Purification Of Immunoglobulin G Research Articles

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 100-250 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.

Dye Ligand Epoxide Chitosan/Alginate: A Potential New Stationary Phase for Human IgG Purification

Cibacron Blue F3GA immobilized as ligand onto epoxide chitosan/alginate composite was prepared and evaluated for the purification of human immunoglobulin G (IgG) isolated from human serum by dye-ligand affinity chromatography. The effect of buffer pH on IgG adsorption, kinetic and isothern adsorption of high-purity IgG were studied by batch adsorption. The adsorption isotherm data were well fitted (using 25 mmol/L sodium phosphate buffer at pH 6.0) by the Langmuir-Freundlich model with a maximum adsorption capacity of 99 mg/g, apparent dissociation constant of 1.2 x 10-6 mol/L and n equal to 4.86. Fixed-bed experiments were performed using high-purity IgG, achieving a recovery rate of 53%. Human serum diluted ten times was also injected into the column. An enrichment of IgG in the elution fraction with traces of human serum albumin was observed. According to the study results, this material can be applied in the purification of human IgG under optimized conditions.

Purification of polyclonal antibodies from Cohn fraction II + III, skim milk, and whey by affinity chromatography using a hexamer peptide ligand

HWRGWV, a peptide that binds specifically to the Fc fragment of human immunoglobulin G (IgG), was used for the purification of IgG from Cohn fraction II + III of human plasma and from bovine skim milk and whey. The concentration of sodium chloride and sodium caprylate in the binding buffer as well as the pH of the elution buffer were optimized to achieve high IgG yield and purity. Under optimized conditions, IgG was recovered from plasma fractions with yield and purity up to 84% and 95%, respectively. IgG was also purified from skim milk with 74% yield and 92% purity and from whey with 85% yield and 93% purity. Purification experiments were also performed with Protein A resin and the results were found to be similar to those obtained with the peptide adsorbent.

Purification of IgG and albumin from human plasma by aqueous two phase system fractionation

The current shortages in human plasma products at global levels justify the development of new, cost effective plasma fractionation methods. We have developed a fractionation process to obtain immunoglobulin G (IgG) and albumin-enriched fractions based on polymer-salt aqueous two phase system (ATPS). A small-scale (0.02 L) ATPS composed of polyethyleneglycol 3350 (PEG), potassium phosphate and sodium chloride, at pH 6.1, was evaluated and subjected to 50-fold scale-up (1 L). Further purification of the fractions was performed using caprylic acid precipitation and ion exchange chromatography. Similar yield and purity were obtained at both small and large scales. IgG precipitated in the PEG rich upper phase at 83% recovery and 2.75-fold purification factor. An 81% pure albumin fraction was obtained in the salt rich bottom phase with a 91% yield. After polishing, IgG was obtained at a recovery of 70% and a purity of 92%. Corresponding values for albumin were 91% and 90%. This IgG and albumin fractionation technology deserves further evaluation as it may represent a potential alternative to conventional plasma fractionation methods.

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.

Enhancing IgG purification from serum albumin containing feedstock with hydrophobic charge-induction chromatography

Hydrophobic charge-induction chromatography (HCIC) with 4-mercaptoethyl-pyridine (MEP) as the ligand is a novel technology for antibody purification, however, the separation selectivity still needs to be improved for the applications, especially for the impurity of serum albumin. In this study, with bovine serum immunoglobulin G (IgG) as the model, the purification of IgG from the serum albumin containing feedstock was developed with the commercial HCIC resin MEP HyperCel, focusing on the optimization of operation pH and salt addition. The adsorption isotherms of IgG and bovine serum albumin (BSA) were investigated at different pHs, and the binding and elution behaviors of two proteins in the column were also studied at varying pHs. In addition, the protein–ligand interactions were investigated with some additives in the buffer. It was found that the conditions of pH 6 with 0.1M NaCl or pH 8 could be used to effectively remove BSA from the MEP resin without the influence on IgG adsorption. Two modes with control of loading or washing buffer were tested to enhance the purification of IgG from BSA containing feedstock, and the purity of IgG was improved to about 95% compared with 62.9% for the control. The results demonstrated that the control of loading pH or the addition of NaCl in the buffer might be an effective method to improve the purification of antibody with the HCIC process.

