Method For Purification Of Antibodies Research Articles

IgG-Binding Peptidomimetic Mixed-Charge Polymer-Modified Resins for Chromatographic Purification of Antibodies.

The process of antibody purification using Fc affinity ligands such as protein A, G, and L faces several challenges including high cost, low stability, and loss of antibody activity due to harsh elution conditions. Here, we describe a chromatographic purification of antibodies utilizing a pH-responsive mixed-charge polymer that mimics the IgG-binding peptide (Z34C) derived from the B domain of protein A. The protein A mimetic resins were prepared by modifying the surface of a TOYOPEARL, methacrylate resin with a polymer that mimics the amino acid sequence of Z34C and the functions of histidine and acidic and neutral amino acids using histamine methacrylamide (HisMA), methacrylic acid, and neutral monomers. The therapeutic monoclonal antibody (mAb), rituximab, was retained on the column at pH 7 and eluted under mildly acidic conditions at pH 5 using a protein A mimetic resin (HisMA20-EEMA) optimized for antibody interaction. The injected antibodies were selectively captured on the column by hydrophobic and electrostatic interactions with the protein A mimetic polymer under neutral conditions and eluted by electrostatic repulsion under acidic conditions. The HisMA20-EEMA column successfully purified mAbs from mixtures with BSA, mouse ascites fluid, and hybridoma cell culture supernatant. In addition, the HisMA20-EEMA column consistently achieved 90% antibody recovery in 100 consecutive purifications from cell culture supernatant. The antibody purification method presented in this study is low cost, highly durable, easy to synthesize, and allows for mild elution conditions. The results demonstrate that the approach of mimicking IgG-binding peptides with mixed-charge polymers is useful for the development of column packing materials for antibody purification.

Stable and reusable calcium-responsive biopolymer for affinity precipitation of therapeutic antibodies.

Affinity precipitation is an attractive method for protein purification due to its many advantages, including the rapid capture of target proteins, simple processing, high specificity, and ease of scale-up. We previously reported a robust antibody purification method using Ca2+-dependent precipitation of ZZ-hCSQ2, a fusion protein of human calsequestrin 2, and the antibody-binding protein ZZ. However, the stability of this fusion protein was not sufficiently high for industrial use because the antibody recovery yield decreased to 60% after being reused 10 times. To identify a more stable calsequestrin (CSQ), we calculated Rosetta energy values for the folding stabilities of various CSQ homologs and selected human CSQ1 (hCSQ1) with lowest energy value (-992.6) as the new CSQ platform. We also identified that the linker sequence between ZZ and CSQ was vulnerable to proteases and alkaline pH by N-terminal protein sequencing. Therefore, we changed the linker to four asparagine (4N) sequences, which were shorter and less flexible than the previous glycine-rich linker. The new version of ZZ-CSQ, ZZ-4N-hCSQ1, was stable in a protease-containing conditioned medium obtained from the cultured Chinese hamster ovary cell or high pH condition (0.1M sodium hydroxide) for more than 5 days and could be reused at least 25 times for antibody purification without loss of recovery yield. The antibodies purified by ZZ-4N-hCSQ1 precipitation also showed greater purity (~33.6-fold lower host cell DNA and ~6.4-fold lower host cell protein) than those purified by protein A chromatography. These data suggest that ZZ-4N-hCSQ1 precipitation is more efficient and can achieve cost-effectiveness of up to 12.5-fold cheaper than previous antibody purification methods and can lower the production costs of therapeutic antibodies.

Comparison of Miniaturized Chromatographic Columns and 96-Well Plates for Automated Antibody Purification under Economic and Sustainable Aspects

Therapeutic antibodies are an area of major interest in treating infectious diseases, autoimmune diseases, and cancer. Efficient purification strategies are required to discover and produce antibodies, representing a significant cost driver. Miniaturization and automation of the chromatographic purification process may contribute to cost reduction. In this article, miniaturized chromatographic columns and 96-well filter plates are used to purify an IgG antibody from cell cultures and are compared in terms of quality, cost, and sustainability. The validation results of the two miniaturized chromatographic antibody purification methods indicate high efficacy and are comparable except for minor differences in yield. The lowest cost per sample can be obtained with the miniaturized chromatographic columns, as annual throughput can be maximized with the miniaturized plates. By evaluating sustainability, significant reductions in laboratory waste produced during antibody purification can be shown, along with an increase in employee safety by using the automation system. Overall, the comparison of results shows that, in the development of new purification strategies, the type of implementation has a decisive influence on the outcomes in terms of recovery, cost-effectiveness, and sustainability.

