Superior magnetic monodisperse particles for direct purification of immunoglobulin G under magnetic field

Monodisperse magnetic acrylate based particles (5.0 µm in diameter) containing histidine were synthesized using a modified suspension polymerization method for the purification of immunoglobulin G from human plasma in a magnetically stabilized fluidized bed. N-methacryloyl-(L)-histidine methyl ester (MAH) was used as pseudo-specific ligand/co-monomer. MAH content of the magnetic particles was calculated as 55.3 µmol MAH/g polymer using elemental analysis. Immunoglobulin G binding amount of the magnetic particles decreased with increase of the flow-rate. The maximum immunoglobulin G binding was observed at pH 7.4 (phosphate buffer). Immunoglobulin G binding amount onto the magnetic poly(ethylene glycol dimethacrylate) [mPEGDMA] particles was found to be almost negligible due to the hydrophilic polymer structure. High binding values were obtained from aqueous solutions (1646 mg/g). Higher immunoglobulin G binding was observed when human plasma was used (2169 mg/g). Purity of the separated immunoglobulin G from human plasma was found to be 87%. Magnetic PEGDMAH particles could be used many times without significant loss in protein binding amount.     

Hydrophobic charge‐induction resin with 5‐aminobenzimidazol as the functional ligand: preparation,protein adsorption and immunoglobulin G purification

A new hydrophobic charge‐induction chromatography resin was prepared with 5‐aminobenzimidazol as functional ligand and polyacrylic ester beads as matrix. Adsorption isotherms and adsorption in columns were investigated using human immunoglobulin G and bovine serum albumin as model proteins, and the influence of pH and NaCl concentration was discussed. Results showed that the ligand density was 195 μmol/mL gel, and protein selectivity can be improved by controlling pH and salt addition. An optimized purification process (sample loading at pH 8.0 with 0.2 M NaCl and elution at pH 5.0) was performed to purify human immunoglobulin G from bovine serum albumin containing feedstock, which resulted in human immunoglobulin G purity of 99.7% and recovery of 94.6%. A similar process was applied for the purification of monoclonal antibody from cell culture supernatant, which showed antibody purity of 94.9% and recovery of 92.5%. The results indicated that the new resin developed had comparable performance as Protein A chromatography and would be suitable for antibody purification from complex feedstock.     

Fast and efficient separation of immunoglobulin M from immunoglobulin G using short monolithic columns

Certain diagnostic, analytical and preparative applications require the separation of immunoglobulin G (IgG) from immunoglobulin M (IgM). In the present work, different ion-exchange methacrylate monoliths were tested for the separation of IgG and IgM. The strong anion-exchange column had the highest dynamic binding capacity reaching more than 20mg of IgM/ml of support. Additionally, separation of IgM from human serum albumin, a common contaminant in immunoglobulin purification, was achieved on the weak ethylenediamino anion-exchange column, which set the basis for the IgM purification method developed on convective interaction media (CIM) supports. Experiments also confirmed flow independent characteristics of the short monolithic columns.     

Improved purification of immunoglobulin G from plasma by mixed‐mode chromatography

Efficient loading of immunoglobulin G in mixed‐mode chromatography is often a serious bottleneck in the chromatographic purification of immunoglobulin G. In this work, a mixed‐mode ligand, 4‐(1H‐imidazol‐1‐yl) aniline, was coupled to Sepharose Fast Flow to fabricate AN SepFF adsorbents with ligand densities of 15–64 mmol/L, and the chromatographic performances of these adsorbents were thoroughly investigated to identify a feasible approach to improve immunoglobulin G purification. The results indicate that a critical ligand density exists for immunoglobulin G on the AN SepFF adsorbents. Above the critical ligand density, the adsorbents showed superior selectivity to immunoglobulin G at high salt concentrations, and also exhibited much higher dynamic binding capacities. For immunoglobulin G purification, both the yield and binding capacity increased with adsorbent ligand density along with a decrease in purity. It is difficult to improve the binding capacity, purity, and yield of immunoglobulin G simultaneously in AN SepFF chromatography. By using tandem AN SepFF chromatography, a threefold increase in binding capacity as well as high purity and yield of immunoglobulin G were achieved. Therefore, the tandem chromatography demonstrates that AN SepFF adsorbent is a practical and feasible alternative to MEP HyperCel adsorbents for immunoglobulin G purification.     

