Phenylboronate Chromatography Research Articles

Mobile-Phase Modulators as Salt Tolerance Enhancers in Phenylboronate Chromatography: Thermodynamic Evaluation of the Mechanisms Underlying the Adsorption of Monoclonal Antibodies.

Phenylboronate chromatography has been employed for bioseparation applications though details concerning the mechanisms of interaction between the ligand and macromolecules remain widely unknown. Here, the phenomena underlying the adsorption of an anti-human interleukin-8 (anti-IL8) monoclonal antibody (mAb) onto an m-aminophenylboronic acid (m-APBA) ligandin the presence of different mobile-phase modulators (NaF/MgCl 2 /(NH 4 ) 2 SO 4 ) and under different pH values (7.5/8.5/9.0) is investigated. Flow microcalorimetry (FMC) is applied to measure instantaneous heat energy transfer, providing insights about the role of specific and nonspecific interactions involved in the adsorptive process. Results show that the adsorption of anti-IL8 mAb to m-APBA is enthalpically driven, corroborating the presence of the reversible esterification reaction between boronic acid or boronates and cis-diol-containing molecules. Nevertheless, for all mobile-phase modulators studied, changes in thermogram profiles are observed as well as reductions in the net heat of adsorption when increasing the pH. Overall, FMC and parallel chromatographic experiments data suggest that ligand salt tolerance could be enhanced using mobile-phase modulators, with all salts studied promoting the specific cis-diol interactions and reducing nonspecific interactions. The last feature is more noticeable at pH values above ligand's pK a , mainly due to the ability of NaF and (NH 4 ) 2 SO 4 to diminish electrostatic interactions when compared to the commonly used NaCl.

Thermodynamics of the adsorption of monoclonal antibodies in phenylboronate chromatography: Affinity versus multimodal interactions

The aim of this work was to investigate the complex phenomena underlying the adsorption of an anti-human IL-8 (anti-IL8) monoclonal antibody (mAb) to m-aminophenylboronate (m-APBA) by Flow Microcalorimetry (FMC) and to understand the role of non-specific interactions in the adsorption process. FMC was exploited as a dynamic on-line method to measure instantaneous heat energy transfers in order to understand the surface phenomena underlying mAb’s adsorption towards the synthetic ligand m-APBA under different pH (7.5, 8.5, 9.0, 9.5 and 10.0) and salt concentrations (0 and 150 mM NaCl). Results showed that the adsorption of anti-IL8 mAb to m-APBA is enthalpically driven (ΔHads<0), as expected for the predominant reversible esterification reaction between boronates and cis-diols-containing molecules. For all the pH conditions studied, thermograms presented a first exothermic peak, characteristic of the reversible esterification reaction between mAb (pI≥9.3) and m-APBA (pKa = 8.8), except at pH 9.0 in the presence of 150 mM NaCl, for which the thermogram presented a first endothermic peak. The heat of adsorption (ΔHads) obtained at conditions where cis-diol interactions were predominant was approximately −243 ± 38 kJ/mol against −82 ± 14 kJ/mol (p-value < 0.05) obtained at pH 9.0 with 150 mM NaCl. The observed shift in the thermogram profile at pH 9.0, 150 mM NaCl, and the consequent decrease of 60–70% in ΔHads were indicative of the promotion of electrostatic interactions between the protein and the ligand. Overall, and whereas the binding of the PBA ligand to mAb molecules has been described for decades as being affinity-based, our study demonstrates the multimodal behaviour of this interaction and contributes towards the understanding of the adsorption thermodynamics.

Phenylboronate chromatography selectively separates glycoproteins through the manipulation of electrostatic, charge transfer, and cis-diol interactions.

