Enhancing protein capacity of rigid macroporous polymeric adsorbent.

A macroporous poly(glycidyl methacrylate-triallyl isocyanurate-divinylbenzene) resin was synthesized and modified with diethylamine to yield an anion-exchange resin suitable for protein adsorption. Efforts were made to enhance protein ion exchange capacity of the resin by investigating the copolymer composition. Different synthesis recipes were attempted, and the resultant resins were characterized by measuring the specific surface area and the adsorption ability using bovine serum albumin (BSA) as a model protein. The intraparticle pore size distribution measured by mercury porosimetry showed that the pores in the range of 40-120 nm took 75% of the total pore volume, indicating that the ion exchanger was favorable for protein adsorption. BSA capacity obtained with an appropriate recipe was as high as 78.6 mg/g wet resin or 50 mg/mL packed volume, which was higher than the capacities of some commercially available ion exchangers. Moreover, by using a pore diffusion model, the effective pore diffusivity of BSA was found to be 5.5 x 10(-12) m(2)/s, similar to those in the commercial ion exchangers.     

Agar-based magnetic affinity support for protein adsorption

Magnetic colloidal particles were prepared by a coprecipitation method. The particles were composed of nanometer-sized superparamagnetic Fe(3)O(4) particles stabilized by lauric acid. Then, magnetic agar gel beads were produced by a water-in-oil emulsification method using a mixture of agar solution and the magnetic colloidal particles as the aqueous phase. A reactive triazine dye, Cibacron blue 3GA (CB), was coupled to the gel to prepare an agar-based magnetic affinity support (MAS) for protein adsorption. The support showed good magnetic responsiveness in a magnetic field. Bovine serum albumin (BSA) was used as a model protein to test adsorption equilibrium and kinetic behavior of the MAS. The adsorption equilibrium of BSA to the MAS was described by the Langmuir-type isotherm. Adsorption capacity of the MAS for BSA was up to 25 mg/mL at a CB coupling density of 1.6 micromol/mL. The effect of ionic strength on BSA adsorption was complex, exhibiting a maximum capacity at an ionic strength of 0.06 mol/L. The adsorption of BSA to the MAS was also influenced by pH. Uptake rate of BSA to the MAS was analyzed using a pore diffusion model. The pore diffusion coefficient was estimated to be 1.75 x 10(-11) m(2)/s. Finally, recycled use of the MAS demonstrated the stability of the MAS in protein adsorption and magnetic responsiveness.     

Synthesis of tentacle-type magnetic beads as immobilized metal-chelate affinity support for cytochrome c adsorption

Magnetic poly(2-hydroxyethylmethacrylate) (mPHEMA) beads with an average diameter of 100-140 microm were produced by suspension polymerization in the presence of magnetite particles (i.e. Fe3O4). Specific surface area and average pore size of the magnetic beads was found to be 50 m2/g and 819 nm, respectively. Ester groups in the mPHEMA structure were converted to imine groups by reacting with poly(ethyleneimine) (PEI) in the presence of NaH. Amino (-NH2) content of PEI-attached mPHEMA beads was determined as 102 mg PEI/g. Then, Cu2+ ions were chelated on the magnetic beads in the range of 20-793 micromol Cu2+/g. Cytochrome c (cyt c) adsorption was performed on the metal chelating beads from aqueous solutions containing different amounts of cyt c at different pHs, Cu2+ loadings and temperatures. Cyt c adsorption on the mPHEMA/PEI beads was 4.6 mg/g. Cu2+ chelation increased the cyt c adsorption significantly (40.1 mg/g). Adsorption capacity increased with Cu2+ loading and then reached a saturation value. Cyt c adsorption decreased with increasing temperature. Cyt c molecules could be reversibly adsorbed and eluted ten times with the magnetic adsorbents without noticeable loss in their cyt c adsorption capacity. The applicability of two kinetic models including pseudo-first order and pseudo-second order model was estimated on the basis of comparative analysis of the corresponding rate parameters, equilibrium capacity and correlation coefficients. Results suggest that chemisorption processes could be the rate-limiting step in the adsorption process. In the last part of this article, cyt c adsorption experiments were performed in a magnetically stabilized fluidized bed (MSFB) system at optimum conditions determined from the batch experiments. The adsorption capacity decreased significantly from 46.8 to 15.4 mg/g polymer with the increase of the flow-rate from 0.5 to 4.0 ml/min. The resulting magnetic chelator beads possessed excellent long-term storage stability.     

