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Robert Van Reis - One of the best experts on this subject based on the ideXlab platform.
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surface extenders and an optimal pore size promote high Dynamic Binding capacities of antibodies on cation exchange resins
Journal of Chromatography A, 2009Co-Authors: David S Hart, Gunnar Malmquist, Andreas Axen, Chithkala Harinarayan, Mandakini Sharma, Robert Van ReisAbstract:Abstract Increased recombinant protein expression yields and a large installed base of manufacturing facilities designed for smaller bulk sizes has led to the need for high capacity chromatographic resins. This work explores the impact of three pore sizes (with dextran distribution coefficients of 0.4, 0.53, and 0.64), dextran surface extender concentration (11–20 mg/mL), and ligand density (77–138 μmol H+/mL resin) of cation exchange resins on the Dynamic Binding capacity of a therapeutic antibody. An intermediate optimal pore size was identified from three pore sizes examined. Increasing ligand density was shown to increase the critical ionic strength, while increasing dextran content increased Dynamic Binding capacity mainly at the optimal pore size and lower conductivities. Dynamic Binding capacity as high as 200 mg/mL was obtained at the optimum pore size and dextran content.
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an exclusion mechanism in ion exchange chromatography
Biotechnology and Bioengineering, 2006Co-Authors: Chithkala Harinarayan, Anders Ljunglof, Jay Mueller, Robert L Fahrner, J Van Alstine, Robert Van ReisAbstract:Protein Dynamic Binding capacities on ion exchange resins are typically expected to decrease with increasing conductivity and decreasing protein charge. There are, however, conditions where capacity increases with increasing conductivity and decreasing protein charge. Capacity measurements on two different commercial ion exchange resins with three different monoclonal antibodies at various pH and conductivities exhibited two domains. In the first domain, the capacity unexpectedly increased with increasing conductivity and decreasing protein charge. The second domain exhibited traditional behavior. A mechanism to explain the first domain is postulated; proteins initially bind to the outer pore regions and electrostatically hinder subsequent protein transport. Such a mechanism is supported by protein capacity and confocal microscopy studies whose results suggest how knowledge of the two types of IEX behavior can be leveraged in optimizing resins and processes.
Chithkala Harinarayan - One of the best experts on this subject based on the ideXlab platform.
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ion exchange chromatography of monoclonal antibodies effect of resin ligand density on Dynamic Binding capacity
Journal of Chromatography A, 2009Co-Authors: Ann Marie Hardin, Gunnar Malmquist, Andreas Axen, Chithkala Harinarayan, Robert Van ReisAbstract:Dynamic Binding capacity (DBC) of a monoclonal antibody on agarose based strong cation exchange resins is determined as a function of resin ligand density, apparent pore size of the base matrix, and protein charge. The maximum DBC is found to be unaffected by resin ligand density, apparent pore size, or protein charge within the tested range. The critical conductivity (conductivity at maximum DBC) is seen to vary with ligand density. It is hypothesized that the maximum DBC is determined by the effective size of the proteins and the proximity to which they can approach one another. Once a certain minimum resin ligand density is supplied, additional ligand is not beneficial in terms of resin capacity. Additional ligand can provide flexibility in designing ion exchange resins for a particular application as the critical conductivity could be matched to the feedstock conductivity and it may also affect the selectivity.
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surface extenders and an optimal pore size promote high Dynamic Binding capacities of antibodies on cation exchange resins
Journal of Chromatography A, 2009Co-Authors: David S Hart, Gunnar Malmquist, Andreas Axen, Chithkala Harinarayan, Mandakini Sharma, Robert Van ReisAbstract:Abstract Increased recombinant protein expression yields and a large installed base of manufacturing facilities designed for smaller bulk sizes has led to the need for high capacity chromatographic resins. This work explores the impact of three pore sizes (with dextran distribution coefficients of 0.4, 0.53, and 0.64), dextran surface extender concentration (11–20 mg/mL), and ligand density (77–138 μmol H+/mL resin) of cation exchange resins on the Dynamic Binding capacity of a therapeutic antibody. An intermediate optimal pore size was identified from three pore sizes examined. Increasing ligand density was shown to increase the critical ionic strength, while increasing dextran content increased Dynamic Binding capacity mainly at the optimal pore size and lower conductivities. Dynamic Binding capacity as high as 200 mg/mL was obtained at the optimum pore size and dextran content.
