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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.

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 25 mg and 2.5 g scales, delivered maximum binding capacities (Qmax) for hen egg white lysozyme in excess of 320 mg g−1, combined with sub-micromolar dissociation constants (0.45–0.69 µm) and “tightness of binding” values greater than 49 L g−1. Two batches of polyacrylic acid-fimbriated magnetic cation exchangers were combined to form a 5 g pooled batch exhibiting Qmax values for lysozyme, lactoferrin, and lactoperoxidase of 404, 585, and 685 mg g−1, 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 2 L 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. Biotechnol. Bioeng. 2013; 110: 1714–1725. © 2013 Wiley Periodicals, Inc.

The loading of a metal-organic framework (MOF), [Cu(3)(btc)(2)xH(2)O] HKUST-1, with europium β-diketonate complexes is studied with the goal to using the porous molecular framework as a photonic antenna. Whereas loading of HKUST-1 powder particles produced via the conventional solvothermal synthesis method was strongly hindered, for HKUST-1 SURMOFs, thin MOF films fabricated using the liquid phase epitaxy method, a high filling factor can be achieved. The optical properties of the HKUST-1-MOFs before and after loading were analysed with the aid of luminescence spectroscopy. Careful analysis of the absorption spectra reveals the presence of an effective energy transfer between the HKUST-1 framework and the Eu(3+) centers.

We describe a method for multiplexed analysis of proteins using fluorescently encoded microbeads. The sensitivity of our method is comparable to the sensitivity obtained by enzyme-linked immunosorbent assay while only 5 µl sample volumes are needed. Streptavidin-coated, 1 µm beads are encoded with a combination of fluorophores at different intensity levels. As a proof of concept, we demonstrate that 27 microbead populations can be readily encoded by affinity conjugation using three intensity levels for each of three different biotinylated fluorescent dyes. Four populations of encoded microbeads are further conjugated with biotinylated capture antibodies and then combined and immobilized in a microfluidic flow cell for multiplexed protein analysis. Using four uniquely encoded microbead populations, we show that a cancer biomarker and three cytokine proteins can be analysed quantitatively in the picogram per millilitre range by fluorescence microscopy in a single assay. Our method will allow for the fabrication of high density, bead-based antibody arrays for multiplexed protein analysis using integrated microfluidic devices and automated sample processing.

Liquid-phase mass transfer in ion exchange controlled by diffusion across a Nernst film was studied for the general case of any arbitrary number of counterions and coions. The Nernst-Planck equations are solved analytically for the momentary fluxes. The solution reduces to all exact solutions available in the literature for special cases and provides an approximation in the general case of arbitrary systems. The quality of the approximation is demonstrated by comparison with the general numerical solution. For most applications the errors can be neglected compared with those caused by the idealising assumptions of the film model. Experimental results obtained with a short column demonstrate the applicability of the solution to multi-component systems, thus illustrating that the electric potential has a strong effect on the fluxes. In extreme cases, one or several species can be transferred against their concentration gradients.

The surface complexation theory considers the sorption of ions as a local equilibrium reaction, which is caused by the amphoteric behaviour of the surface. Application of this theory to exchange equilibria with ordinary and chelating weak-acid ion exchange resins which can release protons has opened the way for a comprehensive description. Binary equilibria are described by a logarithmic equilibrium parameter, which is a linear function of the composition of the resin phase. Multicomponent equilibria are considered as a superposition of several binary equilibria. The approach considers the pH-dependent dissociation of functional groups. Furthermore, chemical reactions in the liquid phase in which exchangeable species are involved, can readily be taken into account. In contrast to cation exchange resins, the behaviour of anion exchangers is characterized by the uptake of protons enabling them to also adsorb anions. As a consequence, anion exchange equilibria can be described in similar way. The evaluation of data demonstrates that similar relationships exist between a logarithmic equilibrium parameter and the resin phase composition. As for cation exchangers, multicomponent equilibria can be predicted with excellent accuracy from binary parameters.