大孔及涂敷硅球作基质的组氨酸拟亲和色谱纯化人免疫球蛋白G

研究用分子组氨酸和配体、大孔硅球为基质的拟亲和色谱分离纯化人免疫蛋白Ｇ（ＩｇＧ），认为键合组氨酸具有半抗原体质而与ＩｇＧ发生免疫亲和作用，以色谱组份重新进样验证了色谱柱对ＩｇＧ亲和专一性，并用包敷Ｄｅｘｔｒａｎ大孔硅球作基质的拟亲和色谱纯化人血清中的ＩｇＧ，减少了色谱峰拖尾，缩短了分离时间。     

Label-free electrochemical impedance spectroscopy biosensor for the determination of human immunoglobulin G

A simple and sensitive biosensor was developed for the determination of human immunoglobulin G (IgG). Protein A was employed as molecular receptor and electrochemical impedance spectroscopy (EIS) was used as detection technique. The biosensor was obtained by self-assembling protein A on a gold electrode. The surface morphology of the self-assembled layer before and after interaction with IgG was studied by atomic force microscopy. Protein A bound specifically to the Fc portion of IgG, and this caused a change in the resistance of the interfacial electron transfer when using a ferrocyanide redox couple as a probe. The increase of the resistance of the electron transfer was linearly related to the concentration of IgG in the range from 10 ng.mL^?1 to 1.0 μg.mL^?1, with a detection limit of 5 ng.mL^?1. The work demonstrates that protein A is a versatile matrix for the immobilization of antibodies.     

Detection of fungal carbohydrate antigens by high-performance immunoaffinity chromatography using a protein A column with covalently linked immunoglobulin G.

Fungal carbohydrate antigens were analysed by high-performance immunoaffinity chromatography (HPIAC) with immunoglobulin G (IgG) antibodies raised against extracellular polysaccharides of Mucor racemosus. The protein A-IgG complex was covalently bound with dimethyl pimelimidate, which enabled the use of strong acidic buffers to release the tightly bound antigens from the column. Prior to pulsed-amperometric detection, an anion-micromembrane suppressor was used to raise the pH of the effluent to above 12 without dilution. The HPIAC system provides a sophisticated method for the rapid and sensitive detection of antigenic oligomeric carbohydrates in biological samples and is proposed as an alternative to quantitative enzyme-linked immunosorbent assay techniques.     

Initial evaluation of protein A modified capillary‐channeled polymer fibers for the capture and recovery of immunoglobulin G

A novel protein A affinity chromatography stationary phase has been developed from polypropylene capillary‐channeled polymer fibers modified with a recombinant protein A ligand for the capture and recovery of immunoglobulin G (IgG) with high specificity and yield. An SPE micropipette tip format was employed so that solvent, protein, and antibody consumption was minimized. The adsorption modification of the fiber surfaces with protein A was evaluated as a function of feed concentration and volume. Optimal modification of the fiber surface with protein A yielded a 5.7 mg/mL (bed volume) ligand capacity with the modified fibers showing stability across numerous solvent environments. Performance was evaluated through exposure to human IgG and myoglobin, individually and as a mixture. Myoglobin was used as a surrogate for host cell proteins common to growth media. The efficacy of the selective binding to the ligand is demonstrated by the 2.9:1 (IgG/protein A) binding stoichiometry. Elution with 0.1 M acetic acid yielded an 89% recovery of the captured IgG based on absorption measurements of the collected eluents. Regeneration was possible with 10 mM NaOH. Protein A modified polypropylene capillary‐channeled polymer fibers show promising initial results as an affinity phase for efficient capture and purification of IgG.     

Selective high-performance liquid chromatographic purification of bispecific monoclonal antibodies.

The recent development of improved production techniques for bispecific monoclonal antibodies (biMAbs) has significantly increased interest in specific purification procedures. In this investigation, a general high-performance liquid chromatographic (HPLC) purification method is proposed that allows highly purified biMAbs to be obtained from mouse ascites fluid containing a mixture of different antibodies, i.e., parental MAbs, active biMAb and a mixture of randomly assembled heavy and light chains. Proteins from ascites fluid were precipitated with ammonium sulphate and applied to a high-performance protein A column to separate the total immunoglobulin fraction. BiMAbs were isolated from other immunoglobulins by two subsequent passages through a high-performance hydroxyapatite (HPHT) column. This purification protocol combines specificity of protein A for immunoglobulin G (IgG) and high selectivity of hydroxyapatite for different IgG idiotypes. All purification steps were performed rapidly and reliably by HPLC. This method was applied to the purification of six different biMAbs with consistently high yields, purity and homogeneity. This general purification method may prove extremely valuable when highly pure preparation of biMAbs is required, as for in vivo use.     

