Effect of the counter-anion type and concentration on the liquid chromatography retention of beta-blockers

Zirconia beads (25-38 microm in diameter) were modified with N,N,N',N'-ethylenediaminetetramethylenephosphonic acid to generate a zirconia based pseudoaffinity support, further referred to as r_PEZ. The influence of pH, salt concentration and temperature on the binding of human immunoglobulin G (hIgG) to r_PEZ was studied. Temperature had no significant impact on the maximum binding capacity (Qmax), and the equilibrium-binding constant (Kd), whereas pH and the salt concentration had a noticeable impact on both Qmax and Kd. The Qmax value of 55 mg hIgG/ml of bead was obtained at a pH of 5.5 and found to decrease with an increase of pH. The modified zirconia support allowed the separation of immunoglobulins (IgG, IgA and IgM) from untreated human serum. Elution was possible under mild conditions with a step salt gradient. Overall protein recoveries in the range of 109-125% were obtained with human serum. Human IgG, human IgA, and human IgM yields of 29.50+/-6.3, 3.22+/-0.7, and 6.84+/-0.7%, respectively, were obtained at a linear velocity of 4.32 cm/min. Purity of products, obtained from a single chromatographic step was estimated to be greater than 89.0+/-2.6%. The utility of r_PEZ in the selective removal of immunoglobulins, as in immunoadsorption was discussed.     

Anti-IgG immobilized controlled-pore glass. Thionyl chloride-activated succinamidopropyl-glass as a covalent immobilization matrix.

Rabbit anti-bovine IgG was covalently immobilized on thionyl chloride-activated succinamidopropyl controlled-pore glass (CPG) beads (3000 A pore diam; 120/200 mesh). Thionyl chloride-activated beads remained stable for over 1 y retaining full capability of immobilizing protein upon recirculation of protein solution. The immobilized anti-bovine IgG is capable of binding IgG with a dissociation constant (Kd) of 9.45 x 10(-7) M and a capacity of 0.85 g/L of matrix. A column of immobilized anti-IgG is able to remove all detectable contaminating IgG from partially purified enzyme preparations of sulfhydryl oxidase and gamma-glutamyltransferase as determined by ELISA and Western blot. The column matrix could be regenerated by washing with 0.1M acetic acid, pH 2.8.     

Superporous agarose anion exchangers for plasmid isolation

Superporous agarose beads have wide, connecting flow pores allowing large molecules such as plasmids to be transported into the interior of the beads by convective flow. The pore walls provide additional surface for plasmid binding thus increasing the binding capacity of the adsorbent. Novel superporous agarose anion exchangers have been prepared, differing with respect to bead diameter, superpore diameter and type of anion-exchange functional group (poly(ethyleneimine) and quaternary amine). The plasmid binding capacities were obtained from breakthrough curves and compared with the binding capacity of homogeneous agarose beads of the same particle size. Significantly, the smaller diameter superporous agarose beads were found to have four to five times higher plasmid binding capacity than the corresponding homogeneous agarose beads. The experimentally determined plasmid binding capacity was compared with the theoretically calculated surface area for each adsorbent and fair agreement was found. Confocal microscopy studies of beads with adsorbed, fluorescently labelled plasmids aided in the interpretation of the results. Superporous poly(ethyleneimine)-substituted beads with a high ion capacity (230 micromol/ml) showed a plasmid binding of 3-4 mg/ml adsorbent. Superporous quaternary amine-substituted beads had a lower ion capacity (81 micromol/ml) and showed a correspondingly lower plasmid binding capacity (1-2 mg/ml adsorbent). In spite of the lower capacity, the beads with quaternary amine ligand were preferred, due to their much better plasmid recovery (70-100% recovery). Interestingly, both capacity and recovery was improved when the plasmid adsorption step was carried out in the presence of a moderate salt concentration. The most suitable superporous bead type (45-75 microm diameter beads; 4 microm superpores; quaternary amine ligand) was chosen for the capture of plasmid DNA from a clarified alkaline lysate. Two strategies were evaluated, one with and one without enzymatic digestion of RNA. The strategy without RNase gave high plasmid recovery, quantitative removal of protein and a 70% reduction in RNA.     

