Multi-cycle recovery of lactoferrin and lactoperoxidase from crude whey using fimbriated high-capacity magnetic cation exchangers and a novel "rotor-stator" high-gradient magnetic separator

Cerium (IV) initiated "graft-from" polymerization reactions were employed to convert M-PVA magnetic particles into polyacrylic acid-fimbriated magnetic cation exchange supports displaying ultra-high binding capacity for basic target proteins. The modifications, which were performed at 25mg and 2.5g scales, delivered maximum binding capacities (Q) for hen egg white lysozyme in excess of 320mgg, combined with sub-micromolar dissociation constants (0.45-0.69μm) and "tightness of binding" values greater than 49Lg. Two batches of polyacrylic acid-fimbriated magnetic cation exchangers were combined to form a 5g pooled batch exhibiting Q values for lysozyme, lactoferrin, and lactoperoxidase of 404, 585, and 685mgg, respectively. These magnetic cation exchangers were subsequently employed together with a newly designed "rotor-stator" type HGMF rig, in five sequential cycles of recovery of lactoferrin and lactoperoxidase from 2L batches of a crude sweet bovine whey feedstock. Lactoferrin purification performance was observed to remain relatively constant from one HGMF cycle to the next over the five operating cycles, with yields between 40% and 49% combined with purification and concentration factors of 37- to 46-fold and 1.3- to 1.6-fold, respectively. The far superior multi-cycle HGMF performance seen here compared to that observed in our earlier studies can be directly attributed to the combined use of improved high capacity adsorbents and superior particle resuspension afforded by the new "rotor-stator" HGMS design. © 2013 Wiley Periodicals, Inc

Integrated system for temperature-controlled fast protein liquid chromatography. II. Optimized adsorbents and 'single column continuous operation'

Continued advance of a new temperature-controlled chromatography system, comprising a column filled with thermoresponsive stationary phase and a travelling cooling zone reactor (TCZR), is described. Nine copolymer grafted thermoresponsive cation exchangers (thermoCEX) with different balances of thermoresponsive (N-isopropylacrylamide), hydrophobic (N-tert-butylacrylamide) and negatively charged (acrylic acid) units were fashioned from three cross-linked agarose media differing in particle size and pore dimensions. Marked differences in grafted copolymer composition on finished supports were sourced to base matrix hydrophobicity. In batch binding tests with lactoferrin, maximum binding capacity (q max) increased strongly as a function of charge introduced, but became increasingly independent of temperature, as the ability of the tethered copolymer networks to switch between extended and collapsed states was lost. ThermoCEX formed from Sepharose CL-6B (A2), Superose 6 Prep Grade (B2) and Superose 12 Prep Grade (C1) under identical conditions displayed the best combination of thermoresponsiveness (q max,50°C/q max,10°C ratios of 3.3, 2.2 and 2.8 for supports 'A2', 'B2' and 'C1' respectively) and lactoferrin binding capacity (q max,50°C ~56, 29 and 45mg/g for supports 'A2', 'B2' and 'C1' respectively), and were selected for TCZR chromatography. With the cooling zone in its parked position, thermoCEX filled columns were saturated with lactoferrin at a binding temperature of 35°C, washed with equilibration buffer, before initiating the first of 8 or 12 consecutive movements of the cooling zone along the column at 0.1mm/s. A reduction in particle diameter (A2→B2) enhanced lactoferrin desorption, while one in pore diameter (B2→C1) had the opposite effect. In subsequent TCZR experiments conducted with thermoCEX 'B2' columns continuously fed with lactoferrin or 'lactoferrin+bovine serum albumin' whilst simultaneously moving the cooling zone, lactoferrin was intermittently concentrated at regular intervals within the exiting flow as sharp uniformly sized peaks. Halving the lactoferrin feed concentration to 0.5mg/mL, slowed acquisition of steady state, but increased the average peak concentration factor from 7.9 to 9.2. Finally, continuous TCZR mediated separation of lactoferrin from bovine serum albumin was successfully demonstrated. While the latter's presence did not affect the time to reach steady state, the average lactoferrin mass per peak and concentration factor both fell (respectively from 30.7 to 21.4mg and 7.9 to 6.3), and lactoferrin loss in the flowthrough between elution peaks increased (from 2.6 to 12.2mg). Fouling of the thermoCEX matrix by lipids conveyed into the feed by serum albumin is tentatively proposed as responsible for the observed drops in lactoferrin binding and recovery

Integrated system for temperature-controlled fast protein liquid chromatography comprising improved copolymer modified beaded agarose adsorbents and a travelling cooling zone reactor arrangement