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ion exchange chromatography of antibody fragments
Biotechnology and Bioengineering, 2007Co-Authors: Anders Ljunglof, Karol Lacki, Chithkala Harinarayan, Robert Van Reis, Jay Mueller, Robert L Fahrner, James Van AlstineAbstract:Effects of pH and conductivity on the ion exchange chromatographic purification of an antigen-bind- ing antibody fragment (Fab) of pI 8.0 were investigated. Normal sulfopropyl (SP) group modified agarose particles (SP Sepharose TM Fast Flow) and dextran modified particles (SP Sepharose XL) were studied. Chromatographic mea- surements including adsorption isotherms and Dynamic breakthrough Binding capacities, were complemented with laser scanning confocal microscopy. As expected static equilibrium and Dynamic Binding capacities were generally reduced by increasing mobile phase conductivity (1-25 mS/ cm). However at pH 4 on SP Sepharose XL, Fab Dynamic Binding capacity increased from 130 to 160 (mg/mL media) as mobile phase conductivity changed from 1 to 5 mS/cm. Decreasing protein net charge by increasing pH from 4 to 5 at 1.3 mS/cm caused Dynamic Binding capacity to increase from 130 to 180 mg/mL. Confocal scanning laser micro- scopystudiesindicate suchincreases wereduetofasterintra- particle mass transport and hence greater utilization of the media's available Binding capacity. Such results are in agreement with recent studies related to ion exchange of whole antibody molecules under similar conditions. Biotechnol. Bioeng. 2007;96: 515-524. 2006 Wiley Periodicals, Inc.
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an exclusion mechanism in ion exchange chromatography
Biotechnology and Bioengineering, 2006Co-Authors: Chithkala Harinarayan, Anders Ljunglof, Jay Mueller, Robert L Fahrner, J Van Alstine, Robert Van ReisAbstract:Protein Dynamic Binding capacities on ion exchange resins are typically expected to decrease with increasing conductivity and decreasing protein charge. There are, however, conditions where capacity increases with increasing conductivity and decreasing protein charge. Capacity measurements on two different commercial ion exchange resins with three different monoclonal antibodies at various pH and conductivities exhibited two domains. In the first domain, the capacity unexpectedly increased with increasing conductivity and decreasing protein charge. The second domain exhibited traditional behavior. A mechanism to explain the first domain is postulated; proteins initially bind to the outer pore regions and electrostatically hinder subsequent protein transport. Such a mechanism is supported by protein capacity and confocal microscopy studies whose results suggest how knowledge of the two types of IEX behavior can be leveraged in optimizing resins and processes.
Xavier Santarelli - One of the best experts on this subject based on the ideXlab platform.
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Comparative study of strong cation exchangers: Structure-related chromatographic performances
Journal of Chromatography B - Analytical Technologies in the Biomedical and Life Sciences, 2018Co-Authors: Vignesh Narasimhan Janakiraman, Charlotte Cabanne, J. Pezzini, Marion Sole, Sophie Maria, Xavier SantarelliAbstract:Chromatographic performances are highly influenced by operational parameters. New ion exchangers have tailored matrices providing low backpressure, thereby allowing high flow velocity. By systematic frontal analysis and selectivity determination at different flow rates, we independently evaluated cation exchangers to facilitate media selection and investigated the relationship between surface modification and chromatographic performances. Structure-extended resins showed higher Binding capacities compared to resins with conventional ligands directly attached to the matrix. Moreover, they maintained high capacities even with high flow velocities. Ligand accessibility was therefore largely enhanced, allowing proteins to interact and bind under harsh conditions with minimal residence/contact time. High throughput resins can be used for purification of high volume and high concentration feedstock in limited time. This results in higher productivity, and could contribute to cost reduction. In this work, we evaluated the Dynamic Binding capacities of various new ion exchange resins at different Binding conductivities for different residence times, and observed that.