Monolithic cryogels made of agarose–chitosan composite and loaded with agarose beads for purification of immunoglobulin G

In order to obtain a novel absorbent with high adsorption capacity for the purification of immunoglobulin G (IgG), continuous supermacroporous agarose beads embedded agarose–chitosan composite monolithic cryogels (agarose–chitosan cryogels) were prepared by cryo-copolymerization of agarose–chitosan blend solutions with glutaraldehyde as the crosslinker in the presence of agarose beads. After coupling 2-mercaptopyridine onto divinylsulfone-activated matrix, the obtained cryogels were used for the purification of IgG. The microstructure morphologies of the cryogels were analyzed by scanning electron microscopy. The results showed that the obtained cryogels possess interconnected pores of 10–100μm size. The specific surface area was 350m2/g with maximum adsorption capacity of IgG 71.4mg/g. The cryogels showed workable stability, and can be reused at least 15 times without significant loss in adsorption capacity. IgG purity after one-step purification from human plasma was monitored by electrophoresis and the average recovery was estimated to be 90%.

Affinity purification and characterisation of human ACPAs

Backgroundand objectivesAutoimmunity in rheumatoid arthritis (RA) is characterised by autoantibodies to citrullinated proteins/peptides (ACPA). These antibodies, present in 60–70% of patients, antedate clinical onset and associate with an erosive disease...

IgG purification by negative chromatography in amine-based ligands: A comparison of l-lysine and poly-l-lysine

Agarose gels with immobilized amine-based ligands l-lysine (Lys) and poly-l-lysine (PLL) exhibited the ability to adsorb human serum proteins and to separate immunoglobulin G (IgG) in nonretained chromatographic fractions (negative chromatography). The effect of the buffer system and pH on IgG purification on Lys-agarose and PLL-agarose showed that PLL was the most selective ligand. PLL-agarose was able to purify IgG from human serum solution diluted in Bis–Tris buffer at pH 6.0 with 79% yield and 88.7% purity (based on total protein concentration and nephelometric analysis of albumin, transferrin, and immunoglobulins A, G, and M). Human serum albumin (HSA) and IgG adsorption data showed that the first followed the Langmuir model whereas the second followed the Langmuir–Freundlich model, due to the presence of a positive cooperativity effect (n=1.60), with the maximum adsorption capacities of 76.4mgmL−1 for HSA and 23.6mgmL−1 for IgG. The ΔGmax values obtained by non-linear regression of HSA and IgG adsorption data of Temkin model were similar (−7.0kcalmol−1), indicating that the process is spontaneous in nature. The joint analysis of these data shows that PLL-agarose can be considered an alternative adsorbent for the isolation of IgG from human serum with the potential to be integrated to a purification process on a large scale.

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–HCl 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–HCl 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.

Influence of protein adsorption kinetics on breakthrough broadening in membrane affinity chromatography

Existing mathematical models developed to describe membrane affinity chromatography are unable to match the complete breakthrough curve when a single Langmuir adsorption isotherm is used, because important deviations from the observed behavior are systematically encountered in the simulation of breakthrough broadening near saturation. The relevant information required to overcome that limitation has been obtained by considering simultaneously both loading and washing curves, thus evaluating the adsorption data at equilibrium and recognizing what are the appropriate adsorption mechanisms affecting the observed behavior. The analysis indicates that a bi-Langmuir binding kinetics is essential for a correct process description up to the saturation of the stationary phase, together with the use of the relevant transport phenomena already identified for the experimental system investigated. The input parameters used to generate the resulting simulations are evaluated from separate experiments, independent from the chromatographic process. Model calibration and validation is accomplished comparing model simulations with experimental data measured by feeding pure human immunoglobulin G (IgG) solutions as well as a cell culture supernatant containing human monoclonal IgG 1 to B14-TRZ-Epoxy2 bio-mimetic affinity membranes. The simulations obtained are in good agreement with the experimental data over the entire adsorption and washing stages, and breakthrough tailing appears to be associated to the reversible binding sites of the bi-Langmuir mechanism. Remarkably, the model proposed is able to predict with good accuracy the purification of IgG from a complex mixture simply on the basis of the results obtained from pure IgG solutions.

Production and purification of polyclonal anti-hamster immunoglobulins in rabbits

Polyclonal antibodies are mixtures of monoclonal antibodies that were produced against different epitops. The goal of this project is to know the production, purification and horseradish peroxidase (HRP) conjugation of polyclonal antibodies against hamster immunoglobulins in rabbits. 300 ig/300 il of ten hamster immunoglobulins was mixed with the same volume (300 il) of adjuvant and injected into three 6-month-old white New Zealand rabbits. Anti hamster rich rabbits serums were isolated from whole blood and precipitated with ammonium sulfate in the final concentration of 50%. The precipitate was dialysed against phosphate buffered saline (PBS) (pH: 7.4) and applied to ion exchange chromatography (IEC) on diethylaminoethyl (DEAE)-sepharose 6B with tris-phosphate (pH: 8.1), and tris-phosphate contain 50 mM NaCl buffer. The purity of produced antibody was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reduced condition. Then purified immunoglobulin G (IgG) was conjugated with HRP. For exact measurement of conjugated IgG titer and evaluating of cross reaction, enzyme linked immunosorbent assay (ELISA) test was designed. Since IEC is a more simple and inexpensive method for the purification of IgG, we obtained a protein with approximate purity of 95%. Produced IgG showed high titer and high specificity in the designed ELISA. Purified antibody and its conjugation with HRP are used in research and diagnosis of hamster disease. Key words : Production, purification, hamster immunoglobulins.