Concanavalin a immobilized affinity adsorbents for reversible use in yeast invertase adsorption

Concanavalin A (Con A) immobilized poly(2-hydroxyethyl methacrylate) (PHEMA) beads were investigated for specific adsorption of yeast invertase from aqueous solutions. PHEMA beads were prepared by a suspension polymerization technique with an average size of 150-200 microm, and activated by epichlorohydrin. Con A was then immobilized by covalent binding onto these beads. The maximum Con A immobilization was found to be 10 mg/g. The invertase-loading capability of the PHEMA/Con A beads was 107 mg/g. The maximum invertase adsorption capacity on the PHEMA/Con A adsorbents was observed at pH 5.0. The values of the Michaelis constant K(m) of invertase were significantly larger upon adsorption, indicating decreased affinity by the enzyme for its substrate, whereas V(max) was smaller for the adsorbed invertase. Adsorption improved the pH stability of the enzyme as well as its temperature stability. Thermal stability was found to increase with adsorption. The adsorbed enzyme activity was found to be quite stable in repeated experiments. Storage stability of adsorbed invertase.     

重组蛋白A亲和填料的制备及其性能评价

为了获得一种优良的抗体纯化介质,制备了重组金黄色葡萄球菌蛋白A(rProtein A)亲和填料,并考察了所制备的亲和填料的纯化性能。利用自行构建的rProtein A工程菌,经诱导表达、纯化获得rProtein A纯品,将其偶联到经环氧氯丙烷活化的Sepharose 4 Fast Flow凝胶上,得到rProtein A亲和填料,并使用兔抗尿酸氧化酶抗体对该填料的性能进行验证。结果显示,在自制的rProtein A亲和填料上rProtein A浓度为1.5×10~4 mol/L。采用Scatchard模型分析,得到其解离常数和最大表观吸附量分别为2.28×10~7 mol/L和20.697 g/L,说明制得的rProtein A亲和填料对抗体有很好的结合能力。将该填料于0.1 mol/L NaOH溶液中浸泡1 h,其色谱性能未见变化。将该填料用于纯化兔抗体,湿胶结合抗体量可达19 mg/mL;一步柱色谱即可得到电泳纯度的抗体样品,回收率高于96%。本研究为rProtein A亲和填料的国产化奠定了基础。     

Evaluation of new high-density ion exchange adsorbents for expanded bed adsorption chromatography

New adsorbents Q HyperZ and CM HyperZ composed of hydrogel-filled porous zirconium oxide particles were evaluated for expanded bed adsorption applications in the present work. The HyperZ adsorbents have wet density of 3.16 g ml(-1), particle size of 44.5-100.8 microm and average sphere diameter of 67 microm. The bed expansion as the function of flow velocity and fluid viscosity was measured and correlated with Richardson-Zaki equation. The suitable expansion factor was considered less than 2.5, while the corresponding flow velocity was about 450 cmh(-1). Liquid mixing in the bed was determined to evaluate the stability of expanded bed. The Bodenstein numbers tested were higher than 40 and the axial mixing coefficients (D(ax)) were between 0.5 and 9.7x10(-6)m(2)s(-1), which demonstrated that a stable expanded bed could be formed under suitable operation conditions. Bovine serum albumin (BSA) and lysozyme were used as model proteins to estimate the adsorption capacities of Q and CM HyperZ, respectively. The maximum equilibrium adsorption of Q and CM HyperZ could reach 45.7 and 27.2 mg g(-1) drained adsorbents, respectively. It was found that yeast cells had little influence on the adsorption capacities of the two adsorbents tested. The dynamic adsorption capacity of BSA at 10% breakthrough with Q HyperZ was 35.9 mg g(-1) drained adsorbent at flow velocity of 100 cm h(-1) for packed bed adsorption. The values for expanded bed adsorption were 34.4 mg g(-1) drained adsorbent at flow velocity of 200 cm h(-1), 33.6 mg g(-1) drained adsorbent at 300 cm h(-1) and 31.7 mg g(-1) drained adsorbent 400 cm h(-1). The results demonstrated that Q HyperZ and CM HyperZ are suitable for expanded bed adsorption of biomolecules.     