TRIM21 chimeric protein as a new molecular tool for multispecies IgG detection

BackgroundThe production of monoclonal antibodies for immunoglobulin detection is not cost-effective, while polyclonal antibody production depends on laboratory animals, raising concerns on animal welfare. The widespread use of immunoglobulins in the pharmaceutical industry and the increasing number and variety of new antibodies entering the market require new detection and purification strategies. The Tripartite motif-containing protein 21 is a soluble intracellular immunoglobulin G receptor that binds to the constant region of immunoglobulin G from various species with high affinity. We hypothesized that using this protein as an antibody-binding module to create immunoglobulin detection probes will improve the portfolio of antibody affinity ligands for diagnostic or therapeutic purposes. ResultsWe created a chimeric protein containing a mutated form of the C-terminal domain of mouse Tripartite motif-containing protein 21 linked to streptavidin to detect immunoglobulin G from various species of mammals. The protein is produced by heterologous expression and consists of an improved molecular tool, expanding the portfolio of antibody-affinity ligands for immunoassays. We also demonstrate that this affinity ligand may be used for purification purposes since imidazole elution of antibodies can be achieved instead of acidic elution conditions of current antibody purification methods. ConclusionData reported here provides an additional and superior alternative to the use of secondary antibodies, expanding the portfolio of antibodies affinity ligands for detection and purification purposes.

Anion species-triggered antibody separation system utilizing a thermo-responsive polymer column under optimized constant temperature

Although the field of antibody drugs has grown larger, the antibody production still faces several challenges. Effective antibody purification is required, but the conventional purification method for antibodies is cost intensive and often causes aggregation problems, indicating the need for new alternative antibody purification methods. In the present study, a constant temperature antibody purification system for use with a thermo-responsive polymer column was developed based on switching of anion species in eluents. By adjusting the temperature for each antibody, the developed column enabled separation of the therapeutic monoclonal antibodies, rituximab and trastuzumab, from contaminants without changing salt concentration or pH of the eluents. The thermo-responsive hydrogel-modified column packing material was synthesized by introducing n-butyl methacrylate, acrylic acid, N,N′-methylenebisacrylamide and N-isopropylacrylamide to the surface of silica beads with an initiator by a graft-from approach. Elution behavior of antibodies with three types of anions, such as citrate, phosphate, and chloride were tested under three different temperature conditions. It was demonstrated that the thermo-responsive hydrogel grafted column showed a switchable antibody retention behavior at constant temperature and salt concentration, with antibody adsorption by NaCl eluent and desorption by citric acid buffer eluent.

Preferential production of IgM-secreting hybridomas by immunization with DNA vaccines coding for Ebola virus glycoprotein: use of protein boosting for IgG-secreting hybridoma production.

PurposeThe goal of this study was to investigate the utility of DNA vaccines encoding Ebola virus glycoprotein (GP) as a vaccine type for the production of GP-specific hybridomas and antibodies.Materials and MethodsDNA vaccines were constructed to express Ebola virus GP. Mice were injected with GP DNA vaccines and their splenocytes were used for hybridoma production. Enzyme-linked immunosorbent assays (ELISAs), limiting dilution subcloning, antibody purification methods, and Western blot assays were used to select GP-specific hybridomas and purify monoclonal antibodies (MAbs) from the hybridoma cells.ResultsTwelve hybridomas, the cell supernatants of which displayed GP-binding activity, were selected by ELISA. When purified MAbs from 12 hybridomas were tested for their reactivity to GP, 11 MAbs, except for 1 MAb (from the A6-9 hybridoma) displaying an IgG2a type, were identified as IgM isotypes. Those 11 MAbs failed to recognize GP. However, the MAb from A6-9 recognized the mucin-like region of GP and remained reactive to the antigen at the lowest tested concentration (1.95 ng/mL). This result suggests that IgM-secreting hybridomas are predominantly generated by DNA vaccination. However, boosting with GP resulted in greater production of IgG-secreting hybridomas than GP DNA vaccination alone.ConclusionDNA vaccination may preferentially generate IgM-secreting hybridomas, but boosting with the protein antigen can reverse this propensity. Thus, this protein boosting approach may have implications for the production of IgG-specific hybridomas in the context of the DNA vaccination platform. In addition, the purified monoclonal IgG antibodies may be useful as therapeutic antibodies for controlling Ebola virus infection.