Proteomics as a tool for optimization of human plasma protein separation

The application of proteomics technology in purification of proteins from human plasma and for characterization of plasma-derived therapeutics has been recently discussed. However, until now, the impact of this technology on the plasma protein fractionation and analysis of the final product has not been realized. In the present work, we demonstrate the use of proteomic techniques the monitoring of the first step of the plasma fractionation by use of anion-exchange chromatography. This chromatographic method is frequently used in the purification scheme for isolation of vitamin K dependent clotting factors II, VII, IX and X, and clotting inhibitors protein C and protein S, as well as inter-alpha inhibitor proteins (IaIp). After the removal of immunoglobulin G and non-binding proteins in the flow-through fraction, albumin and weakly bound proteins were eluted with low concentration of sodium chloride. The proteins that strongly bind to the anion-exchange column were eluted by higher salt concentrations. The fractions of interest were analyzed, and proteins were identified by LC-ESI-MS/MS. By use of this method, not only candidates for therapeutic concentrates, but also some potentially harmful components were identified. This strategy was very helpful for further process optimization, fast identification of target proteins with relatively low abundance, and for the design of subsequent steps in their removal or purification.     

Purification of the pregnancy zone protein by affinity chromatography.

A method for purification of the pregnancy zone protein (PZP) by affinity chromatography was developed. A monospecific immunoglobulin fraction, covalently coupled to Sepharose 4B, was used as binding agent and the elution conditions for PZP are described. The purified protein was shown to have identical properties compared to native PZP with regard to molecular weight, immunodiffusion precipitation and immunosuppressive activity.     

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.     

Determination of immunoglobulin G in bovine colostrum and milk by direct biosensor SPR-immunoassay

An automated biosensor surface-plasmon resonance-based assay was developed for the determination of immunoglobulin G (IgG) in bovine milk and colostrum with either goat or rabbit antibovine IgG or protein G used as detecting molecule. The method is configured as a direct and nonlabeled immunoassay, with quantitation against an authentic IgG calibrant. Whole colostrum or milk is prepared for analysis by dilution into buffer. Analysis conditions, including ligand immobilization, flowrate, contact time, and regeneration, were optimized, and nonspecific binding was evaluated. Performance parameters included working range of 15-10 000 ng/mL, method detection limit of 0.08 mg/mL, overall instrument response reproducibility relative standard deviation (RSD(R)) of 0.47%, mean between-run RSD(R) of 10.5% for colostrum, and surface stability over 200 analyses. The proposed method was compared with independent alternative methods. The technique was applied to the measurement of IgG content during early lactation transition from colostrum to milk, as well as in consumer milk, colostrum, and hyperimmune milk powders.     

Monitoring of mouse immunoglobulin G by flow-injection analytical affinity chromatography

A flow-injection analytical affinity chromatographic (FIAAC) system was developed for the on-line monitoring of mouse immunoglobulin G (IgG). Protein A or anti-mouse IgG antibodies immobilized on oxirane beads were filled in a miniature column. The IgG-containing samples and standards were passed through the column and detected fluorimetrically after elution with citrate buffer (pH 3 or 2.5). The on-line monitoring of mouse IgG 2a during a 7-day cultivation of hybridoma cells in a perfusion reactor by FIAAC is presented. A chemical barrier was used to prevent contamination of the reactor from the FIAAC.     

Avid AL, a synthetic ligand affinity gel mimicking immobilized bacterial antibody receptor for purification of immunoglobulin G.

Avid AL is an affinity gel designed for the purification of immunoglobulin G (IgG). The gel was prepared by first reacting Sepharose with 3,5-dichloro-2,4,6-trifluoropyridine and 4-dimethylaminopyridine and then with 2-mercaptoethanol. The IgG purified by Avid AL is about 95% pure. The binding parameters of Avid AL for the whole IgG, Fab and Fc fragment and the stability of gel were investigated. The IgG bound to Avid AL can be eluted with an acidic buffer or with a novel neutral buffer containing electron donors. The development of such a mild neutral elution buffer is described. Application of Avid AL in a rapid gram-scale IgG purification was demonstrated. The possible mechanism of IgG binding is discussed.     

