Thiophilic Ligand Research Articles

Preparation and characterization of thiophilic cryogels with 2-mercapto ethanol as the ligand for IgG purification

In this study, thiophilic cryogels were prepared by two different approaches and they were used in purification of IgG from aqueous solutions and human plasma. In the first approach, poly(2-hydoxyethyl methacrylate) [PHEMA] cryogel disks were prepared. The PHEMA cryogel disks were activated by divinylsulfone (DVS) and 2-mercapto ethanol was attached. In the second approach, poly(2-hydroxyethyl methacrylate-etylene glycol dimethacrylate) [P(HEMA–EGDMA)] beads were synthesized and 2-mercapto ethanol was attached to the beads as a thiophilic ligand. In order to increase the surface area, P(HEMA–EGDMA)/PHEMA composite cryogel disks were prepared by embedding the P(HEMA–EGDMA) beads into PHEMA cryogels. Both the thiophilic PHEMA (T-PHEMA) cryogel disks and the thiophilic P(HEMA–EGDMA)/PHEMA [T-P(HEMA–EGDMA)/PHEMA] composite cryogel disks were characterized by Fourier transform infrared spectroscopy, surface area measurements, elemental analysis, swelling tests and scanning electron microscopy. The effects of salt concentration, pH, temperature, initial IgG concentration were analysed for IgG adsorption from aqueous solutions in batch mode. When lyotropic salt, Na2SO4, was used, the adsorption capacity was 27.5mg/g and 68.7mg/g for T-PHEMA and the T-P(HEMA–EGDMA)/PHEMA composite cryogel disks, respectively. 1.0M NaCl was used as desorption agent. The change in adsorption capacity was not remarkable for a repetition of adsorption–desorption cycle ten times.Both the thiophilic cryogel disks and the thiophilic composite cryogel disks were also successfully used for IgG isolation from human plasma. The purity assayed by SDS-PAGE was 89%. The adsorption capacities were 74.8mg/g and 137.4mg/g in human plasma samples for the T-PHEMA cryogel disks and the T-P(HEMA–EGDMA)/PHEMA composite cryogel disks, respectively.

Purification antibody by thiophilic magnetic sorbent modified with 2-mercapto-1-methylimidazol

An efficient strategy based on the thiophilic magnetic beads was developed to isolate immunoglobulin G (IgG) from human plasma with high efficiency. Depending on the improved surface modification, the surface density of thiophilic ligand (2-mercapto-1-methylimidazol) reached 1.67mmolg−1 resulting in a high adsorption capacity (qmax=20.7mgg−1) toward IgG. This purification of IgG could be carried out in some physiological conditions due to the heterocyclic structure of 2-mercapto-1-methylimidazol. It is found that the recognition region between the thiophilic ligand and IgG was mainly focused on the Fab fragments of IgG using the technology of enzyme digestion. Furthermore, the bound antibodies could be easily recovered without loss of their bioactivity at low salt concentration. These magnetic carries brought great potentials in large-scale antibody purification from human plasma attributing to their high specificity and efficiency, stable structure, sensitive magnetic response and excellent reusability.

Using thiophilic magnetic beads in purification of antibodies from human serum

The rapid progress of biotechnology, immunology and molecular biology are requiring antibodies with higher purity and stronger activity. Therefore, the development of efficient technology, which is suitable for large-scale purification of antibodies at low cost, becomes much more urgent. In this study, we presented a novel method to isolate immunoglobulin G from human serum based on the utilization of thiophilic magnetic polymer beads. Micron-sized magnetic beads with paramagnetic properties were synthesized by microsuspension polymerization in the presence of modified magnetite. After the thiophilic ligand of 2-mercaptonicotinic acid was modified on the surface, these magnetic beads exhibited a strong specificity towards immunoglobulin G in a salt-independent manner. Then, antibodies could be directly isolated from human serum in batch-wise mode with the assistance of magnetic decantation. The purity of the isolated antibody exceeded 94%. Because isolation was performed in physiological conditions, bioactivity of the antibody was fully preserved (>99%). Prominent advantages of this method, such as strong specificity, rapid processing, mild conditions, conventional equipment and excellent reusability, make this non-chromatographic technology embody great potentialities to isolate the antibodies on a large scale.

Chromatographic and fluorometric study of interactions between thiophilic and hydrophobic ligands and tryptophan peptide homologues

The interactions of tryptophan and its peptide homologues with thiophilic ligands were studied in terms of their chromatographic retention and steady-state fluorescence under various conditions, and compared with non-polar structures typically regarded as pure hydrophobic ligands. The experimental results show that both non-polar and polar interactions are involved in what has been termed “thiophilic adsorption chromatography”.