Phenylboronate chromatography (PBC) has been applied for several years, however details regarding the mechanisms of interactions between the ligand and biomolecules are still scarce. The goal of this work is to investigate the various chemical interactions between proteins and their ligands, using a protein library containing both glycosylated and nonglycosylated proteins. Differences in the adsorption of these proteins over a pH range from 4 to 9 were related to two main properties: charge and presence of glycans. Acidic or neutral proteins were strongly adsorbed below pH 8 although the uncharged trigonal form of phenylboronate (PB) is less susceptible to forming electrostatic and cis-diol interactions with proteins. The glycosylated proteins were only adsorbed above pH 8 when the electrostatic repulsion between the boronate anion and the protein surface was mitigated (at 200 mM NaCl). All basic proteins were highly adsorbed above pH 8 with PB also acting as a cation-exchanger with binding occurring through electrostatic interactions. Batch adsorption performed at acidic conditions in the presence of Lewis base showed that charge-transfer interactions are critical for protein retention. This study demonstrates the multimodal interaction of PBC, which can be a selective tool for separation of different classes of proteins.

Redox proteomics of fat globules unveils broad protein lactosylation and compositional changes in milk samples subjected to various technological procedures

The Maillard reaction between lactose and proteins occurs during thermal treatment of milk and lactosylated β-lactoglobulin, α-lactalbumin and caseins have widely been used to monitor the quality of dairy products. We recently demonstrated that a number of other whey milk proteins essential for nutrient delivery, defense against bacteria/virus and cellular proliferation become lactosylated during milk processing. The extent of their modification is associated with the harshness of product manufacturing. Since fat globule proteins are also highly important for the health-beneficial properties of milk, an evaluation of their lactosylation is crucial for a complete understanding of aliment nutritional characteristics. This is more important when milk is the unique dietary source, as in the infant diet. To this purpose, a sequential proteomic procedure involving an optimized milk fat globule (MFG) preparation/electrophoretic resolution, shot-gun analysis of gel portions for protein identification, selective trapping of lactosylated peptides by phenylboronate chromatography and their analysis by nanoLC-ESI-electron transfer dissociation (ETD) tandem MS was used for systematic characterization of fat globule proteins in milk samples subjected to various manufacturing procedures. Significant MFG protein compositional changes were observed between samples, highlighting the progressive adsorption of caseins and whey proteins on the fat globule surface as result of the technological process used. A significant lactosylation of MFG proteins was observed in ultra-high temperature sterilized and powdered for infant nutrition milk preparations, which well paralleled with the harshness of thermal treatment. Globally, this study allowed the identification of novel 157 non-redundant modification sites and 35 MFG proteins never reported so far as being lactosylated, in addition to the 153 ones ascertained here as present on other 21 MFG-adsorbed proteins whose nature was already characterized. Novel MFG proteins include components involved in nutrient delivery, defense response against pathogens and cellular proliferation/differentiation. Nutritional, biological and toxicological consequences of these findings are here discussed, highlighting their possible impact on children's diet.

Capture of human monoclonal antibodies from a clarified cell culture supernatant by phenyl boronate chromatography

In this work, we investigated the feasibility of using phenyl boronate (PB) chromatography for the direct capture of monoclonal antibodies from a CHO cell supernatant. Preliminary results, using pure protein solutions have shown that PB media can bind to human antibodies, not only at strong alkaline conditions but also at acidic pH values. In fact, antibodies have been found to bind in the pH range 5.5-8.5. On the other hand, insulin and human serum albumin did not bind at alkaline pH but at lower pH, which reflects the importance of non-specific interactions with the matrix. Different binding and eluting buffers were evaluated for the capture of immunoglobulin G (IgG) from a CHO cell supernatant and the most promising results were obtained using 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid at pH 8.5 as binding buffer and 1.5 M Tris-HCl as eluting buffer. Using a step elution, all IgG was recovered in the elution pool with a maximum purification factor of 56. A gradient elution allowed a further increase of the final purity, yet achieving a slightly lower yield. IgG recovery was around 85% and the purification factor was 76. The highest purity was obtained when the pH of the cell supernatant feed was previously adjusted to 8.5. Starting from an initial protein purity of 1.1% and high-performance liquid chromatography (HPLC) purity of 2.2%, after PB adsorption, a final protein purity of 85% and a HPLC purity of 88% was achieved.