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Efficient purification of recombinant proteins fused to maltose-Binding protein by mixed-mode chromatography
Journal of chromatography. A, 2009Co-Authors: Charlotte Cabanne, J. Pezzini, Gilles Joucla, Agnès Hocquellet, Caroline Barbot, Bertrand Garbay, Xavier SantarelliAbstract:Two mixed-mode resins were evaluated as an alternative to conventional affinity resins for the purification of recombinant proteins fused to maltose-Binding protein (MPB). We purified recombinant MBP, MBP-LacZ and MBP-Leap2 from crude Escherichia coli extracts. Mixed-mode resins allowed the efficient purification of MBP-fused proteins. Indeed, the quantity of purified proteins was significantly higher with mixed-mode resins, and their purity was equivalent to that obtained with affinity resins. By using purified MBP, MBP-LacZ and MBP-Leap2, the Dynamic Binding capacity of mixed-mode resins was 5-fold higher than that of affinity resins. Moreover, the recovery for the three proteins studied was in the 50-60% range for affinity resins, and in the 80-85% range for mixed-mode resins. Mixed-mode resins thus represent a powerful alternative to the classical amylose or dextrin resins for the purification of recombinant proteins fused to maltose-Binding protein.
Alois Jungbauer - One of the best experts on this subject based on the ideXlab platform.
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engineering properties of a camelid antibody affinity sorbent for immunoglobulin g purification
Journal of Chromatography A, 2009Co-Authors: Marlene Zandian, Alois JungbauerAbstract:Abstract An IgG-specific camelid antibody matrix (BAC, Naarden, The Netherlands), developed from an immune phage display library, was characterized regarding engineering properties including mass transfer characteristics. Uptake kinetics and equilibrium Binding capacity were determined by a finite bath method. Adsorption kinetic parameters were also determined using a real time biosensor. Slightly different properties to conventional Staphylococcal protein A affinity media were shown; especially a 2–2.5 times lower maximal Binding capacity with a value of 26 mg/ml polyclonal IgG was obtained. Mass transfer could be described by using a film and pore diffusion model ( D e = 5 × 10 −8 cm 2 /s). Determined engineering parameters were used to predict breakthrough behaviour in column mode considering film and pore resistances. The Dynamic Binding capacity at 10% breakthrough did not change when residence time was at least 6 min.
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an immunoaffinity column with a monoclonal antibody as ligand for human follicle stimulating hormone
IEEE Journal of Solid-state Circuits, 2009Co-Authors: Marlene Zandian, Alois JungbauerAbstract:A detailed characterization regarding the chromatographic and biochemical properties of an immunoaffinity column for sample pretreatment using a follicle stimulating hormone (FSH) β-unit specific mAb has been performed. A maximum Binding capacity of approximately 300 μg/mL was determined. A KD value of 4.5×10–8 M could be obtained in batch isotherm measurements. The Dynamic Binding capacity at 10% did not change when residence time was at least 6 min. High selectivity could be demonstrated using crude culture supernatant from FSH producing Chinese hamster ovary (CHO) cells. A great stability of the matrix was confirmed by performing 100 consecutive chromatographic runs without loss of capacity.
