Thermal degradation of cereal straws in air and nitrogen

Multigram quantities (2.5-10 g) of highly purified IgG were obtained within 4 h from serum by using Avid AL packed in a radial-flow column. Avid AL is an affinity gel containing a synthetic, low-mol-wt ligand capable of selectively binding IgG from serum of all animal species tested. By packing the gel in a radial-flow column up to 500 mL, a high flow rate of 50 mL/min can be achieved without adversely affecting the performance of the gel and the purity of the isolated antibody.     

Evaluation of Amino Acid O-Phosphoserine as Ligand for the Capture of Immunoglubulin G from Human Serum

The amino acid ortho-phosphoserine (OPS) immobilized on agarose gel was evaluated as a ligand for adsorption of polyclonal human immunoglobulin G (IgG) from human serum in the presence of low ionic strength buffers. Screening of buffer systems showed sodium phosphate as the buffer that exhibited higher IgG purity values. Through breakthrough curve analysis for agarose-OPS (feeding of 31.93?mg of total protein per mL of gel), a purification factor of 5.4 with an IgG purity of 89?% was obtained (based on IgG, IgM, IgA, HSA, and Trf nephelometric analysis). IgG adsorption equilibrium studies showed that these data followed the Langmuir-Freundlich model, with cooperativity parameter (n) equal to 1.74, indicating the presence of positive cooperativity, probably due to multipoint interactions. The maximum IgG binding capacity was 24.2?mg?mL^?1, near the value for the bioaffinity ligand protein A. The agarose-OPS adsorbent provides an attractive alternative for capturing of IgG from human serum.     

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.     

An ELISA-based approach to optimize elution conditions for obtaining hapten-specific antibodies

The correct choice of the elution conditions to break an affinity interaction is important for the successful purification of biomolecules. The optimal elution buffer liberates the bound substance in a minimum volume and maintains the activity of the purified material. The present study demonstrates an enzyme-linked immunosorbent assay (ELISA)-based approach for selection of specific elution conditions for eluting antibodies against a small molecule (atrazine) from pooled sera. Six different elution conditions were tried for the removal of antibodies from the complex. Large-scale purification of anti-atrazine antibodies from the sera was done with a hapten-specific column using an amino-terminal crosslinked agarose gel. Efficacy in terms of total amount of recovery and binding affinities of eluted antibodies from the column were further investigated by ELISA. Results indicate that the ELISA-based elution approach is ideal for the selection of suitable elution buffer that can subsequently be utilized for affinity purification applications.     

The effect of NaCl on the adsorption of human IgG onto CM-Asp–PEVA hollow fiber membrane-immobilized nickel and cobalt metal ions

Over the past decade, immobilized metal-affinity adsorbents have attracted increasing interest for purification of natural and recombinant immunoglobulin G (IgG). In this work, nickel and cobalt metal ions complexed with CM-Asp (carboxymethylaspartate) immobilized on poly(ethylenevinyl alcohol) (PEVA) hollow fiber membranes were evaluated for purification of human IgG from serum. The buffer system and NaCl had important effects on human serum protein adsorption in both adsorbents. Efficient purification of IgG was accomplished in sodium phosphate buffer without NaCl at pH 7.0. Under this condition, the electrostatic interactions are important for adsorption. The Ni(II)-CM-Asp–PEVA had a protein adsorption capacity of 17.5 mg of IgG mL^?1 fiber when human serum diluted was loaded in crossflow filtration mode and the eluted IgG had a purity of 82.6 % (based on total protein and IgG, IgM, HSA, and Trf nephelometric analysis). Fitting the experimental IgG adsorption data to the Langmuir and Langmuir–Freundlich models showed that Ni(II)-CM-Asp and Co(II)-CM-Asp had Langmuirean and non-Langmuirean behavior, respectively, with positive cooperativity for IgG-Co(II)-CM-Asp binding, probably due to multipoint interactions (n = 2.12 ± 0.31). Thus, these membranes can be considered as alternative adsorbents for the purification or depletion of IgG from human serum.     