Characterization of glycated hemoglobin in diabetic patients: usefulness of electrospray mass spectrometry in monitoring the extent and distribution of glycation

A combination of chromatographic and mass spectrometric techniques was used to evaluate the extent and distribution of glycation within the glycated hemoglobin (GHb) molecule. Studies on quantification of hemoglobin (Hb) glycation by electrospray ionization mass spectrometry (ES-MS) of intact globins employed specimens from 10 diabetic individuals and five normal controls. Detailed structural analysis of the phenylboronate affinity chromatography/ion-exchange (IE) HPLC-separated sub-populations of GHb was performed on a specimen carrying 13.7% GHb. An efficient protocol for mapping glycation sites within α and β globins was developed, e.g., Glu-C/Asp-N proteolytic digestion followed by LC–ES-MS. Relative site occupancy within discrete components of GHb was evaluated. A correlation between the degree of glycation measured at Hb level (by affinity chromatography) and at globin level (measured by ES-MS) was carried out. The above studies led us to conclude that during the process of phenylboronate chromatography GHb dimers, rather than tetramers, are bound to the affinity resin so a fraction of glycated dimers rather than tetramers is measured. This finding implies that a process of glycation affects a much higher number of native Hb tetramers than was previously contemplated. No glycation sites appear to be missed by phenylboronate affinity chromatography. We have found no evidence of the presence of multiple glycations within a single globin chain. While glycation of both globins within a dimer cannot be excluded, it is unlikely to be a significant phenomenon. According to ES-MS data, an equivalent of about one globin per αβ dimer of the affinity chromatography-isolated GHb carried glycation.

26] Bioassay for determining endogenous levels of cyclic ADP-ribose

Publisher Summary This chapter describes a bioassay for cyclic ADP-ribose (ADPR) on the basis of its Ca 2+ -releasing activity in homogenates prepared from sea urchin eggs. Substances that could potentially interfere with the Ca 2+ release bioassay, such as Ca 2+ , inositol 1,4,5-triphosphate, and NAD + , must be removed from the sample. Cyclic ADP-ribose in the extracts is purified by a two-step system consisting of phenylboronate chromatography followed by anion-exchange chromatography on AG MP-1. Strongylocentrotus purpuratus eggs are obtained by stimulating ovulation of a female sea urchin with a 1-ml injection of 0.5 M KCI and washed once in artificial seawater, twice in Ca 2+ -free seawater containing 1 m M ethylene glycol tetraacetic acid (EGTA), twice in Ca 2+ -free seawater without EGTA, once with the homogenization buffer, and resuspended with the same medium to 25%. The sensitivity of the bioassay can be increased by pretreating the homogenate with caffeine, which is known to potentiate cADPR-induced Ca 2+ release.

Plasmodium falciparum and Plasmodium chabaudi: Characterization of glycosylphosphatidylinositol-degrading activities

Merozoites of malaria parasites have a membrane-bound serine protease whose solubilization and subsequent activity depend on a parasite-derived glycosylphosphatidylinositol-phospholipase C (GPI-PLC). The GPI-degrading activities from both Plasmodium falciparum and Plasmodium chabaudi have been characterized and partially purified by phenylboronate chromatography. They are membrane-bound, developmentally regulated, calcium-independent enzymes and as such they resemble GPI-PLC of Trypanosoma brucei. Furthermore, a T. brucei GPI-PLC-specific monoclonal antibody (mAT3) immunoprecipitates the plasmodial GPI-degrading activity. Thin-layer chromatography is suggestive of two activities: a GPI-PLC and a phospholipase A.

Studies on the localization and properties of rat duodenal HCO 3−-ATPase with special relation to alkaline phosphatase

Brush-border membrane fractions were isolated from rat duodenum. Purity and integrity of the fraction was confirmed by electron microscopy, enzymic analysis and demonstration of Na +-dependent glucose uptake. The membranes were enriched 15-fold in alkaline phosphatase and α-glucosidase and 6-fold in HCO 3 −-ATPase activities. Assays of latent activity indicated that these enzymes were predominantly localised to the external aspect of the microvillus membrane. The enzymes were solubilised and subjected to analysis by gel filtration, ion exchange and phenylboronate chromatography. No separation of alkaline phosphatase and HCO 3 −-ATPase was obtained and it is suggested that they reflect the same enzyme activity. The apparent activation by HCO 3 − was investigated, and was found to be due to shifts in the pH dependency of the activity due to changes in ionic strength.