Hemoglobin binding from human blood hemolysate with poly(glycidyl methacrylate) beads

Metal-chelating affinity beads have attracted increasing interest in recent years for protein purification. In this study, iminodiacetic acid (IDA) was covalently attached to the poly(glycidyl methacrylate) [PGMA] beads (1.6 μm in diameter). Cu(2+) ions were chelated via IDA groups on PGMA beads for affinity binding of hemoglobin (Hb) from human blood hemolysate. The PGMA beads were characterized by scanning electron microscopy (SEM). The PGMA-Cu(2+) beads (628 μmol/g) were used in the Hb binding-elution studies. The effects of Hb concentration, pH and temperature on the binding efficiency of PGMA-Cu(2+) beads were performed in a batch system. Non-specific binding of Hb to PGMA beads in the absence of Cu(2+) ions was very low (0.39 mg/g). The maximum Hb binding was 130.3 mg/g. The equilibrium Hb binding increased with increasing temperature. The negative change in Gibbs free energy (ΔG°<0) indicated that the binding of Hb on the PGMA-Cu(2+) beads was a thermodynamically favorable process. The ΔS and ΔH values were 102.2 J/mol K and -2.02 kJ/mol, respectively. Significant amount of the bound Hb (up to 95.8%) was eluted in the elution medium containing 1.0 M NaCl in 1 h. The binding followed Langmuir isotherm model with monolayer binding capacity of 80.3-135.7 mg/g. Consecutive binding-elution experiments showed that the PGMA-Cu(2+) beads can be reused almost without any loss in the Hb binding capacity. To test the efficiency of Hb depletion from blood hemolysate, eluted portion was analyzed by fast protein liquid chromatography. The depletion efficiency for Hb was above 97.5%. This study determined that the PGMA-Cu(2+) beads had a superior binding capacity for Hb compared to the other carriers within this study.     

Macromolecule-macromolecule interaction in drug distribution. II. Effect of alpha-globulin on saturable uptake of fractionated [3H]heparin by rat parenchymal hepatocytes in primary culture.

The uptake of fractionated [3H]heparin was investigated in rat parenchymal hepatocytes in primary culture. The initial uptake of fractionated [3H]heparin was found to be saturable with the maximum uptake velocity (Vmax) of 10.1 +/- 1.46 pmol/min/mg protein and the Michaelis constant (Km) of 284 +/- 47.9 nM. The effect of alpha-globulin, the major protein binding to fractionated [3H]heparin, on the saturable uptake profile of fractionated [3H]heparin was also investigated. The uptake clearance was reduced, depending on the concentration of fractionated [3H]heparin, by the addition of 1 mg/ml alpha-globulin. We assumed that fractionated 3H-heparin bound to alpha-globulin was not available for uptake and that the reduction in the uptake clearance was solely attributable to the saturable binding of fractionated [3H]heparin to alpha-globulin. The uptake clearance versus concentration profile was analyzed to obtain the dissociation constant (Kd) of 31.8 nM and the capacity (n) of 0.047 for the binding of fractionated [3H]heparin to alpha-globulin. The saturable binding of fractionated [3H]heparin to alpha-globulin was supported by in vitro binding experiments using gel chromatography, in which bound fractionated [3H]heparin decreased with the concentration of fractionated [3H]heparin in the presence of alpha-globulin. In conclusion, the present study demonstrated the saturable uptake of fractionated [3H]heparin by rat parenchymal hepatocytes and the saturable binding of fractionated [3H]heparin to alpha-globulin. The saturable uptake may suggest the involvement of a specific transport system such as receptor-mediated endocytosis.     

Equilibrium and kinetics of protein binding on ion-exchange cellulose membranes with grafted polymer layer

The performance of weak and strong anion- and cation-exchange membrane adsorbents with a grafted gel layer (Sartobind Q, D, S, and C) was investigated using six proteins: bovine serum albumin, human serum albumin, α-lactalbumin, β-lactoglobulin, lysozyme, and myoglobin. Static binding experiments were used to assess the effect of pH and buffer concentration and to determine the adsorption isotherms for selected membrane/protein combinations. The equilibrium data were duly described either by the Langmuir or Freundlich isotherms. Dynamic binding experiments were carried out for the same membrane/protein combinations in a broad range of linear flow velocity. Both the dynamic binding capacity at 10 % breakthrough and the final binding capacity at complete breakthrough were independent of the flow velocity despite strong dispersion of the adsorption zone. A good match between the equilibrium data from static and dynamic experiments was obtained for the anion exchangers. The correlation between the dynamic binding capacity and protein molecule size was observed for the strong cation exchanger. This was due to the different accessibility of the gel layer for the protein molecules.     