Abstract 2476: Bispecific redirected T-cell immunotherapy targeting P-cadherin expressing tumors

Abstract Introduction: Strong evidence exists supporting the important role T-cells play in the immune response against tumors. Still, the ability to initiate tumor specific immune responses remains a challenge. We have developed a Dual-Affinity Re-Targeting (DART®) protein engineered with enhanced pharmacokinetic properties to extend in vivo half-life, and designed to engage and activate endogenous polyclonal T cell populations via the CD3 complex in the presence of P-cadherin expressing tumors. We designate this bispecific redirecting T-cell molecule as P-cadherin LP-DART. P-cadherin up-regulation has been reported in various tumors, including breast, gastric, endometrial, colorectal and pancreatic cancers, and is correlated with poor survival of patients. Methods: The P-cadherin LP-DART was stably expressed by CHO cells and purified to homogeneity via standard antibody-purification methods. Functional in vitro studies were performed with a panel of human cancer cell lines and human T cells isolated from healthy donors. In vivo tumor growth inhibition studies were performed in immunodeficient athymic nude or NOD-scid IL2Rgammanull (NSG) mice bearing human tumor cell line- or patient derived-xenografts and human T-cells, and treated with P-cadherin LP-DART. Results: P-cadherin LP-DART exhibited binding to a broad panel of cancer cell lines expressing various levels of endogenous cell surface P-cadherin, and comparable binding to a number of human donor derived T-cells expressing CD3. In the presence of T-cells, this bispecific molecule elicited P-cadherin expression level dependent cytotoxic T-lymphocyte (CTL) responses against the different tumor cell lines, and induced antigen dependent T-cell activation and cytokine release. P-cadherin LP-DART also demonstrated potent in vivo anti-tumor activity against implanted tumor xenografts. Significant tumor growth inhibition was observed across a range of potential indications that express P-cadherin. Measurement of in vivo pharmacodynamic markers support P-cadherin LP-DART mediated CTL infiltration and killing as the mechanism of tumor inhibition. Conclusions: P-cadherin LP-DART displays potent in vitro and in vivo redirected T-cell activity against a broad panel of cancer cell lines expressing a range of cell surface P-cadherin levels. These data support further investigation of P-cadherin LP-DART as a potential novel therapeutic treatment for cancers expressing P-cadherin. Citation Format: Timothy S. Fisher, Adam Root, Bryan Peano, Allison Rohner, Justin Lucas, Mark Elliott, Konstantinos Tsaparikos, Hui Wang, Jonathan Golas, Maria Gavriil, Susan Benard, Tao He, Tracey Clark, Nahor Haddish-Berhane, Ralph Alderson, Yinhua Yang, Syd Johnson, Paul Moore, Lioudmila Tchistiakova, Hans-Peter Gerber, Chad May. Bispecific redirected T-cell immunotherapy targeting P-cadherin expressing tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2476. doi:10.1158/1538-7445.AM2015-2476

Rapid Purification of Anti-SARS-CoV Protein N Monoclonal Antibody by Using Magnetic Beads

A rapid and simple antibody purification method is reported for the isolation of monoclonal antibody IgG1 from SARS-CoV N protein rabbit serum by using surface-modified magnetic beads. First magnetic poly (methacrylate-divinylbenzene) (PMA-DVB) beads were prepared and modified with aldehyde end group through surface chemical reactions. Then protein A were covalently bound to aldehyde-activated magnetic beads and further used to purify monoclonal antibody IgG1 from rabbit serum directly. This rapid magnetic carrier technology proves to be efficient and rapid for antibody purification and has potential to massive production of monoclonal antibody.