Purification to apparent homogeneity of a factor stimulating the growth of multiple lineages of hemopoietic cells.

A glycoprotein that stimulates the proliferation of multiple hemopoietic stem and progenitor cell types was purified to apparent homogeneity. The factor, termed P cell-stimulating factor (PSF), was assayed by its ability to support the growth of murine factor-dependent hemopoietic cell lines operationally termed persisting cells (P cells). PSF was purified 50,000-fold from serum-free medium conditioned by the myelomonocytic cell line WEHI-3B by sequential ammonium sulfate precipitation, phenyl boronate chromatography, gel filtration on Sephadex G-100, neuraminidase treatment, Mono Q anion exchange chromatography, reverse phase high performance liquid chromatography on a C18 silica column, and two steps of high performance gel permeation chromatography on a TSK 3000 SW column operated under first neutral and then acidic solvent conditions. Although purified PSF could not be detected on sodium dodecyl sulfate-polyacrylamide gels stained with silver, following electrophoresis of purified PSF labeled with iodine-125, autoradiography showed only a single broad band of Mr = 30,000. This labeled band corresponded to the profile of PSF activity eluted from polyacrylamide gel slices. After reduction, labeled PSF had a slightly higher Mr of 32,000, although reduction resulted in loss of 98% of PSF activity, thus suggesting that the integrity of internal disulfide bond(s) was required for activity. When purified PSF was chromatographed on a TSK 3000 SW column under denaturing conditions in 0.1% sodium dodecyl sulfate, the single peak of absorbance at 280 nm coincided with a sharp peak of biological activity. The following unique NH2-terminal amino acid sequence of the purified PSF was obtained: NH2ALA -SER-Ile-Ser-X-X-Asp-Thr-His-Arg-Leu-Thr-Arg-. The concentration of PSF required for half-maximal stimulation of P cell growth was estimated as 1.3 X 10(-13) M or 4 pg/ml. The availability of purified PSF will allow rigorous examination of the hypothesis that a single molecule acts on multiple hemopoietic cell lineages.

Selective Stimulation by Mouse Spleen Cell Conditioned Medium of Human Eosinophil Colony Formation

Stimulation of unfractionated or nonadherent human marrow cells in agar culture by pokeweed-mitogen-stimulated BALB/c mouse spleen cell conditioned medium (SCM) led, in most cultures, to the exclusive formation of eosinophil colonies. The culture system exhibited linearity of eosinophil colony formation with varying numbers of cells cultured, and the absolute numbers and size of SCM-stimulated eosinophil colonies approximated those in cultures stimulated by human placental conditioned medium. The active factor in SCM for human eosinophil colony formation was not clearly separable from the factors stimulating granulocyte-macrophage and eosinophil colony formation by mouse marrow cells on ammonium sulfate and phenyl boronate chromatography, but was of larger size than the mouse-active factors and separable from them by phenyl sepharose chromatography. This selective culture system for eosinophil colony formation should be of value for studies on human eosinophil progenitor and maturing cell populations in a variety of disease states.

Selective stimulation by mouse spleen cell conditioned medium of human eosinophil colony formation

Stimulation of unfractionated or nonadherent human marrow cells in agar culture by pokeweed-mitogen-stimulated BALB/c mouse spleen cell conditioned medium (SCM) led, in most cultures, to the exclusive formation of eosinophil colonies. The culture system exhibited linearity of eosinophil colony formation with varying numbers of cells cultured, and the absolute numbers and size of SCM-stimulated eosinophil colonies approximated those in cultures stimulated by human placental conditioned medium. The active factor in SCM for human eosinophil colony formation was not clearly separable from the factors stimulating granulocyte-macrophage and eosinophil colony formation by mouse marrow cells on ammonium sulfate and phenyl boronate chromatography, but was of larger size than the mouse-active factors and separable from them by phenyl sepharose chromatography. This selective culture system for eosinophil colony formation should be of value for studies on human eosinophil progenitor and maturing cell populations in a variety of disease states.