Engineering unnatural nucleotide specificity to probe G protein signaling

G proteins comprise approximately 0.5% of proteins encoded by mammalian genomes. To date, there exists a lack of small-molecule modulators that could contribute to their functional study. In this report, we present the use of H-Ras to develop a system that answers this need. Small molecules that allow for the highly specific inhibition or activation of the engineered G protein were developed. The rational design preserved binding of the natural substrates to the G protein, and the mutations were functionally innocuous in a cellular context. This tool can be used for isolating specific G protein effectors, as we demonstrate with the identification of Nol1 as a putative effector of H-Ras. Finally, the generalization of this system was confirmed by applying it to Rap1B, suggesting that this method will be applicable to other G proteins.     

The effective and specific isolation of antibodies from human serum by using thiophilic paramagnetic polymer beads

A rapid and effective method to specifically isolate immunoglobulin G (IgG) from human serum by thiophilic paramagnetic polymer beads was developed. The thiophilic paramagnetic beads were synthesized with vinyl acetate and divinylbenzene by microsuspension polymerization in the presence of magnetite nanoparticles. Divinylsulfone and 2-mercapto-benzothiazole were subsequently used to modify the surface of these beads, resulting in thiophilic particles that exhibited a high specificity to the antibodies in serum at low salt concentration. The adsorbed IgG was eluted by 0.8 M KCl and 72% of the IgG in the serum was recovered. The purity of the isolated IgG reached 98.4% and the bioactivity was fully maintained (>99%). The high efficiency, mild conditions and simplicity make this technology suitable for the economic purification of antibodies in a large scale.     

Development of a Novel Affinity Membrane Purification System for Deoxyribonuclease

A membrane based affinity purification system was developed for the purification of the DNA specific nuclease, DNase I. Single stranded DNA was bound to unmodified polyvinylidene fluoride (PVDF) membranes which were used to purify DNase I from a solution of bovine serum albumin. Using coated membranes, a 6-fold increase in specific activity was achieved with 80 % enzyme recovery. This method provides a simple yet effective way to purify DNase I and can be very useful for the purification of other DNA specific enzymes.     

Simultaneous separation and quantitation of the major bovine whey proteins including proteose peptone and caseinomacropeptide by reversed-phase high-performance liquid chromatography on polystyrene-divinylbenzene

A precise, sensitive and reliable RP-HPLC method was developed to enable not only unequivocal determination of alpha-lactalbumin and beta-lactoglobulin in bovine whey samples, but also simultaneous measurement of proteose peptone, caseinomacropeptide, bovine serum albumin and immunoglobulin G. The optimised method on the Resource RPC column allowed separation of the proteins in 30 min and could be applied to the analysis of soluble proteins in a variety of commercial and laboratory whey products. Furthermore, some qualitative information on protein heterogeneity and quality could be derived from the RP-HPLC analyses with additional data available from on-line electrospray mass spectrometry. Within- and between-day repeatability over a wide range of concentrations was excellent (RSD< or =5%) for all proteins except immunoglobulin G and bovine serum albumin where RSD was 7-10%. Analysis of grouped data from whey protein concentrate and whey protein isolate samples gave a limit of detection of < or =0.3% powder mass and a limit of quantitation of < or =1.0% powder mass for all proteins except immunoglobulin G. Limits of detection and quantitation were 0.6% and 2.0%, respectively, for this protein. Quantitative data obtained by the RP-HPLC method compared very favourably with data obtained by alternative methods of whey protein analysis.     

Purification of human immunoglobulin G by thermoseparating aqueous two-phase systems

The purification of human immunoglobulin G (IgG) from a Chinese hamster ovary (CHO) cells supernatant was studied using an aqueous two-phase system (ATPS) composed of ethylene oxide/propylene oxide (UCON) and dextran. In UCON/dextran systems IgG partitions preferentially to the less hydrophobic dextran-rich phase (Kp<1). The addition of triethylene glycol-diglutaric acid (TEG-COOH) shifted the IgG partition into the upper phase showing significant improvements in both the recovery yields and purity. The purification of IgG from a CHO cell supernatant with UCON 2000/dextran/TEG-COOH system was optimised using a central composite design. Using an ATPS composed of 8% UCON, 6% dextran and 20% TEG-COOH, IgG was purified, in two steps, with a global yield of 85% and 88% purity. Statistical valid models were obtained to predict the effect of the experimental conditions on the IgG yield and purity, for both extraction and back-extraction steps. A system composed of 10% UCON, 5.5% dextran and 20% TEG-COOH was identified as the best compromise between final purity and yield.