Depletion of high-abundance proteins from human plasma using a combination of an affinity and pseudo-affinity column

Human serum albumin (HSA) and immunoglobulin G (IgG) represent over 75% of all proteins present in human plasma. These high-abundance proteins prevent the detection of low-abundance proteins which are potential markers for various diseases. The depletion of HSA and IgG is therefore essential for further proteome analysis. In this paper we describe the optimization of conditions for selective depletion of HSA and IgG using affinity and pseudo-affinity chromatography. A BIA Separations CIM (convective interaction media) Protein G disk was applied for the removal of IgG and the Mimetic Blue SA A6XL stationary phase for the removal of HSA. The binding and the elution buffer for CIM Protein G disk were chosen on the basis of the peak shape. The dynamic binding capacity was determined. It was shown to be dependent on the buffer system used and independent of the flow rate and of the concentration of IgG. Beside the binding capacity for the IgG standard, the binding capacity was also determined for IgG in human plasma. The Mimetic Blue SA A6XL column was characterized using human plasma. The selectivity of the depletion was dependent on the amount of human plasma that was loaded on the column. After the conditions on both supports had been optimized, the Mimetic Blue SA A6XL stationary phase was combined with the CIM Protein G disk in order to simultaneously deplete samples of human plasma. A centrifuge spin column that enables the removal of IgG and HSA from 20 μL of human plasma was designed. The results of the depletion were examined using sodium dodecyl sulfate polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis.     

An electrochemical quartz crystal impedance study on anti-human immunoglobulin G immobilization in the polymer grown during dopamine oxidation at an Au electrode

The polymeric film grown during dopamine oxidation at an Au electrode was studied as a novel matrix for immobilizing anti-human immunoglobulin G (IgG) via the electrochemical quartz crystal impedance analysis (EQCIA) method. The growth of the polymeric films at Au electrodes during dopamine oxidation in neutral phosphate buffer (pH 7.4) and the immobilization of anti-human IgG into the polymeric films during their growth have been traced at real time. Lysozyme control experiments suggested that anti-human IgG was electrostatically incorporated into the polymeric film. Also, the porosity of the polymeric films has been discussed by measuring the "wet" and "dry" frequency shifts. Compared with a polypyrrole film immobilized with anti-human IgG, the proposed matrix possessed a larger amount of specific binding sites for human IgG by subsequent immunoreaction tests. The association constant of the anti-human IgG immunoreaction was obtained with satisfactory results.     

Analysis of proteins copurifying with the cd4/lck complex using one-dimensional polyacrylamide gel electrophoresis and mass spectrometry: comparison with affinity-tag based protein detection and evaluation of different solubilization methods

Mass spectrometry-based identification of the components of affinity purified protein complexes after polyacrylamide gel electrophoresis (PAGE) and in-gel digest has become very popular for the detection of novel protein interactions. As an alternative, the entire protein complex can be subjected to proteolytic cleavage followed by chromatographic separation of the peptides. Based on our earlier report of a method using affinity tag-mediated purification of cysteine-containing peptides to analyse proteins present in an affinity purification of the CD4/lck receptor complex, we here evaluated the use of one-dimensional polyacrylamide gel electrophoresis for analysis of the same receptor complex purification. Using electrospray and tandem mass spectrometry analyses of tryptic peptides from in-gel digested proteins we identified the components of the CD4 receptor complex along with 23 other proteins that were all likely to be non-specifically binding proteins and mainly different from the proteins detected in our previous study. We compare the alternative strategy with the affinity tag-based method that we described earlier and show that the PAGE-based method enables more proteins to be identified. We also evaluated the use of a more stringent lysis buffer for the CD4 purification to minimise non-specific binding and identified 52 proteins along with CD4 in three independent experiments suggesting that the choice of lysis buffer had no significant effect on the extent of non-specific binding. Non-specific binding was inconsistent and involved various types of proteins underlining the importance of reproducibility and control experiments in proteomic studies.     

Purification of IgG from Sera of Rabbit and Guinea Pig by Flow-Through Mode Ion-Exchange Chromatography using DEAE Sepharose Fast Flow Column

A one-step purification process using flow-through mode ion-exchange chromatography (Ft-IEC) was evaluated for the purification of Immunoglobulin G (IgG) from rabbit and guinea pig serum. A simple protein precipitation with saturated ammonium sulfate was used to pretreated plasma samples. Chromatographic separation was carried out on DEAE Sepharose Fast Flow (F.F.) column at a flow rate of 1 mL min^?1. Ft-IEC using Tris?CHCl buffer at pH 7.0 allowed the recovery of 22% of the loaded IgG with purity of 95% existed in flowthrough and washing effluents but not in elution effluents. To be compared, bind-elute mode IEC using Tris?CHCl buffer at pH 8.5 based on the routine protocol showed that the recovery of 15% of the loaded IgG with purity of 80% existed in elution effluents. These results indicate that the Ft-IEC is an effective method for purifying IgG from sera of rabbit and guinea pig and may also be suitable for other animal sera by adjusting pH at equilibrium buffer for chromatography column.     