Classical purification of the glycoprotein equine chorionic gonadotropin (eCG) from serum includes pH fractionation with metaphosphoric acid, two ethanol precipitation steps as well as dialysis followed by fixed-bed chromatography. A simplified process requiring only 1/3 of the solvent and improving the yield from 53 to 65% has been developed. The process comprises an ultra-/diafiltration step after the first ethanol precipitation, directly followed by an adsorption/desorption procedure based on magnetic microadsorbents with N,N-diethyl-ammonium functionalization. The process reaches an overall purification factor of eCG of more than 1800 and an average product activity of 1300 IU(ELISA)/mg. After adapting the parameters of the fractionation and the type of magnetic microadsorbents, the new concept is likely to be transferable to other serum proteins.

Increasing interest in biological surfactants has led to intensified research directed at more cost-efficient production of biosurfactants, relative to traditional surface-active components based on petrochemical feedstocks. This publication will focus on a new integrated process for continuous rhamnolipid (RL) production. RL was synthesized by Pseudomonas aeruginosa DSM 2874 and was continuously removed in situ by foam fractionation. To prevent loss of the biocatalyst through foaming, bacteria were entrapped in magnetic alginate beads. Immobilizates were retained from the foam by high-gradient magnetic separation and back-flushed in the bioreactor at constant intervals. It was demonstrated that continuous RL production in a 10-L bioreactor over several cycles with intermediate growth periods is feasible. Complete separation of RLs from the production medium with an average enrichment ratio of 15 in the collapsed foam was demonstrated, yielding a final RL amount of 70 g after four production cycles.

Downstream processing using magnetic adsorbent particles, so called magnetic beads, is a promising technology for future bioseparation challenges for high-value as well as for mid-priced products. Magnetic adsorbent based separation currently applies the same ligand technologies as chromatographic separation. After binding, magnetic forces are used for the separation of protein loaded beads from crude bio suspensions. The concept has been proven in many proof of principle examples but few pilot scale processes, due to two main hurdles: Improved, cheaper magnetic adsorbents are required, as are improved magnetic separators. This review examines where magnetic bead based separations can fit into a downstream process before studying several state of the art synthesis processes for magnetic beads and their magnetic and sorptive properties. An overview of magnetic separator technology is given with special focus on bioprocessing. Examples of the separation and purification of proteins applying magnetic beads to a biosuspension is shown.

During the last decades, whole-cell immobilization has been used successfully in many bioprocesses. In particular, it is aimed at implementing continuous production processes, reaching higher production rates, and reusing the biocatalyst. In some cases, effective retention of immobilizates in the bioprocess is not feasible by membranes or sieves due to pore plugging or undesired losses of immobilizates. In the present publication, it is reported about the investigation of magnetic immobilizates of Pseudomonas aeruginosa for application in continuous biosurfactant production of rhamnolipids by foam fractionation and retention of entrained immobilizates by high-gradient magnetic separation from foam. Different materials and methods were tested with respect to important parameters, such as stability, diffusion properties or magnetic separation. Good magnetic separation of immobilizates was achieved at 5% (w/w) magnetite loading. Best results in terms of homogeneous embedding, good diffusion properties, and stability enhancement vis-a-vis pure alginate beads was achieved with alginate beads with embedded Bayoxide® magnetite or MagPrep® silica particles. Although polyurethane immobilizates showed higher stabilities compared with alginate beads, rhamnolipid diffusion in immobilizates was superior in magnetic alginate beads. Regarding bead production, smaller immobilizates were achieved with suspension polymerization compared to droplet extrusion by the JetCutting® technology. In total, magnetic immobilizates are a promising tool for an easier handling of biocatalysts in a continuous biological production process, but they have to be adapted to the current production task.© 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009

Molecular patterning processes taking place in biological systems are challenging to study in vivo because of their dynamic behavior, subcellular size, and high degree of complexity. In vitro patterning of biomolecules using nanolithography allows simplification of the processes and detailed study of the dynamic interactions. Parallel dip-pen nanolithography (DPN) is uniquely capable of integrating functional biomolecules on subcellular length scales due to its constructive nature, high resolution, and high throughput. Phospholipids are particularly well suited as inks for DPN since a variety of different functional lipids can be readily patterned in parallel. Here DPN is used to spatially pattern multicomponent micro- and nanostructured supported lipid membranes and multilayers that are fluid and contain various amounts of biotin and/or nitrilotriacetic acid functional groups. The patterns are characterized by fluorescence microscopy and photoemission electron microscopy. Selective adsorption of functionalized or recombinant proteins based on streptavidin or histidine-tag coupling enables the semisynthetic fabrication of model peripheral membrane bound proteins. The biomimetic membrane patterns formed in this way are then used as substrates for cell culture, as demonstrated by the selective adhesion and activation of T-cells.