Experimental and Theoretical Investigation of Effect of Spacer Arm and Support Matrix of Synthetic Affinity Chromatographic Materials for the Purification of Monoclonal Antibodies

The aim of this study was to elucidate the influence of each material component-the support, the spacer, and the surface chemistry-on the overall material performance of an affinity type purification media for the capture of immunoglobulin G (IgG). Material properties were investigated in terms of an experimental evaluation using affinity chromatography as well as computer modeling. The biomimetic triazine-based A2P affinity ligand was chosen as a fixed point, while spacer and support were varied. The investigated spacers were 1-2-diaminoethane (2LP), 1,3-propanedithiol (SS3), 3,6-dioxo-1,8-octanedithiol (DES), and a 1,4-substituted [1,2,3]-triazole spacer (TRZ). The support media considered were the agarose (AG) resins, PuraBead, the polyvinylether, Fractoprep, the polymethacrylate, Fractogel, and the porous silica, Fractosil. All materials were tested with pure IgG standard solution, with a mock feed solution as well as real cell culture supernatant. The interaction between IgG and A2P linked through the investigated spacers to AG was studied using molecular dynamics. The effect of a modification of the support chemical structure or of the protein-ligand binding site on the material performances was studied through target oriented simulations. Dynamic binding experiments (DBC) revealed that the performances of materials containing 2LP spacers were significantly decreased in the presence of Pluronic F68. The simulations indicated that this is probably determined by the establishment of intermolecular interactions between the 2LP charged amino group and the ether oxygen of Pluronic F68. The spacer giving the highest IgG dynamic binding capacity when Pluronic F68 was present in the feed was TRZ. The simulations showed that, among the investigated spacers, TRZ is the only one that prevents the adsorption of A2P on the support surface, thus suggesting that the mobility and lack of interaction of the ligand with the support is an important property for an affinity material. Both experiments and calculations agree that the chemistry of the support surface can have a significant impact on IgG binding, either affecting the IgG DBC, as found experimentally for materials having similar ligand densities and spacer arms but different supports, or competing with the affinity ligand when hydrophobic groups are added to the model surface, as was computationally predicted.

A new process of IgG purification by negative chromatography: Adsorption aspects of human serum proteins onto ω-aminodecyl-agarose

The adsorbent ω-aminodecyl-agarose was evaluated as to its feasibility for the adsorption of human serum and plasma proteins, aiming at the purification of immunoglobulin G (IgG). The contribution of electrostatic and hydrophobic interactions (mixed-mode) and the effects of buffer system on the adsorption of serum proteins were also studied. The adsorption isotherm parameters of human serum albumin (HSA) and IgG were evaluated, pointing to the existence of cooperative effects in the process. A positive ( n = 2.30 ± 0.38) and negative cooperativity ( n = 0.63 ± 0.12) were observed for IgG and HSA binding, respectively. High purity IgG was obtained (based on total protein concentration and nephelometric analysis of HSA, transferrin, and immunoglobulins A, G, and M) with a 75% recovery in Hepes 25 mmol L −1 pH 6.8 feeding human serum. These results indicate that the use of ω-aminodecyl-agarose is a potential technique for purification 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 Cu 2+, Ni 2+ and Zn 2+ 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 Cu 2+ was used as ligand. CIM-IDA disk showed dynamic binding capacity in the range of 14–16 mg/ml with Cu 2+ and 7–9 mg/ml with Ni 2+ for human IgG with MMA buffer. In the case of CIM-IDA-Zn 2+ 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.