An integrated approach to temperature-controlled chromatography, involving copolymer modified agarose adsorbents and a novel travelling cooling zone reactor (TCZR) arrangement, is described. Sepharose CL6B was transformed into a thermoresponsive cation exchange adsorbent (thermoCEX) in four synthetic steps: (i) epichlorohydrin activation; (ii) amine capping; (iii) 4,4′-azobis(4-cyanovaleric acid) immobilization; and ‘graft from’ polymerization of poly(N-isopropylacrylamide-co-N-tert-butylacrylamide-co-acrylic acid-co- N,N′-methylenebisacrylamide). FT-IR, 1H NMR, gravimetry and chemical assays allowed precise determination of the adsorbent’s copolymer composition and loading, and identified the initial epoxy activation step as a critical determinant of ‘on-support’ copolymer loading, and in turn, protein binding performance. In batch binding studies with lactoferrin, thermoCEX’s binding affinity and maximum adsorption capacity rose smoothly with temperature increase from 20 to 50 ºC. In temperature shifting chromatography experiments employing thermoCEX in thermally-jacketed columns, 44 – 51% of the lactoferrin adsorbed at 42 ºC could be desorbed under binding conditions by cooling the column to 22 ºC, but the elution peaks exhibited strong tailing. To more fully exploit the potential of thermoresponsive chromatography adsorbents, a new column arrangement, the TCZR, was developed. In TCZR chromatography, a narrow discrete cooling zone (special assembly of copper blocks and Peltier elements) is moved along a bespoke fixed-bed separation columnfilled with stationary phase. In tests with thermoCEX, it was possible to recover 65% of the lactoferrin bound at 35 ºC using 8 successive movements of the cooling zone at a velocity of 0.1 mm/s; over half of the recovered protein was eluted in the first peak in more concentrated form than in the feed. Intra-particle diffusion of desorbed protein out of the support pores, and the ratio between the velocities of the cooling zone and mobile phase were identified as the main parameters affecting TCZR performance. In contrast to conventional systems, which rely on cooling the 3 whole column to effect elution and permit only batch-wise operation, TCZR chromatography generates sharp concentrated elution peaks without tailing effects and appears ideally suited for continuous operation

Design of a Magnetic Field-Controlled Chromatography Process for Efficient and Selective Fractionation of Rare Earth Phosphors from End-of-Life Fluorescent Lamps

Rare earth-containing materials are essential for a wide range of modern technologies and have significant technological and economic importance. Therefore, the development of efficient separation and purification processes for these materials is crucial for environmental sustainability and resource conservation. In this study, we investigated the potential of magnetic field-controlled chromatography for the fractionation of different rare earth-containing phosphors from end-of-life fluorescent lamps. The results demonstrate that with the intrinsic magnetization of the phosphor particles and a careful choice of process parameters, we can control the separation outcome. An optimized gradient shape resulted in purities of up to 95.3% at recoveries of 93.6% (LaPO4:Ce3+,Tb3+). The aqueous eluent consumption was found to be quite modest at 4.1 L/g, and it contained only minimal quantities of nontoxic and biodegradable solvent. This study could have significant implications for the development of efficient and effective purification processes for rare earth-containing materials, which are primarily driven by economic concerns and environmental considerations. The possibility of scaling up the process by increasing the column size or transferring it to continuous processing methods could further enhance its practical applicability in industry

Partitioning Behavior of Silica-Coated Nanoparticles in Aqueous Micellar Two-Phase Systems: Evidence for an Adsorption-Driven Mechanism from QCM‑D and ATR-FTIR Measurements

Quartz crystal microbalance with dissipation (QCM-D), attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), and total organic carbon detection (TOC) are employed to examine the cause of the differences in the partitioning of silica-coated nanoparticles in an aqueous micellar two-phase system based on nonionic surfactant Eumulgin ES. The particles partition into the micelle-rich phase at pH 3 and into the micelle-poor phase at pH 7. Our results clearly show that the nonionic surfactants are adsorbed to the silica surface at pH 3. Above the critical temperature, a stable surfactant bilayer forms on the silica surface. At pH 7, the surfactants do not adsorb to the particle surface; a surfactant-loaded particle is therefore drawn to the micelle-rich phase but otherwise repelled from it. These results suggest that the partitioning in aqueous micellar two-phase systems is mainly driven by hydrogen bonds formed between the surfactants and the component to be partitioned