葡萄球菌肠毒素B分子印迹聚合物的制备及其与蛋白质的结合性能

以葡萄球菌肠毒素B(SEB)蛋白为模板分子,以聚苯乙烯微球为基质,采用表面分子印迹法制备了SEB分子印迹聚合物.利用平衡吸附试验分析了SEB聚合物对目标蛋白的吸附能力及对类似底物的选择性;分析了该聚合物的吸附动力学,并利用扫描电镜观察了其形貌特征和颗粒尺寸.结果表明,经Scatchard模型分析求得的标题聚合物的最大表观结合量Qmax为3.23mg/g;所制备的SEB分子印迹聚合物呈微球形,粒径约为1²μm,对SEB蛋白具有较好的吸附性和特异选择性.     

Fractionation factors for stable isotopes of N and O during N2O reduction in soil depend on reaction rate constant

Nitrous oxide (N(2)O) is a major greenhouse gas that is mainly produced but also reduced by microorganisms in soils. We determined factors for N and O isotope fractionation during the reduction of N(2)O to N(2) in soil in a flow-through incubation experiment. The absolute value of the fractionation factors decreased with increasing reaction rate constant. Reaction rates constants ranged from 1.7 10(-4) s(-1) to 4.5 10(-3) s(-1). The minimum, maximum and median of the observed fractionation factors were for N -36.0 per thousand, -1.0 per thousand and -9.3 per thousand and for O -74.0 per thousand, -6.9 per thousand and -26.3 per thousand, respectively. The ratio of O isotope fractionation to N isotope fractionation was 2.4 +/- 0.3 and it was independent from the reaction rate constants. This leads us to conclude that fractionation factors are variables while their ratio in this particular reaction might be a constant.     

Solid-state (14)N MAS NMR of ammonium ions as a spy to structural insights for ammonium salts

The high resolution offered by magic-angle spinning (MAS), when compared to the static condition in solid-state NMR of powders, has been used to full advantage in a (14)N MAS NMR study of some ammonium salts: CH(3)NH(3)Cl, (NH(4))(2)(COO)(2) x H(2)O, (CH(3))(3)(C(6)H(5)CH(2))NCl, (CH(3))(3)(C(6)H(5))NI, [(n-C(4)H(9))(4)N](2)Mo(2)O(7), (NH(4))(2)HPO(4), and NH(4)H(2)PO(4). It is shown that the high-quality (14)N MAS NMR spectra, which can be obtained for these salts, allow determination of the (14)N quadrupole coupling parameters, i.e. C(Q) (the quadrupole coupling constant) and eta(Q) (the asymmetry parameter), with very high precision. In particular, it is shown that precise C(Q), eta(Q) parameters can be determined for at least two different (14)N sites in case the individual spinning-sideband (ssb) intensities arise from a single manifold of ssbs, i.e. the ssbs for the two sites cannot be resolved. This feature of (14)N MAS NMR, which is the first demonstration for manifolds of ssb in MAS NMR without the potential information from a central transition, becomes especially useful at the slow spinning frequencies (nu(r) = 1000-1500 Hz) applied to some of the ammonium salts studied here. The detection of the number of sites has been confirmed by the corresponding crystal structures determined from single-crystal X-ray diffraction (XRD), either in this work for the unknown structure of benzyl trimethylammonium chloride or from reports in the literature. The magnitudes of the (14)N quadrupole coupling constants for the ammonium salts studied here are in the range from C(Q) approximately 20 kHz to 1 MHz while the asymmetry parameters span the full range 0 < or = eta(Q) < or = 1. Clearly, the (14)N quadrupole coupling parameters (C(Q), eta(Q)) for ammonium ions appear highly sensitive toward crystal structure and therefore appreciably more informative for the characterization of ammonium salts in comparison to the isotropic (14)N (or (15)N) chemical shifts.     