Immobilization of lipase on methyl-modified silica aerogels by physical adsorption

In this work, methyl-modified silica aerogels, a new kind of macro-porous material with high porosity, were used as carriers to immobilize lipase by adsorption. SEM, TEM, nitrogen adsorption device, and thermogravimetric analysis were used to characterize the properties of modified aerogels. The surface area was 395.6 m(2)/g, and the average pore diameter was 68.72 nm. The contact angle of aerogel particles increased from 20.9 degrees to 99.2 degrees after methyl modification. Reaction characteristics of the material after enzyme loading were also discussed. The results showed that adsorption capacity could reach 67.42 mg/g; and the maximal enzyme activity was 19.87 micromol min(-1)mg(-1) (protein), and activity retention could reach 56.44%. It is worth mentioning that the amount of modified aerogels added had significant effects on the diameter of droplets and the mass transfer behavior of substrates in the reaction emulsion. Online microscope was used to visualize the droplets in the emulsion, where the aerogel particles were observed locating at the interface of oil and water. The average diameter of droplets reached the minimum when 0.06 g of modified aerogels was added into the reaction emulsion which contained 10 ml of oil and 10 ml of phosphate buffer solution. The phenomenon was resulted from the wettability of methyl-modified silica aerogels.     

Direct observation of intraparticle equilibration and the rate-limiting step in adsorption of proteins in chromatographic adsorbents with confocal laser scanning microscopy

The adsorption of different proteins in a single biospecific and hydrophobic adsorbent particle for preparative protein chromatography has been observed directly by confocal laser scanning microscopy as a function of time at a constant bulk concentration c(b). The bulk concentration was in the non-linear part of the adsorption isotherm. At all times the concentration of free protein at the particle surface was almost equal to the bulk content indicating that external mass transfer resistance is not rate limiting for the adsorption under these conditions. Inside the particles a distinct maximum in adsorbed and free protein concentration that moved inside to a distance of approximately 0.2 R (R particle radius) from the particle surface, was observed. This is due to a decreasing solid-phase density and adsorptive capacity in the particle between 0.8 R and R indicating that the fraction of macropores (or void space) is larger in the outer than in the inner part of the adsorbent particles. By increasing the bulk concentration by a factor of 10 the equilibration time was reduced by about the same magnitude. This is in agreement with the concentration dependence of the effective pore diffusion coefficient D(p,eff)=D(p)/[epsilon(p)[1+nK/(K +c)(2)]] derived from the mass conservation relations describing the adsorption process. The time dependence protein adsorption up to approximately 90% of the equilibration value q* could be described by a bilinear free driving force model. The rapid equilibration in the outer part of the particle with a half-life time of approximately 100 s in the studied systems accounted for 0.3-0.4 q*. The slower equilibration with a up to ten times longer half-life time, was the adsorption in the inner part of the particle that outside 0.5 R accounts for 0.5-0.6 q*. These data were compared with literature data for batch adsorption of proteins in biospecific, hydrophobic and ion-exchange adsorbents. They could also be described by a bilinear free driving force model, with about the same quantitative results as obtained for similar conditions in the single particle experiments. The static adsorption parameters, maximum binding site concentration n, and dissociation constant for the protein binding to a binding site K, were determined from Scatchard plots. For the same protein-adsorbent system the plots changed from linear to non-linear with increasing n. This change occurred when the average distance between adjacent binding sites become of the same order of magnitude as the size of the binding site or adsorbed protein. This causes a shielding of free binding sites increasing with n and the concentration of adsorbed protein, yielding a concentration dependence in K. These results show that for a high throughput and rapid adsorption in preparative chromatography, the adsorption step should be carried out in the non-linear part of the adsorption isotherm with concentrations up to c(b) where q*/c(b)>/=10 to obtain high protein recoveries. To avoid tailing due to the flow of adsorbed proteins in the inner part of the particles further into the particles at the start of the desorption, and to speed up desorption rates, protein adsorption in the particle within 0.5 R from the particle center should be avoided. This requires the further development of suitable pellicular particles for preparative protein chromatography that meet this requirement.     