Purification of a human immunoglobulin G1 monoclonal antibody from transgenic tobacco using membrane chromatographic processes

Efficient purification of protein biopharmaceuticals from transgenic plants is a major challenge, primarily due to low target protein expression levels, and high impurity content in the feed streams. These challenges may be addressed by using membrane chromatography. This paper discusses the use of cation-exchange and Protein A affinity-based membrane chromatographic techniques, singly and in combination for the purification of an anti- Pseudomonas aerugenosa O6ad human IgG1 monoclonal antibody from transgenic tobacco. Protein A membrane chromatography on its own was unable to provide a pure product, mainly due to extensive non-specific binding of impurities. Moreover, the Protein A membrane showed severe fouling tendency and generated high back-pressure. With cation-exchange membrane chromatography, minimal membrane fouling and high permeability were observed but high purity could not be achieved using one-step. Therefore, by using a combination of the cation-exchange and Protein A membrane chromatography, in that order, both high purity and recovery were achieved with high permeability. The antibody purification method was first systematically optimized using a simulated feed solution. Anti- P. aeruginosa human IgG1 type monoclonal antibody was then purified from transgenic tobacco juice using this optimized method.

Magnetic bead purification as a rapid and efficient method for enhanced antibody specificity for plant sample immunoblotting and immunolocalization

Molecular analysis of plant tissues with antibodies has traditionally been hindered by the presence of high non-specific binding by plant cell walls and other components along with significant contaminants within sera that retard identification of specific plant tissue targets. Methods which rely on immobile solid supports conjugated with high-affinity molecular entities, have been used to purify sera. Despite their wide use, traditional antibody purification methods can result in low yields or activity and can produce significant levels of secondary contaminants, resulting in high non-specific background and dilution of tissue-specific signals. Mobile support matrixes like magnetic beads conjugated with high-affinity antisera have recently become an efficient alternative method for isolating and identifying diverse molecular targets. In this study, rabbit anti-calreticulin (CRT) immunoglobulin G (IgG) was isolated from whole anti-CRT sera with magnetic beads and tested by Western blot and immunocytochemistry for CRT localization in Pistia stratiotes plant tissues. IgG protein quantitation and purity was compared between purified and non-purified pre-immune and anti-CRT sera using spectrophotometric, reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and fluorescence staining followed by quantitative densitometry analysis. Anti-CRT IgG binding specificity after purification was determined by Western blot of total soluble protein extract. Purified and non-purified pre-immune and anti-CRT samples were subsequently utilized for CRT immunogold localization in Pistia tissue sections and visualized with confocal microscopy. The results demonstrate that magnetic bead purified anti-CRT IgG from whole serum shows enhanced specificity and reduced background. The ease of use and speed of this IgG purification technique should find widespread use in the plant biology field.

Influence of the Antibody Purification Method on Immunoassay Performance: Hapten–Antibody Binding in Accordance with the Structure of the Affinity Column Ligand

The effects of ligands for immunoaffinity chromatography on the immunoassay were investigated with three goat anti-methamphetamine (anti-MA) antibodies (Abs). An N-4-aminobutyl derivative of methamphetamine (4-ABMA) was conjugated with proteins and used as immunogens. All the antisera produced were purified by affinity chromatography with various ligands of 4-ABMA–proteins and of haptens as well as protein G: 4-ABMA–bovine serum albumin (4-ABMA–BSA), 4-ABMA–keyhole limpet hemocyanine (4-ABMA–KLH), 4-ABMA–ovalbumin (4-ABMA–OVA), MA, 4-ABMA, and amphetamine were used as ligands. Enzyme-linked immunosorbent assay (ELISA) was conducted to examine characteristics of the purified Abs with the 4-ABMA–OVA competitor coated. The results obtained revealed that characters of the purified Abs were closely related with chemical structures of ligands used. The Abs from the MA and the amphetamine columns showed better sensitivities than those from the others in each antiserum. Particularly, the Ab from the amphetamine column gave the best results in terms of sensitivity and specificity. The recognition or the affinity of the Ab selected was considered to be affected by the structure of the ligand concerned. These results suggest that the Ab purification method should be considered as an important parameter which has great influence on the performance of immunoassays with polyclonal Abs.