One-step purification of histone deacetylase from Escherichia coli cell-lysate by counter-current chromatography using aqueous two-phase system

Aqueous-aqueous two-phase (AATP) systems composed of polyethylene glycol (PEG) (molecular mass, M(r):1000-8000) and dextran (M(r):40,000) were evaluated for purification of maltose binding protein tagged-histone deacetylase (MBP-HDAC) by counter-current chromatography (CCC). CCC purification of an MBP-HDAC from Escherichia coli cell-lysate was successfully demonstrated with a 7.0% PEG 3350-10% dextran T40 system containing 10 mM potassium phosphate buffer at pH 9.0. After CCC purification, both polymers in the CCC fractions were easily removed by ultrafiltration in a short period of time. The collected fractions containing target protein were analyzed by an HPLC-based in vitro assay as well as sodium dodecyl sulfate polyacrylamide gel electrophoresis. MBP tag was digested from fusion HDAC during the CCC separation and native HDAC was purified by one-step operation with well preserved deacetyl enzyme activity.     

Performance of hexamer peptide ligands for affinity purification of immunoglobulin G from commercial cell culture media

Previous work has reported on the identification and characterization of the hexapeptide ligands HWRGWV, HYFKFD, and HFRRHL for the affinity capture of IgG through specific binding to its Fc fragment. This paper addresses issues related to the successful application of these ligands, on a commercial methacrylate chromatographic resin, for the purification of IgG from mammalian cell culture fluids. The concentrations of sodium chloride and sodium caprylate in the binding buffer were optimized to maximize the purity and yield of IgG upon elution. Screening of several regeneration conditions found that either 2M guanidine-HCl or a combination of 0.85% phosphoric acid followed by 2M urea resulted in complete recovery of the IgG adsorption capacity and that the column could be reused over many cycles. The hexapeptide ligands were used for the purification of humanized and chimeric monoclonal antibodies from two commercial CHO cell culture fluids. The chimeric MAb of IgG1 subclass was purified using the HWRGWV resin whereas the humanized MAb of IgG4 subclass was purified using the HWRGWV, HYFKFD and HFRRHL resins. The purities and yields obtained for both the MAbs were found to be higher than 94% and 85% respectively. These results compare well with the yields and purities obtained using Protein G columns. The residual DNA and host cell protein reduction obtained by the HWRGWV resin was in the range of 4 log reduction value (LRV) and 2 LRV respectively, comparable to those reported for Protein A resins. The dynamic binding capacity of all three peptide resins for the humanized monoclonal antibody was in the range of 20mg/mL.     

Isolation of ellagic acid from pomegranate peel extract by hydrophobic interaction chromatography using graphene oxide grafted cotton fiber adsorbent

Ellagic acid, a natural polyphenol, was isolated from pomegranate peel extract by hydrophobic interaction using graphene oxide grafted cotton fiber as a stationary adsorbent. The grafted graphene oxide moieties served as hydrophobic interaction‐binding sites for ellagic acid adsorption. The graphene oxide grafted cotton fiber was made into a membrane‐like sheet in order to complete ellagic acid purification by using a binding–elution mode. The effects of operational parameters, such as the composition of the binding buffer/elution buffer, buffer pH, and buffer concentration, on the isolation process were investigated. It was found that 5 mmol/L sodium carbonate aqueous solution is a proper‐binding buffer, and sodium hydroxide aqueous solution ranging from 0.04 to 0.06 mol/L is a suitable elution solution for ellagic acid purification. Under the optimized condition, the purity of ellagic acid increased significantly from 7.5% in the crude extract to 75.0–80.0%. The pH value was found to be a key parameter that determines the adsorption and desorption of ellagic acid. No organic solvent is involved in the entire purification process. Thus, a simple and environmentally friendly method is established for ellagic acid purification using a graphene oxide‐modified biodegradable and bio‐sourced fibrous adsorbent.     