Protein adsorption and transport in cation exchangers with a rigid backbone matrix with and without polymeric surface extenders

We compare the properties and protein adsorption characteristics of two polymeric cation exchangers: UNOsphere S, which has an open macroporous architecture, and Nuvia S, which is based on a very similar backbone matrix but contains sulfonated polymeric surface extenders. A monoclonal IgG and lysozyme were used as model adsorbates. The characteristic pore sizes, determined by inverse size exclusion chromatography, were about 140 nm for UNOsphere S, and only about 10 nm for Nuvia S, indicating that the polymeric extenders occupy a substantial portion of the base matrix pores. Greater exclusion limits were found for Nuvia S in 1 M NaCl and for a similar matrix containing uncharged surface extenders, suggesting that the polymeric extenders collapse partially at high ionic strength or when they are uncharged. Large equilibrium binding capacities were obtained for Nuvia S, approaching 320 ± 10 mg/mL of particle volume for both proteins in comparison with the UNOsphere S values of 170 ± 10 and 120 ± 10 mg/mL for lysozyme and IgG, respectively. Much higher adsorption rates were also found for Nuvia S, and the rate was nearly independent of protein concentration in solution. Confocal laser scanning microscopy showed very sharp intraparticle protein concentration profiles for UNOsphere S, consistent with a pore diffusion mechanism but diffuse concentration profiles for Nuvia S, consistent with a solid diffusion mechanism. The improved capacity and transport afforded by the polymeric extenders provide substantial potential benefits for bioprocess applications without sacrificing the desirable flow properties of the backbone matrix.     

Particle-by-particle quantification of protein adsorption onto poly(ethylene glycol) grafted surfaces

The use and advantage of flow cytometry as a particle-by-particle, low sampling volume, high-throughput screening technique for quantitatively examining the non-specific adsorption of proteins onto surfaces is presented. The adsorption of three proteins: bovine serum albumin (BSA), immunoglobulin gamma (IgG) and protein G, incubated at room temperature for 2 h onto organosilica particles modified with poly(ethylene glycol) (PEG) of increasing MW (2000, 3400, 6000, 10,000 and 20,000 g mol(-1)) and grafted amounts (0.14-1.4 mg m(-2)) was investigated as a model system. Each protein exhibited Langmuir-like, high affinity monolayer limited adsorption on unmodified particles with the proteins reaching surface saturation at 1.8, 4.0 and 2.5 mg m(-2) for BSA, IgG and protein G, respectively. Protein adsorption on PEG-modified surfaces was found to decrease with increasing amounts of grafted polymer. PEG grafting amounts >0.6 mg m(-2) effectively prevented the adsorption of the larger two proteins (BSA and IgG) while a PEG grafting amount >1.3 mg m(-2) was required to prevent the adsorption of the smaller protein G.     

Structural and performance characteristics of representative anion exchange resins used for weak partitioning chromatography

Weak partitioning chromatography (WPC) has been proposed for the purification of monoclonal antibodies using an anion exchange (AEX) resin to simultaneously remove both acidic and basic protein impurities. Despite potential advantages, the relationship between resin structure and WPC performance has not been evaluated systematically. In this work, we determine the structure of representative AEX resins (Fractogel® EMD TMAE HiCap, Q Sepharose FF, and POROS 50 HQ) using transmission electron microscopy and inverse size exclusion chromatography and characterize protein interactions while operating these resins under WPC conditions using two mAb monomers, a mAb dimer, mAb multimers, and BSA as model products and impurities. We determine the isocratic elution behavior of the weakly bound monomer and dimer species and the adsorptive and mass transfer properties of the strongly bound multimers and BSA by confocal laser scanning microscopy. The results show that for each resin, using the product K_p value as guidance, salt, and pH conditions can be found where mAb multimers and BSA are simultaneously removed. Isocratic elution and adsorption mechanisms are, however, different for each resin and for the different components. Under WPC conditions, the Fractogel resin exhibited very slow diffusion of both mAb monomer and dimer species but fast adsorption for both mAb multimers and BSA with high capacity for BSA, while the Sepharose resin, because of its small pore size, was unable to effectively remove mAb multimers. The POROS resin was instead able to bind both multimers and BSA effectively, while exhibiting a greater resolution of mAb monomer and dimer species. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:425–434, 2017     

Porous devices derived from co-continuous polymer blends as a route for controlled drug release