Comparison of affinity membranes for the purification of immunoglobulins

The purification of immunoglobulins was studied by comparing 10 different affinity membranes, prepared by coupling various affinity ligands to different microfiltration membranes. Membranes carrying the synthetic peptide TG19318, histidine, the thiophilic ligand and iminodiacetic acid complexed with Zn(II) showed a weak affinity for human IgG, as expressed by apparent association constants ( K A) in the order of 10 5 M −1. Human IgM and rat IgG bound with high affinity to TG19318 membranes, thus, demonstrating the potential of this sorbent for the purification of immunoglobulins other than human IgG. When carrying Protein-A ligands, membranes based on Nylon 66 coated with low-molar-mass dextran or poly(vinylalcohol), as well as commercial pre-activated polysulfone (Ultrabind ®) and regenerated cellulose (Sartobind ®) membranes, showed high affinity for human IgG ( K A≈10 6 M −1). In contrast, a nylon membrane coated with high-molar-mass dextran yielded only K A≈10 5 M −1, which was attributed to a low accessibility of the immobilized ligand. Besides the high association constants, Protein-A adsorbers based on polysulfone and regenerated cellulose membranes showed several other advantages, such as enhanced charge-to-charge consistency, simpler preparation procedure, membrane sterilisability, good selectivity for IgG purification from cell culture supernatant and good stability throughout repeated adsorption–elution cycles.

Separation of IgG from human plasma using thiophilic hollow fiber membranes

To replace conventional bioaffinity ligand like protein A or protein G, pseudobioaffinity ligands seem to be an interesting alternative for the purification of IgG. In this study, thiophilic ligand (T-gel) was immobilized on a copolymer of ethylene and vinyl alcohol (PEVA) based hollow fiber membrane to obtain an affinity support for the purification of IgG from human plasma. Microporous hollow fibers have been modified via divinyl sulfone activation and coupled by 2-mercaptoethanol (T-PEVA). The affinity module allowed the purification of IgG from human plasma with high efficiency (85% of IgG loaded were adsorbed) with a very small contamination of albumin (<1%). The enrichment was quite high (IgG/albumin) as shown by a final ratio of 39.3/0.27 before loading. The binding capacity could allow the separation of 1.5 g of IgG from human plasma with a functionalized membrane surface area of 1 m 2. We tried to minimize sample preparation by decreasing the salt concentration, the sample dilution and by optimizing the rinsing and elution steps. This thiophilic derivatized hollow fiber module proved that it could be used as a very efficient tool for the purification of IgG provided that high concentration of salt was added.

Salt-independent binding of antibodies from human serum to thiophilic heterocyclic ligands

Several thiophilic adsorbents with mercaptoheterocyclic ligands have been analyzed for their ability to bind human serum proteins in a salt-independent way. In contrast to 2-mercaptopyrimidine, 2-mercaptopyridine derived ligands show a group-selective binding of immunoglobulins and α 2-macroglobulin, not only in the presence of high concentrations of sodium sulphate but in buffers with low ionic strength. The binding is restricted to thiophilic gels obtained by coupling 2-mercaptopyridine to a vinylsulphone-activated matrix and is not achieved on epichlorohydrin-activated gels. A novel thiophilic ligand based on mercaptonicotinic acid, containing a carboxylic group together with the thiophilic pattern of thioaromatic adsorbents, is demonstrated to be useful as an alternative purification scheme for antibodies.

Thiophilic nature of divinylsulphone cross-linked agarose

We present data to suggest that divinylsulphone-activated matrix (DVS-gel) purifies immunoglobulins with the same protein-ligand interaction as the thiophilic ligand, T-gel. Divinylsulphone-activated matrix purifies IgG (>90% pure) from blood serum with a binding capacity of 6.3 mg/ml, compared to 6.9 mg/ml with T-gel. It also separates IgG and sIgA from cheese whey, with the same dependency on water-structuring salt concentration as T-gel. We offer an explanation for these results in terms of the polymerisation of divinylsulphone and, therefore, offer a new structure for the so-called thiophilic ligand.

Purification of monoclonal antibodies on dextran-coated silica support grafted by thiophilic ligand.

Coated silica beads are promising supports for high-performance liquid chromatography (HPLC) of proteins; they combine the excellent mechanical properties of silica with minimal non-specific interactions with proteins in solution due to the presence of hydrophilic dextran polymers adsorbed at the silica surface. So, dextran-coated porous silica beads can be grafted with beta-mercaptoethanol by using divinylsulfone as coupling reagent to obtain new thiophilic supports usable in HPLC. The affinity of monoclonal IgG subclasses from mouse ascitic fluids for the active phases can be analysed. These dextran-coated silica supports grafted with thiophilic ligands allow a one-step purification of these antibodies. Moreover, the chromatographic separation of two subclasses, immunoglobulin G1 (IgG1) and IgG3, is observed and can be correlated to the high resolution of these new HPLC thiophilic supports.

Novel heterocyclic ligands for the thiophilic purification of antibodies.

Novel thiophilic ligands based on mercaptoheterocycles were synthesized for use in one-step purification of antibodies. In order to better characterize these new structures, affinity constants were measured, as well as the influence of pH and salt on adsorption and elution. The ligand concentration was optimized for efficient and fast adsorption and elution of antibodies from ascites and serum. The purification of antibodies from cell culture supernatant proved difficult due the indicator phenol red of the growth media.

Novel sulfone-based thiophilic ligands for the high-performance liquid chromatographic purification of antibodies

Novel thiophilic ligands based on sulfone structures were synthesized for the fast high-performance liquid chromatographic (HPLC) purification of antibodies. In order to better characterize these new structures, affinity constants were measured as well as the influence of pH and salt on adsorption and elution. Efficient and fast purification of antibodies from ascites and serum was achieved. The purification of antibodies from cell culture supernatant proved difficult due the indicator pheno-red of the growth media. Two thiophilic structures turned out to be protein adsorbents, which adsorbed all proteins loaded and from which no protein could be eluted under normal conditions.