During the last few decades, increasing interest in biological surfactants led to an intensification of research for the cost-efficient production of biosurfactants compared with traditional petrochemical surface-active components. The quest for alternative production strains also is associated with new demands on biosurfactant analysis. The present paper gives an overview of existing analytical methods, based on the example of rhamnolipids. The methods reviewed range from simple colorimetric testing to sophisticated chromatographic separation coupled with detection systems like mass spectrometry, by means of which detailed structural information is obtained. High-performance liquid chromatography (HPLC) coupled with mass spectrometry currently presents the most precise method for rhamnolipid identification and quantification. Suitable approaches to accelerate rhamnolipid quantification for better control of biosurfactant production are HPLC analysis directly from culture broth by adding an internal standard or Fourier transform infrared attenuated total reflectance spectroscopy measurements of culture broth as a possible quasi-online quantification method in the future. The search for alternative rhamnolipid-producing strains makes a structure analysis and constant adaptation of the existing quantification methods necessary. Therefore, simple colorimetric tests based on whole rhamnolipid content can be useful for strain and medium screening. Furthermore, rhamnolipid purification from a fermentation broth will be considered depending on the following application.

Circular dichroism (CD) was used to characterize the secondary structure of penicillin G acylase upon covalent immobilization on silica nanoparticles. Covalent immobilization was achieved by functionalizing the silica nanoparticles with glutardialdehyde and coupling to the free NH(2) groups of the enzyme (lysine and arginine side chains). The loading of the covalently bound enzyme was increased up to saturation, which was reached at 54.6 mg immobilized enzyme per g silica nanobeads. For structural characterization of the commercially available enzyme its exact molecular mass was determined by mass spectrometry in order to enable precise evaluation of the CD data. The fraction of secondary structure elements of the free and immobilized enzyme were estimated from the respective CD spectra using standard algorithms (CONTINLL, CDSSTR, SELCON3). The fractions obtained by the different algorithms for the free enzyme agreed well with one another and also with data from X-ray diffraction described in the literature. Interestingly, the secondary structure fractions found for the immobilized enzyme were very similar to the free enzyme and nearly constant over all experiments. These results indicate that even a loading of up to 55.8 mg/g (enzyme per silica nanoparticles) causes only slight structural changes. However, the specific activity determined by a kinetic assay decreased by around 60%, when increasing the loading from 14.9 to 55.8 mg/g. Because of the fact that we found no major changes in the secondary structure, diffusion limitation seems to be the main reason for the decline of the specific activity.

High-gradient magnetic separation based processing is demonstrated for the semi-continuous multicycle re-use of a lipase immobilised on magnetic microparticles. The lipase of Candida antarctica A-type (CALA) was immobilised on polyvinyl alcohol coated magnetic particles (1-2mum diameter) with epoxy functionalisation. The immobilised CALA was used to hydrolyse a model oil-water 2-phase-system composed of a phosphate buffer with tributyrin at up to 3l scale. The immobilised enzyme was subsequently recovered in a magnetic filter using high-gradient magnetic separation and reapplied in repeated cycles of hydrolysis and recovery. Two different temperatures of 30 and 50 degrees C, tributyrin concentrations (0.12 and 35gl(-1)) and reaction times were tested. In each case the reaction was followed by pH titration using NaOH, as well as by HPLC analysis. Consecutive cycles were conducted for each reaction condition and in total the immobilised CALA was subjected to 20 recovery and re-use cycles, after which approximately 14% of the initial specific activity still remained.