Antibody purification methods.

Antibodies (Abs) from the sera of patients with autoimmune diseases are reported to have different catalytic functions. Their recovery by efficient purification methods is, therefore, a crucial step. This article reviews different available methods for their recovery and emphasizes a new approach, namely adsorbents with immobilized histidine, which allows a good purification both in yield and purity of Abs, with the additional advantage of using gentle elution conditions. This, in turn, will ensure the recovery of intact (nondenatured) catalytically functional Abs, directly from the sera.

Simple affinity purification of antibodies using in vivo biotinylation of a fusion protein

We describe a method for affinity purification of antibodies using gene fusions to the antigen. Fusions are made to a protein domain which is recognized in vivo by biotin ligase and biotinylated at a unique position in the fusion protein. When expressed in E. coli, the fusion protein can be captured from a whole cell lysate using an avidin resin to generate an affinity column which is useful for antibody purification. The procedure is simple, rapid and does not require chemical biotinylation or prior purification of the target antigen.

The optimization of ELISA for methamphetamine determination: the effect of immunogen, tracer and antibody purification method on the sensitivity

To obtain more sensitive immunoassay for methamphetamine (MA) determination, the optimum condition of enzyme-linked immunosorbent assay (ELISA) was investigated in regard to immunogens, antibody purification methods and coating tracers. Activated MA, N-(4-aminobutyl)methamphetamine (4-ABMA), was conjugated with bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH) and used as immunogen. The antibodies were purified by protein G chromatography or various immunoaffinity chromatography-linked MA-protein ligands, such as MA-BSA, MA-KLH or MA-ovalbumin (OVA). Each purified antibody was characterized by means of sensitivity and cross-reactivity using the three MA-protein coating tracers, MA-BSA, MA-KLH and MA-OVA. The best sensitivity of each antibody was acquired with the MA-OVA tracer although the tracer concentration and the antibody titer level at optimum condition were varied. The antibody with high titer level did not always yield good sensitivity. At optimum condition, immunoaffinity chromatography-purified antibodies were better for sensitivity and for specificity than protein G-purified antibodies. The cross-reactivity of the purified antibodies seemed to be affected by immunogen structure and showed somewhat different patterns according to the immunoaffinity ligand utilized. These data show that the antibody purification method as well as choice of coating tracer and immunogen is essential for the sensitivity and specificity of EIA; the optimum condition for assay should be discovered using various methods and combinations.

Differential distribution of N-acetylaspartylglutamate and N-acetylaspartate immunoreactivities in rat forebrain

Contradictory immunohistochemical data have been reported on the localization of N-acetylaspartylglutamate in the rat forebrain, using different carbodiimide fixation protocols and antibody purification methods. In one case, N-acetylaspartylglutamate immunoreactivity was observed in apparent interneurons throughout all allocortical and isocortical regions, suggesting possible colocalization with GABA. In another case, strong immunoreactivity was observed in numerous pyramidal cells in neocortex and hippocampus, suggesting colocalization with glutamate or aspartate. Reconciling these disparate findings is crucial to understanding the role of N-acetylaspartylglutamate in nervous system function. Antibodies to N-acetylaspartylglutamate and a structurally related molecule, N-acetylaspartate, were purified in stages, and their cross-reactivities with protein conjugates of N-acetylaspartylglutamate and N-acetylaspartate were monitored at each stage by solid-phase immunoassay. Reduction of the cross-reactivity of the anti-N-acetylaspartylglutamate antibodies of N-acetylaspartate-protein conjugates to about 1% eliminated significant staining of most pyramidal neurons in the rat forebrain. Utilizing highly purified antibodies, the distributions of N-acetylaspartylglutamate and N-acetylaspartate were examined in several major telencephalic and diencephalic regions of the rat, and were found to be distinct. N-acetylaspartylglutamate-immunoreactivity was observed in specific neuronal populations, including many groups thought to use GABA as a neurotransmitter. Among these were the globus pallidus, ventral pallidum, entopeducular nucleus, thalamic reticular nucleus, and scattered non-pyramidal neurons in all layers of isocortex and allocortex. N-acetylaspartate-immunoreactivity was more broadly distributed than N-acetylaspartylglutamate-immunoreactivity in the rat forebrain, appearing strongest in many pyramidal neurons. Although N-acetylaspartate-immunoreactivity was found in most neurons, it exhibited a great range of intensities between different neuronal types.