Cascade ultrafiltration bioreactor-separator system for continuous production of F(ab′) 2 fragment from immunoglobulin G

We discuss a continuous two-stage cascade ultrafiltration bioreactor-separator system for fragmentation of immunoglobulin G (IgG) by pepsin and purification of F(ab′) 2 fragment thus generated. F(ab′) 2 is frequently preferred to intact IgG in therapeutic applications due to lower immunogenicity and faster onset of action. The 10 kDa MWCO membrane of the first stage bioreactor retained pepsin, IgG and F(ab′) 2 while allowing degraded Fc sub-fragments through and the 70 kDa membrane of the second stage retained both IgG and F(ab′) 2 while allowing pepsin through. Due to this differential retention behavior, the pepsin concentration within the first stage was significantly higher than that in the feed. The first bioreactor therefore primarily carried out IgG digestion while the second stage primarily served as a separator for pepsin and F(ab′) 2. The two-stage system was first assessed using pure equine IgG as feed. Under optimized feed and sweep flow rates, 97% IgG conversion with 93% pure F(ab′) 2 product were obtained. A mathematical model for the bioreactor system was developed and simulated and experimental data were found to be in good agreement. When the bioreactor system was operated with unpurified equine serum as feed, close to 95% IgG conversion was observed. The F(ab′) 2 product obtained from serum showed a single band on SDS-PAGE. The results demonstrated the suitability of the two-step cascade membrane bioreactor for production of F(ab′) 2 from both IgG and unpurified equine serum.

The full amino acid repertoire is superior to serine/tyrosine for selection of high affinity immunoglobulin G binders from the fibronectin scaffold

The design of combinatorial libraries for molecular recognition requires extensive diversity to provide high affinity binding to myriad epitopes while maintaining a high degree of functionality to enable inclusion of binders in the limited screenable library size. In the current work, we directly compare minimal and maximal amino acid diversity libraries in the context of the 10th type III domain of human fibronectin. Libraries with either serine/tyrosine or full 20 amino acid diversity were created, pooled and screened for binding to rabbit and goat immunoglobulin G (IgG), and affinity matured by directed evolution. Multiple picomolar binders to rabbit IgG and nanomolar binders to goat IgG were engineered with peak affinities of 51 +/- 4 pM and 1.2 +/- 0.4 nM, respectively. Sequence analysis reveals that 93% of the selected BC and FG loops, including those from the highest affinity clones, originate from the full diversity library. Thus, with a modest initial library size (approximately 1 x 10(8)) and an efficient affinity maturation scheme, more extensive diversity is superior to a binary serine/tyrosine code for the generation of picomolar to low nanomolar binders in the fibronectin domain. The highest affinity binders demonstrated utility in affinity purification of IgG from serum and as detection reagents in flow cytometry.

Purification of human IgG by negative chromatography on ω-aminohexyl-agarose

The ω-aminohexyl diamine immobilized as ligand on CNBr- and bisoxirane-activated agarose gel was evaluated for the purification of human immunoglobulin G (IgG) from serum and plasma by negative affinity chromatography. The effects of matrix activation, buffer system, and feedstream on recovery and purity of IgG were studied. A one-step purification process using Hepes buffer at pH 6.8 allowed a similar recovery (69–76%) of the loaded IgG in the nonretained fractions for both matrices, but the purity was higher for epoxy-activated gel (electrophoretically homogeneous protein with a 6.5-fold purification). The IgG and human serum albumin (HSA) adsorption equilibrium studies showed that the adsorption isotherms of IgG and HSA obeyed the Langmuir–Freundlich and Langmuir models, respectively. The binding capacity of HSA was high (210.4 mg mL −1 of gel) and a positive cooperativity was observed for IgG binding. These results indicate that immobilizing ω-aminohexyl using bisoxirane as coupling agent is a useful strategy for rapid purification of IgG from human serum and plasma.

The Getting, the Purification and the Characterization of Species’Immunoglobulin G (IgG) and Anti-immunoglobulin G Serum for Dog,Goat and Rabbit

The research aimed at preparing, purifying and characterizing the immunoglobulin G, for dog, goat and rabbit, and the anti-IgG serum for the same species. The IgG species purification was done through precipitation, using ammonium sulfate solution and ion exchange chromatography (DEAE-cellulose). Checking the IgG purity was done by immunoelectrophoresis (IEF), for the corresponding anti-species serum, and by polyacrylamide gel electrophoresis in denaturant system (SDS-PAGE). Using IEF, we obtained a precipitation arch with cathodic migration, whereas using SDS-PAGE, we obtained a fraction with a molecular mass of 150 KDa. Dog and goat anti-IgG serum was prepared on rabbit, and the rabbit anti-IgG serum on goat, by IgG hyperimmunisation concealed in Freund adjuvant at a 1 to 1 ratio. Sensitivity and specificity testing of antisera was performed by immunoenzymatic test (ELISA). There were highlighted the high titers of antibodies to the dilution of 1:500 for anti-IgG rabbit and goat sera, and 1:400 for anti-IgG dog serum. Specificity testing of dog and rabbit anti-IgG in the heterologous system showed results which are in the negative range. The DO values were high for goat anti-IgG serum, sheep IgG serum and sheep anti-IgG serum and goat anti-IgG serum, due to antigenic kinship between these species. The reagents prepared will be used in great specificity and sensitivity immunodiagnostic tests.