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。     

Effects of peptide density and elution pH on affinity chromatographic purification of human immunoglobulins A and M

A family of linear hexamer peptide ligands HWRGWV, HYFKFD and HFRRHL, initially identified for their affinity to the Fc portion of human immunoglobulin G (hIgG), also have potential for use in the purification of human immunoglobulins A (hIgA) and M (hIgM). HWRGWV demonstrated the strongest binding affinity to hIgM, followed by hIgA and hIgG respectively. The effects of N-terminal acetylation of the peptide, as well as elution buffer pH, on the chromatographic elution of human IgG, IgA and IgM from HWRGWV resins at various peptide densities (0.04-0.55 meq/g) were investigated. Over 80% recovery and 90% purity were achieved for human IgG and IgA isolation from complete minimum essential medium (cMEM) using HWRGWV resin at optimum peptide densities. For human IgM, 75.7% recovery and 86.0% purity were achieved by using HWRGWV at a low peptide density of 0.04 meq/g. Although HYFKFD and HFRRHL exhibited their ability for isolation of human IgG, IgA and IgM from cMEM as well, HWRGWV is the best option among them for large-scale purification of human IgG, IgA and IgM based on conditions tested.     

Study of interaction between drug enantiomers and human serum albumin by flow injection-capillary electrophoresis frontal analysis

Flow injection (FI)-CE coupled with frontal analysis (FA) was applied to the study of stereoselectivity binding of amlodipine (AL) to HSA. Under protein-drug binding equilibrium, the unbound concentrations of drug enantiomers were measured by plateau height. The stereoselectivity of AL binding to HSA was proved by the different free fractions of two enantiomers. In physiological phosphate solution (pH 7.4, ionic strength 0.17) when 200 microM (+/-)AL was equilibrated with 300 microM HSA, the concentration of unbound R-AL was about 1.5 times higher than that of its antipode. The binding constants of two enantiomers, KR-AL and KS-AL, were 9910-11200 and 90200-104000 M(-1), respectively. The results obtained by the method were compared with those determined by conventional equilibrium dialysis (ED)-CE and fluorescence spectra. Hydroxypropyl-beta-CD (HP-beta-CD) (10 mM) was used as a chiral selector in pH 3.7 phosphate buffer. L-tryptophan (L-try) and ketoprofen (Ket) were used as displacement reagents to investigate the binding sites of AL to HSA. A binding synergism effect between hydrochlorothiazide (QL) and AL was observed and the results suggested that QL can destroy binding equilibrium of R-AL and S-AL toward HSA and they can occupy the same binding site of HSA (site I). The reproducibility was confirmed by RSD (RSD<1.5%) of the plateau height determined by FI-CE frontal analysis (FI-CE-FA). The FI-CE-FA was a good method to study protein-drug interaction.     

Chemical Modification of Ethyl Cellulose-Based Highly Porous Membrane for the Purification of Immunoglobulin G

The chemical modification of developed ethyl cellulose-based membrane was carried out to make it suitable for bioseparation. The different reagents were used for the modification of membrane to couple protein A (PA) to study the purification of immunoglobulin G (IgG) from blood. The chemical modification was carried out using relatively simple and mild reaction conditions. The attenuated total reflectance Fourier transform infrared analysis of chemically modified membrane showed new peak at 1,596.06 and 1,716.49 cm^?1. The scanning electron microscopy of PA-coupled membrane, which was used for IgG purification showed open pores and 950?±?21.5 LMH (L?m^?2?h^?1) operational flux at 0.5-bar out pressure. The flux of unmodified membrane was 1,746?±?18.5 LMH at 0.5-bar out pressure. The equilibrium adsorption concentration (318.5?±?5.9 μg?cm^?2) was obtained at 3 h. The adsorption character of PA-coupled membrane was consistent with the Langmuir adsorption model and the non-specific binding was 67.08?±?1.3 μg?cm^?2. The sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis showed similar purification pattern for purified IgG from human serum and commercial preparation of IgG. All the results have suggested a high potential of PA-coupled ethyl cellulose-based membrane for large-scale purification of IgG.     