Dual-Gated Microparticles for Switchable Antibody Release

We pioneer the design of dual-gated microparticles, both responsive to changes in temperature and pH, for stimuli-responsive chromatography targeted at the efficient separation of antibodies. Dual-gated microspheres were synthesized by introducing RAFT-based thiol-terminal block copolymers of poly­(<i>N</i>-isopropylacrylamide-<i>b</i>-4-vinylpyridine) (P­(NIPAM-<i>b</i>-4VP, 4800 ≤ <i>M</i>_n/Da ≤ 10 000, featuring block length ratios of 29:7, 29:15, and 29:30, respectively) by thiol-epoxy driven ligation to the surface of poly­(glycidyl methacrylate) (PGMA) microparticles (10–12 μm), whereby the 4-vinylpyridine units within the lateral chain enable protein binding. The switchable protein release abilities of the resulting microparticle resins are demonstrated by adsorption of immunoglobulins at 40 °C and pH 8 and their release at 5 °C or pH 3, respectively. We demonstrate that P­(NIPAM₂₉-<i>b</i>-4VP₃₀)-grafted PGMA particles show a maximum adsorption capacity for immunoglobulins of 18.9 mg mL^–1 settled resin at 40 °C/pH 8, whereas the adsorption capacity decreased to 7.5 mg mL^–1 settled resin at 5 °C while retaining the pH value, allowing the unloading of the chromatographic column by a facile temperature switch. Critically, regeneration of the dual-gated microspheres became possible by lowering the pH to 3

New Approaches for Bottom-Up Assembly of Tobacco Mosaic Virus-Derived Nucleoprotein Tubes on Defined Patterns on Silica- and Polymer-Based Substrates

The capability of some natural molecular building blocks to self-organize into defined supramolecular architectures is a versatile tool for nanotechnological applications. Their site-selective integration into a technical context, however, still poses a major challenge. RNA-directed self-assembly of tobacco mosaic virus-derived coat protein on immobilized RNA scaffolds presents a possibility to grow nucleoprotein nanotubes in place. Two new methods for their site-selective, bottom-up assembly are introduced. For this purpose, isothiocyanate alkoxysilane was used to activate oxidic surfaces for the covalent immobilization of DNA oligomers, which served as linkers for assembly-directing RNA. Patterned silanization of surfaces was achieved (1) on oxidic surfaces via dip-pen nanolithography and (2) on polymer surfaces (poly­(dimethylsiloxane)) via selective oxidization by UV-light irradiation in air. Atomic force microscopy and X-ray photoelectron spectroscopy were used to characterize the surfaces. It is shown for the first time that the combination of the mentioned structuring methods and the isothiocyanate-based chemistry is appropriate (1) for the site-selective immobilization of nucleic acids and, thus, (2) for the formation of viral nanoparticles by bottom-up self-assembly after adding the corresponding coat proteins

Redox Interfaces for Electrochemically Controlled Protein–Surface Interactions: Bioseparations and Heterogeneous Enzyme Catalysis

Redox-active materials are an attractive platform for engineering specific interactions with charged species by electrochemical control. We present nanostructured redox-electrodes, functionalized with poly­(vinyl)­ferrocene embedded in a carbon nanotube matrix, for modulating the adsorption and release of proteins through electrochemical potential swings. The affinity of the interface toward proteins increased dramatically following oxidation of the ferrocenes, and, due to the Faradaic nature of the organometallic centers, the electrodes were maintained at sufficiently low overpotentials to ensure the preservation of both protein structure and catalytic activity. Our system was selective for various proteins based on size and charge distribution, and exhibited fast kinetics (<120 s for a charge–discharge cycle) and high uptake capacities (>200 mg/g) under moderate overpotentials (+0.4 V vs Ag/AgCl), as well as remarkable stability for binding under ferrocene oxidation conditions. The preservation of bioactivity and protein structure at the interface indicates the potential for these redox-mediated surfaces to be used as heterogeneous supports for enzyme catalysis. This work draws on the molecular selectivity of ferrocene-functionalized materials toward organic anion groups, and demonstrates that these smart redox-active materials can be used for modulation of the macroscopic affinity of surfaces for charged biomacromolecules to enhance processes such as bioseparations, electrochemically controlled protein purification, biocatalysis, and electrochemically mediated drug release

Hierarchically Functionalized Magnetic Core/Multishell Particles and Their Postsynthetic Conversion to Polymer Capsules

The controlled synthesis of hierarchically functionalized core/multishell particles is highly desirable for applications in medicine, catalysis, and separation. Here, we describe the synthesis of hierarchically structured metal–organic framework multishells around magnetic core particles (magMOFs) <i>via</i> layer-by-layer (LbL) synthesis. The LbL deposition enables the design of multishell systems, where each MOF shell can be modified to install different functions. Here, we used this approach to create controlled release capsules, in which the inner shell serves as a reservoir and the outer shell serves as a membrane after postsynthetic conversion of the MOF structure to a polymer network. These capsules enable the controlled release of loaded dye molecules, depending on the surrounding media