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adsorption of pdna on microparticulate charged surface
Journal of Biotechnology, 2009Co-Authors: Christina Paril, David Horner, Robert Ganja, Alois JungbauerAbstract:Plasmid DNA purification strategies are often based on chromatographic processes, which often suffer from low Dynamic Binding capacities and poor productivity. The limitations of conventional chromatography media for adsorption of pDNA led us to the investigation of irregular polymeric microparticles with quaternary amine functionality. They have an average particle size of 8-10 microm. The adsorption properties of microparticles are comparable to monolithic supports, i.e. the adsorption process is accomplished within 15 min and the Binding capacity is in the range of 15 mg/mL. Desorption with NaCl renders nearly theoretical yield. Due to the small particle size the mode of operation is rather batch adsorption than column chromatography. The phase separation is facilitated by formation of reversible flocs of pDNA and microparticles. The implementation of microparticles in a lab-scale capture step out of processed E. coli lysates demonstrated their potential as a high speed and high capacity material for pDNA purification.
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hydrophobic interaction chromatography of proteins ii Binding capacity recovery and mass transfer properties
Journal of Chromatography B, 2003Co-Authors: Rainer Hahn, Karin Deinhofer, Christine Machold, Alois JungbauerAbstract:Hydrophobic interaction chromatography media suited for large scale separations were compared regarding Dynamic Binding capacity, recovery and mass transfer properties. In all cases, pore diffusion was the rate limiting step. Reduced heights equivalent to a theoretical plate for bovine serum albumin derived from breakthrough curves at reduced velocities between 60 and 1500 ranged from 10 to 700. Pore diffusion coefficients were derived from pulse response experiments for the model proteins α-lactalbumin, lysozyme, β-lactoglobulin, bovine serum albumin and immunoglobulin G. Diffusivity of lysozyme did not follow the trend of decreasing diffusivity with increasing molecular mass, as observed for the rest of the proteins. In general, mass transfer coefficients were smaller compared to ion-exchange chromatography. Dynamic Binding capacities for the model protein bovine serum albumin varied within a broad range. However, sorbents based on polymethacrylate showed a lower Dynamic capacity than media based on Sepharose. Some sorbents could be clustered regarding Binding capacity affected by salt. These sorbents exhibited a disproportional increase of Binding capacity with increasing ammonium sulfate concentration. Recovery of proteins above 75% could be observed for all sorbents. Several sorbents showed a recovery close to 100%.
Gary P Drobny - One of the best experts on this subject based on the ideXlab platform.
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studies of Dynamic Binding of amino acids to tio2 nanoparticle surfaces by solution nmr and molecular Dynamics simulations
Langmuir, 2020Co-Authors: Mengjun Xue, Janani Sampath, Rachel N Gebhart, Havard J Haugen, Petter S Lyngstadaas, Jim Pfaendtner, Gary P DrobnyAbstract:Adsorption of biomolecules onto material surfaces involves a potentially complex mechanism where molecular species interact to varying degrees with a heterogeneous material surface. Surface adsorption studies by atomic force microscopy, sum frequency generation spectroscopy, and solid-state NMR detect the structures and interactions of biomolecular species that are bound to material surfaces, which, in the absence of a solid-liquid interface, do not exchange rapidly between surface-bound forms and free molecular species in bulk solution. Solution NMR has the potential to complement these techniques by detecting and studying transiently bound biomolecules at the liquid-solid interface. Herein, we show that dark-state exchange saturation transfer (DEST) NMR experiments on gel-stabilized TiO2 nanoparticle (NP) samples detect several forms of biomolecular adsorption onto titanium(IV) oxide surfaces. Specifically, we use the DEST approach to study the interaction of amino acids arginine (Arg), lysine (Lys), leucine (Leu), alanine (Ala), and aspartic acid (Asp) with TiO2 rutile NP surfaces. Whereas Leu, Ala, and Asp display only a single weakly interacting form in the presence of TiO2 NPs, Arg and Lys displayed at least two distinct bound forms: a species that is surface bound and retains a degree of reorientational motion and a second more tightly bound form characterized by broadened DEST profiles upon the addition of TiO2 NPs. Molecular Dynamics simulations indicate different surface bound states for both Lys and Arg depending on the degree of TiO2 surface hydroxylation but only a single bound state for Asp regardless of the degree of surface hydroxylation, in agreement with results obtained from the analysis of DEST profiles.