In this study we examine the release profile of bovine serum albumin (BSA) from a porous polymer matrix derived from a co-continuous polymer blend. The porosity is generated through the selective extraction of one of the continuous phases. This is the first study to examine the approach of using morphologically tailored co-continuous polymer blends as a template for generating porous polymer materials for use in controlled release. A method for the preparation of polymeric capsules is introduced, and the effect of matrix pore size and surface area on the BSA release profile is investigated. Furthermore, the effect of surface charge on release is examined by surface modification of the porous substrate using layer-by-layer deposition techniques. Synthetic, nonerodible polymer, high-density polyethylene (HDPE), was used as a model substrate prepared by melt blending with two different styrene-ethylene-butylene copolymers. Blends with HDPE allow for the preparation of porous substrates with small pore sizes (300 and 600 nm). A blend of polylactide (PLA) and polystyrene was also used to prepare porous PLA with a larger pore size (1.5 microm). The extents of interconnectivity, surface area, and pore dimension of the prepared porous substrates were examined via gravimetric solvent extraction, BET nitrogen adsorption, mercury porosimetry, and image analysis of scanning electron microscopy micrographs. With a loading protocol into the porous HDPE and PLA involving the alternate application of pressure and vacuum, it is shown that virtually the entire porous network was accessible to BSA loading, and loading efficiencies of between 80% and 96% were obtained depending on the pore size of the carrier and the applied pressure. The release profile of BSA from the microporous structure was monitored by UV spectrophotometry. The influence of pore size, surface area, surface charge, and number of deposited layers is demonstrated. It is shown that an effective closed-cell structure in porous PLA can be prepared, effectively eliminating all short-term BSA release.     

Biosorption of Cr (VI) from aqueous solution by Rhizopus nigricans

The study was aimed to quantify the Cr sorption ability of powdered biomass of Rhizopus nigricans at the best operating conditions. The influence of solution pH, agitation, Cr (VI) concentration, biomass dosage, contact time, biomass particle size and temperature were studied. The optimum pH for biosorption of Cr (VI) was found to be 2.0. Higher adsorption percentage was noted at lower initial concentrations of Cr ions, while the adsorption capacity of the biomass increased with increasing concentration of ions. Optimum biomass dosage was observed as 0.5% (w/v). More than 75% of the ions were removed within 30 min of contact and maximum removal was obtained after 8 h. Biomass particles of smaller size (90 microm) gave maximum adsorption (99.2%) at 100 mg/l concentration. The adsorption capacity increased with increase in temperature and agitation speed and the optimum were determined as 45 degrees C at 120 rpm. Freundlich and Langmuir isotherms were used to evaluate the data and the regression constants were derived. The adsorption rate constant values (Kad) were calculated for different initial concentration of Cr ions and the sorption was found to be higher at lower concentration (100 mg/l) of metal ion.     

Preparation and preliminary evaluation of macroporous magnetic agarose particles for bioseparation

Macroporous magnetic agarose particles (MMAPs) were prepared with calcium carbonate as the porogent by the water-in-oil suspension thermal regeneration method. MMAPs with good sphericity and appropriate particle size were obtained. The physical properties of the beads were determined and it was found that the water content (92.1%), porosity (94.4%) and mean pore diameter (120.1 nm) of the MMAPs were higher than those for the normal magnetic particles, indicating successful generation of macropores after calcium carbonate addition. Compared with normal magnetic particles, the mass transfer of biomolecules in MMAPs was remarkably enhanced. Finally, MMAPs were modified with 5-amino-benzimidazol (ABI) ligand and the adsorption capacity of IgG reached 153 mg/mL, higher than that of the normal magnetic particles (126 mg/mL). Moreover, adsorption behavior of MMAPs to IgG was little changed after twenty-five recycled use. Hence, MMAPs prepared herein showed great potential for bioseparation.     

Studies on adsorption isotherms of endotoxin and BSA using an affinity column

In this paper, an adsorbent with dimethylamine ligand for endotoxin removal was prepared and used to study the adsorption isotherms of endotoxin and BSA. The results showed that as the introducing of endotoxin, the maximum adsorption capacity q_m of BSA increased from 7.24 to 7.74 mg/mL and the apparent association constant K_A of BSA decreased from 14.06 to 11.48 mL/mg. The adsorption isotherms of BSA changed from Langmuir model to Multilayer model. All these gave the direct evidences to construct the adsorption process. By comparing adsorption isotherms of endotoxin with BSA, it was found that the apparent association constant K_A of endotoxin was much higher than that of BSA.     

Light scattering from suspensions of membrane fragments derived from sonication of beef heart mitochondria.