Recombinant proteins L and LG: efficient tools for purification of murine immunoglobulin G fragments.

In order to improve antibody purification methods, recombinant proteins L and LG were tested in the purification of murine monoclonal immunoglobulin G (IgG) and its fragments. After affinity constant evaluation in different buffer systems, high-performance affinity chromatographic columns were prepared by coupling the proteins to Affi-prep 10 resin and tested with eight different murine monoclonal antibodies and their fragments of different isotypes. Affinity chromatographic experiments confirmed radioimmunoassay results showing that protein L bound 75% of the tested antibody fragments whereas protein LG had affinity for all the tested fragments. These results demonstrate that protein LG is the most powerful Ig-binding tool so far described.

5202253 Monoclonal antibody specific for protein C and antibody purification method: Charles T Esmon, Naomi Esmon, Lund, assigned to Oklahoma Medical Research Foundation

5202253 Monoclonal antibody specific for protein C and antibody purification method: Charles T Esmon, Naomi Esmon, Lund, assigned to Oklahoma Medical Research Foundation

5202253 Monoclonal antibody specific for protein C and antibody purification method: Charles T Esmon, Naomi Esmon, Drongen, assigned to Oklahoma Medical Research Foundation

5202253 Monoclonal antibody specific for protein C and antibody purification method: Charles T Esmon, Naomi Esmon, Drongen, assigned to Oklahoma Medical Research Foundation

High performance liquid chromatography immunoaffinity purification of antibodies and antibody fragments

A rapid antibody purification method is described which combines high performance liquid chromatography (HPLC) resolution with affinity chromatography specificity. The antigen used as immobilized ligand is bound to the HPLC column matrix by reacting the amino groups of the protein with the active epoxy groups of the latter. Once the reaction has finished, the unreacted groups of the column are saturated with an appropriate scavenger. Unrelated proteins are then washed out and the specific antibodies recovered by lowering the pH of the elution buffer.

Identification, developmental regulation, and response to heat shock of two antigenically related forms of a major nuclear envelope protein in Drosophila embryos: application of an improved method for affinity purification of antibodies using polypeptides immobilized on nitrocellulose blots.

An affinity-purification method has been developed for the rapid, efficient, and precise elution of antibodies specifically bound to antigens immobilized on nitrocellulose after blot transfer from SDS polyacrylamide gels. The applicability of this technology has been demonstrated using antisera raised against the nuclear matrix-pore complex-lamina fraction prepared from Drosophila melanogaster embryos. In so doing, we have established the existence in whole embryo lysates, of two nearly identical forms of the predominant 74-kilodalton polypeptide previously identified in lower resolution studies of the nuclear matrix-pore complex-lamina fraction. These species, distinguishable on the basis of a slight difference in SDS PAGE mobilities on low concentration polyacrylamide gels, are immunochemically cross-reactive and have been localized exclusively to the nuclear periphery (nuclear envelope) by indirect immunofluorescence analyses of cryosections. The steady-state levels of these two polypeptides have been examined in total embryo lysates both as a function of embryogenesis and in response to heat shock. The larger species is not detectable in early embryos but approaches levels approximately equal to that of the smaller form by about the temporal midpoint of embryonic development. In response to heat shock, this larger form appears to be converted nearly quantitatively into the lower molecular weight polypeptide. These results, as well as the general reliability of the nitrocellulose blot immunoaffinity-purification methodology, have been substantiated through the use of monoclonal antibodies.