Characterization of metal chelate methacrylate monolithic disk for purification of polyclonal and monoclonal immunoglobulin G

Dynamic binding capacity (DBC) of commercial metal-chelate methacrylate monolith-convective interaction media (CIM) was performed with commercial human immunoglobulin G (IgG) (Cohn fraction II, III). Monoliths are an attractive stationary phase for purification of large biomolecules because they exhibit very low back pressure even at high flow rates and flow-unaffected binding properties. Adsorption of IgG onto CIM-IDA disk immobilized with Cu²⁺, Ni²⁺ and Zn²⁺ were studied with Tris-acetate (TA), phosphate-acetate (PA) and MMA (MES, MOPS and acetate) buffer systems at different flow rates. Adsorption and elution of IgG varied with different buffers and adsorption of IgG was maximum with MMA buffer. Adsorption of human IgG from Cohn fractions (II, III) was high when Cu²⁺ was used as ligand. CIM-IDA disk showed dynamic binding capacity in the range of 14–16 mg/ml with Cu²⁺ and 7–9 mg/ml with Ni²⁺ for human IgG with MMA buffer. In the case of CIM-IDA-Zn²⁺ column, the binding capacity was only about 0.5 mg/ml of support. Different desorption strategies like lowering of pH and increasing of competitive agent were also studied to achieve maximum recovery. Chromatographic runs with human serum and mouse ascites fluid were also carried out with metal chelate methacrylate monolithic disk and the results indicate the potential of this technique for polyclonal human IgG and monoclonal IgG purification from complex biological samples.     

Separation of enantiomers on anion exchangers modified with heparin in liquid chromatography

Hepatitis B virus surface antigen (HBsAg) particles were efficiently adsorbed (retained) on a Sulfate-cellulose (S-C) bead column, and then desorbed with sodium chloride solutions (0.5-3.0 M). The HBsAg particles were not efficiently retained onto either sulfopropyl-agarose (SP-A) or quaternary amine-agarose (Q-A) at pH 4.5, 6 and 8. The size-exclusion curve showed that proteins of molecular mass higher than ca. 20,000 cannot penetrate into the pores of S-C beads. The dynamic binding capacity (DBC) values of lysozyme (ca. 7 mg/ml-gel) and of gamma-globulin (ca. 3 mg/ml gel) for S-C did not depend on the flow velocity while the DBC of gamma-globulin for SP-A decreased sharply with an increase in flow velocity. These results indicated that very large molecules are adsorbed only onto the surface of S-C, which resulted in fast adsorption-desorption rates although the equilibrium adsorption capacity is lower than conventional porous gel beads. Because of the rapid adsorption rate, the DBC values of gamma-globulin for S-C at high flow-rate regions are similar to those for SP-A. Bovine serum albumin was not adsorbed onto S-C. As this can not be explained by a simple electrostatic interaction mechanism, molecular recognition of S-C might be different from the agarose-based ion-exchange beads.     

Characterisation of grafted weak anion-exchange methacrylate monoliths

A weak ion-exchange grafted methacrylate monolith was prepared by grafting a methacrylate monolith with glycidyl methacrylate and subsequently modifying the epoxy groups with diethylamine. The thickness of the grafted layer was determined by measuring permeability and found to be approximately 90nm. The effects of different buffer solutions on the pressure drop were examined and indicated the influence of pH on the permeability of the grafted monolith. Protein separation and binding capacity (BC) were found to be flow-unaffected up to a linear velocity of 280cm/h. A comparison of the BC for the non-grafted and grafted monolith was performed using beta-lactoglobulin, bovine serum albumin (BSA), thyroglobulin, and plasmid DNA (pDNA). It was found that the grafted monolith exhibited 2- to 3.5-fold higher capacities (as compared to non-grafted monoliths) in all cases reaching values of 105, 80, 71, and 17mg/ml, respectively. It was determined that the maximum pDNA capacity was reached using 0.1M NaCl in the loading buffer. Recovery was comparable and no degradation of the supercoiled pDNA form was detected. Protein z-factors were equal for the non-grafted and grafted monolith indicating that the same number of binding sites are available although elution from the grafted monolith occurred at higher ionic strengths. The grafted monolith exhibited lower efficiency than the non-grafted ones. However, the baseline separation of pDNA from RNA and other impurities was achieved from a real sample.     