The intensity of light scattering from suspensions of membrane fragments prepared by sonication of beef heart mitochondria in the presence of EDTA at alkaline pH (ESMP) was determined at 45, 90, and 135 degrees with light of wavelength 546 nm. The dissymmetry ratio Z = I45 degrees c/I135 degrees c, where I45 degrees c and I135 degrees c are the scattering intensities at 45 and 135 degrees extrapolated to zero particle concentration and corrected for reflectance effects, was used to calculate particle size from the Rayleigh-Gans-Debye theory. An average particle diameter D of 184-190 nm was obtained, within the range of particle diameter 50-300 nm determined previously by electron microscopy. This average diameter determined by light scattering is a useful parameter for characterization of ESMP particle size. We propose the term: light scattering average particle diameter, DLS, for this parameter. The refractive index of ESMP was determined to be 1.443 by measurement of scattering intensity in buffer solutions of varying sucrose concentration. The value of Z was independent of sucrose concentration in this determination, showing that the particles are osmotically inactive toward sucrose. The values of average particle diameter DLS and of refractive index fall within the range of validity of the Rayleigh-Gans-Debye theory, for which light scattering changes are attributable solely to dimension change, rather than to change in particle refractive index. Uptake of water accompanying energy-linked salt uptake in ESMP was calculated from light scattering changes to be 0.18 mul of H2O/mg of protein, compared with 0.49 mul of H2O/mg of protein measured by dextran inaccessibility. Measurement of light scattering changes provides a rapid and sensitive method for determining volume changes of ESMP. The magnitude of the volume change observed during energy-linked water and salt uptake and the initial degree of hydration suggests that ESMP are analogous to polyelectrolyte gels with regard to sorption of strong electrolytes and that the Donnan formulation for ion exchange equilibria may be usefully applied to these processes in ESMP.     

Circular dichroism studies on conformational changes in protein molecules upon adsorption on ultrafine polystyrene particles

The conformational changes in well-characterized model proteins [bovine ribonuclease A (RNase A), horseradish peroxidase, sperm-whole myoglobin, human hemoglobin, and bovine serum albumin (BSA)] upon adsorption on ultrafine polystyrene (PS) particles have been studied using circular dichroism (CD) spectroscopy. These proteins were chosen with special attention to molecular flexibility. The ultrafine PS particles were negatively charged and have average diameters of 20 or 30 nm. Utilization of these ultrafine PS particles makes it possible to apply the CD technique to determine the secondary structure of proteins adsorbed on the PS surface. Effects of protein properties and adsorption conditions on the extent of the changes in the secondary structure of protein molecules upon adsorption on ultrafine PS particles were studied. The CD spectrum changes upon adsorption were significant in the "soft" protein molecules (myoglobin, hemoglobin, and BSA), while they were insingnificant in the "rigid" proteins (RNase A and peroxidase). The soft proteins sustained a marked decrease in alpha-helix content upon adsorption. Moreover, the native alpha-helix content, which is given as the percentage of the alpha-helix content in the free proteins, of adsorbed BSA was found to decrease with decreasing pH and increase with increasing adsorbed amount. These observations confirm some well-known hypotheses for the confirmational chages in protein molecules upon adsorption. (c) 1992 John Wiley & Sons, Inc.     

Fouling of an anion exchange chromatography operation in a monoclonal antibody process: Visualization and kinetic studies

Fouling of chromatographic resins over their operational lifetimes can be a significant problem for commercial bioseparations. In this article, scanning electron microscopy (SEM), batch uptake experiments, confocal laser scanning microscopy (CLSM) and small‐scale column studies were applied to characterize a case study where fouling had been observed during process development. The fouling was found to occur on an anion exchange (AEX) polishing step following a protein A affinity capture step in a process for the purification of a monoclonal antibody. Fouled resin samples analyzed by SEM and batch uptake experiments indicated that after successive batch cycles, significant blockage of the pores at the resin surface occurred, thereby decreasing the protein uptake rate. Further studies were performed using CLSM to allow temporal and spatial measurements of protein adsorption within the resin, for clean, partially fouled and extensively fouled resin samples. These samples were packed within a miniaturized flowcell and challenged with fluorescently labeled albumin that enabled in situ measurements. The results indicated that the foulant has a significant impact on the kinetics of adsorption, severely decreasing the protein uptake rate, but only results in a minimal decrease in saturation capacity. The impact of the foulant on the kinetics of adsorption was further investigated by loading BSA onto fouled resin over an extended range of flow rates. By decreasing the flow rate during BSA loading, the capacity of the resin was recovered. These data support the hypothesis that the foulant is located on the particle surface, only penetrating the particle to a limited degree. The increased understanding into the nature of the fouling can help in the continued process development of this industrial example. Biotechnol. Bioeng. 2013; 110:2425–2435. © 2013 Wiley Periodicals, Inc.     