Determine Conditions for Adsorbent Use in HGMF . . . . . . . . . 99 3.3 Design and Set-Up of Magnetic Separator Systems . . . . . . . . . . . . . . . . . . 99 3.4 Parameters Affecting System Performance . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1023.4.1 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102 3.4.2 Simplified Yield Estimation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104 3.4.3 Multi-Component Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106 3.4.4 Case Study III: Optimization of the Capacity Ratio Used. . . . 109 3.4.5 Process Productivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110 3.4.6 Case Study IV: Influence of Washing and Elution Steps . . . . . 114 3.4.7 Adsorbent Reuse . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115 3.4.8 Case Study V: Pilot Plant Efficiencies . . . . . . . . . . . . . . . . . . . . . . . . 117SHUKLA: “dk3347_c003” — 2006/5/23 — 17:54 — page 84 — #2. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119Magnetic adsorbents possess a very powerful and unique handle that permits their selective manipulation within and from most (if not all) kinds of biological feedstock, simply through the application of a magnetic field. This ease of manipulation stands in stark contrast to all other adsorbents, and has been exploited in bioprocessing to develop a first capture step for proteins and other species from crude feedstocks known as high-gradient magnetic fishing (HGMF) [1-14].

The application of functionalised magnetic adsorbent particles in combination with magnetic separation techniques has received considerable attention in recent years. The magnetically responsive nature of such adsorbent particles permits their selective manipulation and separation in the presence of other suspended solids. Thus, it becomes possible to magnetically separate selected target species directly out of crude biological process liquors (e.g. fermentation broths, cell disruptates, plasma, milk, whey and plant extracts) simply by binding them on magnetic adsorbents before application of a magnetic field. By using magnetic separation in this way, the several stages of sample pretreatment (especially centrifugation, filtration and membrane separation) that are normally necessary to condition an extract before its application on packed bed chromatography columns, may be eliminated. Magnetic separations are fast, gentle, scaleable, easily automated, can achieve separations that would be impossible or impractical to achieve by other techniques, and have demonstrated credibility in a wide range of disciplines, including minerals processing, wastewater treatment, molecular biology, cell sorting and clinical diagnostics. However, despite the highly attractive qualities of magnetic methods on a process scale, with the exception of wastewater treatment, few attempts to scale up magnetic operations in biotechnology have been reported thus far. The purpose of this review is to summarise the current state of development of protein separation using magnetic adsorbent particles and identify the obstacles that must be overcome if protein purification with magnetic adsorbent particles is to find its way into industrial practice.

The kinetic, selective and stereoselective properties of enzyme immobilised on magnetic polymer beads with diameters in the range 1 microm was studied with penicillin amidase from E. coli. The enzyme was immobilised on epoxy and glutaraldehyde-activated poly(vinyl alcohol), poly(methylmetacrylate) and poly(vinyl acetate-divinylbenzene) magnetic beads. The amount of covalently bound active protein was dependent on the chemical modification of the matrix and increased at higher ionic strength of the immobilisation buffer. The small size of the magnetic beads, that reduces mass transfer limitations, and the decreased charge density in the electric double layer resulted in lower apparent Km values and higher efficiency for benzylpenicillin hydrolysis, higher stereoselectivity in condensation of R-phenylglycine amide with S- and R-Phe and in hydrolysis of racemic phenylacetyl-Phe and higher selectivity in kinetically controlled synthesis of cephalexin compared to the enzyme immobilised on larger and porous carriers.

The applicability of high gradient magnetic separation (HGMS) for the removal of heavy metals from mixed solutions of para- and diamagnetic ions (nickel and zinc) was studied. The heavy metals were precipitated as phosphates by the addition of Na3PO4. The susceptibilities of the precipitations showed the expected linear dependence with respect to the mass fraction of nickel. However, the separation experiments with a 1.8 Tesla magnetic separator demonstrated that even small fractions of nickel result in separation efficiencies comparable to those of pure nickel phosphate. The experimental results were compared with theoretical filter calculations based on deep filter theories and single wire models.

a Biosystems Department, Riso National Laboratory for Sustainable Energy, Technical University of Denmark-DTU, Building 330, P.O. Box 49, DK-4000 Roskilde, Denmark b Engler-Bunte-Institute, Chair of Water Chemistry, University of Karlsruhe (TH), Engler-Bunte-Ring 1, D-76131 Karlsruhe, Germany c Research Center Karlsruhe, Institute of Technical Chemistry, Hermann-von-Helrnholz-Platz 1, D-76344 Eggenstein-Leopoldshafen, Germany d Institute of Engineering in Life Science, Technical Biology Unit, University of Karlsruhe (TH), Engler-Bunte-Ring 1, D-76131 Karlsruhe, Germany