Metal-Chelating Nanopolymers for Antibody Purification from Human Plasma

The purification of immunoglobulin G (IgG) from human plasma was performed by using a novel metal-chelated adsorbent with nano size. The non-porous nanoparticles were produced by surfactant free emulsion polymerization of ethylene glycol dimethacrylate (EDMA) and 2-methacryloylamidohistidine (MAH). Then, Cu(II) ions were chelated on the nanoparticles. The nano-poly(EDMA-MAH) nanoparticles were characterized by Fourier transform infrared, scanning electron microscope, atomic force microscope and elemental analysis. The non-porous nanoparticles were spherical form and have 100?C250?nm size distribution. The maximum IgG adsorption capacity of the Cu(II) chelated nanoparticles was found to be 463?mg/g polymer at pH 7.0 in HEPES buffer. Desorption of IgG was performed by 1.0?M NaCl and desorption rate was found to be 97?%. IgG was obtained from human plasma with purity of 94?% (up to 578?mg/g polymer). The non-porous nanoparticles allowed one-step purification of IgG from human plasma.     

Simple method for the isolation and purification of hemoglobin components.

A simple method for the isolation and purification of hemoglobin components from starch gel by electrophoresis is described. An inverted bottle with a cut off bottom is used. Pieces of gel containing the hemoglobin are embedded in potato starch, wetted with buffer, and placed in the neck of the inverted bottle, the mouth of which has been closed with filter paper and tape. Connection with the negative tank buffer is achieved by inserting a layer of starch gel between the potato starch and the negative tank buffer. By electrophoresis, the hemoglobin migrates and accumulates in a dialysing tube tied around the mouth of the bottle and hanging into the positive tank buffer.     

Quantification of immunoglobulin G and characterization of process related impurities using coupled Protein A and size exclusion high performance liquid chromatography

The present work describes two HPLC-UV methods for multi-protein quantification using (i) only a Protein A sensor cartridge (Protein A HPLC) and (ii) the same Protein A cartridge in combination with a size exclusion HPLC column (PSEC-HPLC). The possibility to simultaneously quantify immunoglobulin G (IgG) besides a non-binding protein such as bovine serum albumin (BSA) increases the applicability of Protein A HPLC. Its most pronounced feature is its independence of the buffer system, pH-value and salt content of the investigated sample solvent, which includes cell media. A comparison with the state-of-the-art, the photometrical Bradford method, shows that Protein A HPLC is as sensitive as Bradford, but that it comes with an extended linear range of 4 orders of magnitude, ranging from 0.15 [μg abs] to 1 [mg abs] absolute injected protein amount. The applicability of the PSEC-HPLC method is demonstrated for the analysis of real cell culture feed samples. While Protein A binds IgG, the SEC-column distributes the feed impurities by their molecular weight. The peak area ratios of IgG and the feed impurities of interest are then plotted against the collected sample fraction. These Protein A-Size-Exclusion-Chromatographic diagrams (PSEC-plot) combine the performance information of feed impurities and IgG in a single plot. Further it is shown that both methods are suitable for the performance evaluation of antibody purification media using static as well as dynamic binding experiments performed on DEAE-Fractogel and Capto Adhere. The investigated test samples were “mock” protein solutions with increasing complexity ranging from simple PBS buffer to serum free cell media and “real” cell culture feed solutions.     

Purification of ascitic fluid-derived murine monoclonal antibodies by anion-exchange and size-exclusion high-performance liquid chromatography

Ascitic fluid-derived murine monoclonal antibodies (MoAbs) of immunoglobulin (Ig) M and IgG isotypes (IgG1 and IgG2a subisotypes) were previously prepared against an isolate of Actinobacillus sp (CAs8C) for the purpose of identifying and characterizing outer membrane antigens on this bacterium. An attempt was made to purify these MoAbs by anion-exchange and size exclusion high-performance liquid chromatography (HPLC). Hybridomas producing the IgG1 and IgG2a MoAbs posed unique difficulties in that they also secreted irrelevant IgG2b MoAbs that were present in the ascitic fluids. Anion-exchange chromatography (Protein-Pak DEAE-5PW column), with a simultaneous change in gradients of pH and ionic strength, was used to purify IgG and as a first step in the purification of IgM. There was good separation of IgG2b from IgG2a, but not from IgG1. Size-exclusion chromatography (Protein-Pak 300 SW column) was required to complete the purification of IgM. The presence of MoAbs in the HPLC fractions was confirmed by discontinuous gradient polyacrylamide gel electrophoresis (denatured and either reduced or non-reduced conditions) and the enzyme-linked immunosorbent assay. HPLC-purified MoAbs were free from transferrin and albumin and retained their specificity for As8C.