Dye-ligand poly(GMA–TAIC–DVB) affinity adsorbent for protein adsorption

Macroporous poly(glycidyl methacrylate-triallyl isocyanurate-divinylbenzene) was prepared by a radical suspension copolymerization. Reaction of the copolymer with 2-hydroxyethyl amine was employed to obtain a hydrophilic matrix. An affinity dye, Cibacron blue 3GA, was then coupled covalently to prepare a novel macroporous affinity adsorbent. The surface and pore structure of the affinity adsorbent were examined by scanning electron micrography (SEM). SEM observations showed that the affinity adsorbent abounded in macropores. Bovine serum albumin (BSA) and lysozyme (Lys) were used as samples to examine the adsorption properties of the adsorbent. Under appropriate conditions, the affinity adsorbent had a capacity of 15.5 mg BSA/g and 22.3 mg Lys/g (wet adsorbent weight). The adsorbed proteins could be desorbed by increasing liquid phase ionic strength or by using a NaOH solution, and the adsorbent could be recycled for protein adsorption.     

One-pot synthesis and characterization of cross-linked quaternized chitosan microspheres as protein adsorbent

The one-pot synthesis and characterization of cross-linked quaternized chitosan microspheres (CQCM) as a protein adsorbent are presented. First of all, chitosan particles were prepared by spray drying method, and then they were quaternized and cross-linked in turn with glycidyltrimethylammonium (GTMAC) chloride and glutaraldehyde in isopropanol containing 10% water in one-pot. The effect of the reaction temperature, reaction time and the amounts of added GTMAC and glutaraldehyde on the protein adsorption ability of CQCM was investigated. The adsorption behavior of the CQCM prepared in the optimum synthetic conditions was well described by the Langmuir isotherm with maximum adsorption capacity equal to 1424 mg BSA/g dry weight. The particle size ranged from 7.6 to 48.9 μm. The mechanism of adsorption-desorption of BSA to the CQCM was ion-exchange. Finally, the extraction of soybean peroxidase from crude soybean peroxidase solution using the CQCM was performed.     

Adsorption of trypsin on commercial silica gel

The immobilization of trypsin onto various commercial silica gels was studied. Silica gels were used directly and characterized by mercuric porosimetry. Agitation rates (100–740 rpm) and particles size (35–75 to 250–500 μm) of silica gels did not affect the trypsin immobilization capacity. The pore size (3 to 15 nm) is a limiting factor of the trypsin adsorption onto the mesopores structure of silica gels. The adsorption of trypsin was determined as a function of their initial concentration and multilayer formed at high trypsin concentration.     

Tailoring size and structural distortion of Fe3O4 nanoparticles for the purification of contaminated water

Fe₃O₄ magnetic nanoparticles with different particle sizes were synthesized using two methods, i.e., a co-precipitation process and a polyol process, respectively. The atomic pair distribution analyses from the high-energy X-ray scattering data and TEM observations show that the two kinds of nanoparticles have different sizes and structural distortions. An average particle size of 6–8 nm with a narrow size distribution was observed for the nanoparticles prepared with the co-precipitation method. Magnetic measurements show that those particles are in ferromagnetic state with a saturation magnetization of 74.3 emu g⁻¹. For the particles synthesized with the polyol process, a mean diameter of 18–35 nm was observed with a saturation magnetization of 78.2 emu g⁻¹. Although both kinds of nanoparticles are well crystallized, an obviously higher structural distortion is evidenced for the co-precipitation processed nanoparticles. The synthesized Fe₃O₄ particles with different mean particle size were used for treating the wastewater contaminated with the metal ions, such as Ni(II), Cu(II), Cd(II) and Cr(VI). It is found that the adsorption capacity of Fe₃O₄ particles increased with decreasing the particle size or increasing the surface area. While the particle size was decreased to 8 nm, the Fe₃O₄ particles can absorb almost all of the above-mentioned metal ions in the contaminated water with the adsorption capacity of 34.93 mg/g, which is ∼7 times higher than that using the coarse particles. We attribute the extremely high adsorption capacity to the highly-distorted surface.     

Ion exchange chromatography of antibody fragments

Effects of pH and conductivity on the ion exchange chromatographic purification of an antigen-binding antibody fragment (Fab) of pI 8.0 were investigated. Normal sulfopropyl (SP) group modified agarose particles (SP Sepharosetrade mark Fast Flow) and dextran modified particles (SP Sepharose XL) were studied. Chromatographic measurements 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 microscopy studies indicate such increases were due to faster intra-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.