae from their medium and the separate recovery of all materials (algae, salts, and nanoparticles). The current lack of profitable harvesting processes for microalgae demands innovative approaches to encourage further development. This application of magnetic nanoparticles is an example of the prospects that nanobiotechnology offers for biomass exploitation.
ABSTRACT
Inspired by nature, many applications and new materials benefit from the interplay of inorganic materials and biomolecules. A fundamental understanding of complex organic-inorganic interactions would improve the controlled production of nanomaterials and biosensors to the development of biocompatible implants for the human body. Although widely exploited in applications, the interaction of amino acids and peptides with most inorganic surfaces is not fully understood. To date, precisely characterizing complex surfaces of inorganic materials and analyzing surface-biomolecule interactions remain challenging both experimentally and computationally. This article reviews several approaches to characterizing biomolecule-surface interactions and illustrates the advantages and disadvantages of the methods presented. First, we explain how the adsorption mechanism of amino acids/peptides to inorganic surfaces can be determined and how thermodynamic and kinetic process constants can be obtained. Second, we demonstrate how this data can be used to develop models for peptide-surface interactions. The understanding and simulation of such interactions constitute a basis for developing molecules with high affinity binding domains in proteins for bioprocess engineering and future biomedical technologies.
ABSTRACT
Magnetic nanoparticles (MNP) are intensively investigated for applications in nanomedicine, catalysis and biotechnology, where their interaction with peptides and proteins plays an important role. However, the characterisation of the interaction of individual amino acids with MNP remains challenging. Here, we classify the affinity of 20 amino acid homo-hexamers to unmodified iron oxide nanoparticles using peptide arrays in a variety of conditions as a basis to identify and rationally design selectively binding peptides. The choice of buffer system is shown to strongly influence the availability of peptide binding sites on the MNP surface. We find that under certain buffer conditions peptides of different charges can bind the MNP and that the relative strength of the interactions can be modulated by changing the buffer. We further present a model for the competition between the buffer and the MNP's electrostatically binding to the adsorption sites. Thereby, we demonstrate that the charge distribution on the surface can be used to correlate the binding of positively and negatively charged peptides to the MNP. This analysis enables us to engineer the binding of MNP on peptides and contribute to better understand the bio-nano interactions, a step towards the design of affinity tags for advanced biomaterials.
Subject(s)
Magnetite Nanoparticles/chemistry , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Adsorption , Binding Sites , Particle Size , Static Electricity , Surface PropertiesABSTRACT
Iron oxide nanoparticles represent a promising low-cost environmentally-friendly material for multiple applications. Especially hematite (α-Fe2O3) nanoparticles demonstrate great possibilities in energy storage and photoelectrochemistry. A hydrothermal one-pot synthesis can be used to synthesise hematite nanoparticles. Here, the particle formation, nucleation and growth of iron oxide nanoparticles using a FeCl3 precursor over time is monitored. The formation of 6-line ferrihydrite seeds of 2-8 nm which grow with reaction time and form clusters followed by a phase transition to ~15 nm hematite particles can be observed with ex situ X-ray diffraction (XRD), transmission electron microscopy (TEM), Raman and UV/Vis spectroscopy. These particles grow with reaction time leading to 40 nm particles after 6 hours. The changes in plasmon and electron transition patterns, observed upon particle transition and growth lead to the possibility of tuning the photoelectrochemical properties. Catalytic activity of the hematite nanoparticles can be proven with visible light irradiation and the use of silver nitrate as scavenger material. The generation of elementary silver is dependent on the particle size of iron oxide nanoparticles while only slight changes can be observed in the oxygen generation. Low-cost nanoscale hematite, offers a range of future applications for artificial photosynthesis.
ABSTRACT
Electrochemically modulated liquid chromatography is a special form of ion exchange chromatography in which the separation process is controlled by applying an electric potential to the stationary phase. This form of chromatography has so far only been applied in research studies. The present study shows that multiwalled carbon nanotubes are an effective resin material for an electrochemically modulated chromatography process. The experiments are carried out in a newly designed column that enables the packing of nanomaterials. We investigate the influence of the applied potential on the retention and elution of maleic acid, determine the dynamic binding capacity, and calculate the utilization degree of the electrical charge in the adsorption process. Moreover, the stability of the resin and the membrane over more than 200 working hours are presented. In addition to the stability, their sturdiness and inexpensive price are important qualities that make multiwalled carbon nanotubes interesting for application as the stationary phase in an electrochemically driven process. The investigated chromatography technique represents a promising separation process for future applications as a preparative step in biotechnology as well as other life science fields.