A new study of the degradation of hyaluronic acid by hyaluronidase using quartz crystal impedance technique

A new quartz crystal impedance sensing technique for the assay of hyaluronidase (HAse) activity is presented. It is based on the changes in viscosity and density during the enzymatic hydrolysis of hyaluronic acid (HA) by HAse. The variations of equivalent circuit parameters of piezoelectric quartz crystal (PQC) during the enzymatic degradation are discussed. The motional resistance shift curves indicate that the viscosity of the test solutions decreases during the hydrolysis process. The initial hydrolysis rates of HA are obtained from changes in viscosity and density as a function of incubation time. Kinetic parameters (the Michaelis constant K(m) and the maximum hydrolysis rate V(max)) of the degradation reaction are estimated by using a linear Lineweaver-Burk plot in this work. The K(m) was 0.44+/-0.03 mg.ml(-1) and the V(max) was -(5.29+/-0.36)x10(-3) kg.m(-2).s(-1/2).min(-1).     

Quantitation of N,N,N',N'-tetrakis (2-hydroxypropyl)-ethylenediamine in plasma by gas chromatography

A wide-bore capillary gas chromatographic method with nitrogen-selective thermionic detection is described for the quantitative analysis of N,N,N',N'-tetrakis (2-hydroxypropyl)ethylenediamine (Quadrol) in plasma. N,N,N',N'-tetrakis (2-hydroxybutyl)ethylenediamine is used as an internal standard. Rat or human plasma samples (0.5 ml) are mixed with internal standard, adjusted to alkaline pH and subjected to a single extraction with dichloromethane. Quadrol recovery from plasma typically exceeds 90%. The method is linear over the range 1.0-50 micrograms/ml. The working detection limit is 0.5 microgram/ml and the analysis time is under 7 min. The procedure has been used to obtain plasma concentration versus time data for the evaluation of Quadrol pharmacokinetics in rats.     

An immobilized immuno-stirrer for the determination of creatine kinase-mb isoenzyme in blood serum

An immobilized immuno-stirrer is described for the determination of creatine kinase-MB isoenzyme in blood serum. The IgG antibodies are immobilized on alkylamine glass beads using glutaraldehyde as cross-linking reagent, and the beads are packed into a rotating porous cell. After incubation with stirring, the CK-M isoenzymes in the blood serum sample are inhibited and are bound to the antibodies inside the stirrer. The residual CK-B isoenzyme activity is then determined spectrophotometrically or electrochemically. The binding capacity of the immuno-stirrer to CK-M isoenzyme was estimated to be 800 Ul^-1 with an average inhibitory efficiency of 97.8%. The within-day and day-to-day coefficients of variation were 5% and 4%, respectively, over a period of 52 days. An immuno-stirrer loaded with antibodies attached to cyanogen bromide-activated cellulose beads was also characterized, but the antibodies were not as stable as on glass beads.     

Bead injection for biomolecular assays: Affinity chromatography enhanced by bead injection spectroscopy

Selective capture of target biomolecules by ligands immobilized on a solid support is a cornerstone of two seemingly unrelated techniques: micro-Affinity Chromatography (microAC) and micro-Bead Injection Spectroscopy (microBIS). This work shows, for the first time, how these techniques can be carried out using the same instrument and how the data obtained this way complement each other, yielding complete information on retention and elution of target biomolecules. Biomolecular association and dissociation were investigated by microAC and microBIS, using computer-controlled programmable flow and the same instrument for automated bead transport, packing of a micro-column, assay of the analyte, and bead disposal. The absorbance of the analyte was monitored within the fiber optic flow cell configured either for monitoring directly on the beads or post-column after elution. The separation, binding, and elution of immunoglobulins (human IgG, rabbit IgG, and horse IgG) on protein G-coated Sepharose beads were studied as model systems. The limit of detection of the microAC technique was determined to be 5 ng microL(-1) IgG, and that of the microBIS technique was 50 ng microL(-1) IgG.     

Microarrays of tagged combinatorial triazine libraries in the discovery of small-molecule ligands of human IgG

A novel and highly diverse tagged triazine library incorporating a triethylene glycol-based linker was synthesized using an orthogonal combinatorial approach on the solid phase and covalently immobilized on a glass substrate as a small molecule microarray (SMM). The SMM was screened with a fluorophore-conjugated human IgG, and 4 novel binders from a library of 2688 compounds were identified from the fully spatially addressable array without the need for compound decoding. Using surface plasmon resonance (SPR) analysis, binding seen on the array was confirmed, and a binding constant as low as Kd = 2.02 x10(-6) M was measured.