Liquid Affinity Chromatography Research Articles

Benchmarking glycoform-resolved affinity separation - mass spectrometry assays for studying FcγRIIIa binding.

The antibody- FcγRIIIa interaction triggers key immunological responses such as antibody dependent cellular cytotoxicity (ADCC), making it highly important for therapeutic mAbs. Due to the direct glycan-glycan interaction with FcγRIIIa receptor, differences in antibody glycosylation can drastically influence the binding affinity. Understanding the differential binding of mAb glycoforms is a very important, yet challenging task due to the co-existence of multiple glycoforms in a sample. Affinity liquid chromatography (AC) and affinity capillary electrophoresis (ACE) hyphenated with mass spectrometry (MS) can provide glycoform-resolved affinity profiles of proteins based on their differences in either dissociation (AC) or equilibrium (ACE) constants. To cross-validate the affinity ranking provided by these complementary novel approaches, both techniques were benchmarked using the same FcγRIIIa constructs. Both approaches were able to assess the mAb - FcγRIIIa interaction in a glycoform selective manner and showed a clear increase in binding for fully versus hemi-fucosylated mAbs. Also, other features, such as increasing affinity with elevated galactosylation or the binding affinity for high mannose glycoforms were consistent. We further applied these approaches to assess the binding towards the F158 allotype of FcγRIIIa, which was not reported before. The FcγRIIIa F158 allotype showed a very similar profile compared to the V158 receptor with the strongest increase in binding due to afucosylation and only a slight increase in binding with additional galactosylation. Both techniques showed a decrease of the binding affinity for high mannose glycoforms for FcγRIIIa F158 compared to the V158 variant. Overall, both approaches provided very comparable results in line with orthogonal methods proving the capabilities of separation-based affinity approaches to study FcγR binding of antibody glycoforms.

Fast and efficient identification of hyaluronidase specific inhibitors from Chrysanthemum morifolium Ramat. using UF-LC-MS technique and their anti-inflammation effect in macrophages

The purpose of the study was to establish a rapid analytical strategy to screen potential anti-inflammatory compounds from Flos Chrysanthemum flower. The enzyme assay was conducted to prescreen botanical extracts, in which Chrysanthemum morifolium aqueous extract (CME) displayed hyaluronidase (HAase) inhibitory activity in a dose-dependent manner with the values of 8.31, 24.25, and 66.51% at concentrations of 1.00, 2.00, and 4 0.00 mg/mL, respectively. Eight potential compounds targeting HAase (compounds 9, 10, 11, 13, 15, 17, 20 and 21) from CME were screened using ultrafiltration affinity liquid chromatography coupled with mass spectrometry (UF-LC-MS) technology. The well-known inhibitor, dipotassium glycyrrhizinate (DG), was used as a positive control and competitive ligand to eliminate false positives. Then, four of these potential components (compounds 9, 10, 17, and 21), namely eriodictyol-7-O-glucoside, luteoloside, apigenin-7-O-glucoside and diosmetin-7-O-glucoside, were distinguished as potent HAase specific inhibitor candidates with high BD and CBD values. The enzyme inhibitory activities of candidate compounds were verified using enzyme inhibition assay. At a concentration of 1000 μM, compounds 9, 10, 17, and 21 showed 40.15, 44.85, 18.04, and 24.15% inhibition of HAase, respectively. Furthermore, all the four compounds significantly decreased the production of nitric oxide (NO) and IL-6, and significantly suppressed the mRNA expression of inducible NO synthase (iNOS) and IL-1β in both murine and human macrophages.

Extending the performance of FcRn and FcγRIIIa affinity liquid chromatography for protein biopharmaceuticals

Affinity liquid chromatography using FcRn and FcγRIIIa columns can provide important information on the drug effector functions and the unique PK/PD properties of therapeutic mAbs. In this study, we propose a unique strategy to improve the performance of affinity chromatography by applying pH-gradient programs that incorporate multi-isocratic and negative gradient segments. These alternative gradient programs are known to greatly improve the separation of large solutes that follow a “bind-and-elute” type retention behavior. First, judicious optimization of the mobile phase compositions was performed to obtain a linear pH response. Then, with the developed strategy using multi-isocratic analysis conditions, the FcRn affinity separation selectivity for the analysis of oxidized mAb species was greatly improved. Furthermore, the introduction of negative gradient segments after each eluted peak improved the resolution between multiple glycosylated mAb species on the FcγRIIIa column. Therefore, this work provides a new strategy to improve the performance of affinity chromatography with mAb species, and could assist in the development of more accurate binding assays for important critical quality attributes related to FcRn and FcγRIIIa binding.

Enantioresolution and Binding Affinity Studies on Human Serum Albumin: Recent Applications and Trends

The interaction between proteins and drugs or other bioactive compounds has been widely explored over the past years. Several methods for analysis of this phenomenon have been developed and improved. Nowadays, increasing attention is paid to innovative methods, such as high performance affinity liquid chromatography (HPALC) and affinity capillary electrophoresis (ACE), taking into account various advantages. Moreover, the development of separation methods for the analysis and resolution of chiral drugs has been an area of ongoing interest in analytical and medicinal chemistry research. In addition to bioaffinity binding studies, both HPALC and ACE al-low one to perform other type of analyses, namely, displacement studies and enantioseparation of racemic or enantiomeric mixtures. Actually, proteins used as chiral selectors in chromatographic and electrophoretic methods have unique enantioselective properties demonstrating suitability for the enantioseparation of a large variety of chiral drugs or other bioactive compounds. This review is mainly focused in chromatographic and electrophoretic methods using human serum albumin (HSA), the most abundant plasma protein, as chiral selector for binding affinity analysis and enantioresolution of drugs. For both analytical purposes, updated examples are presented to highlight recent applications and current trends.

Detailed Characterization of Monoclonal Antibody Receptor Interaction Using Affinity Liquid Chromatography Hyphenated to Native Mass Spectrometry.

We report on the online coupling of FcRn affinity liquid chromatography (LC) with electrospray ionization mass spectrometry (ESI-MS) in native conditions to study the influence of modifications on the interaction of recombinant mAbs with the immobilized FcRn receptor domain. The analysis conditions were designed to fit the requirements of both affinity LC and ESI-MS. The mobile phase composition was optimized to maintain the proteins studied in native conditions and enable sharp pH changes in order to mimic properly IgGs Fc domain/FcRn receptor interaction. Mobile phase components needed to be sufficiently volatile to achieve native MS analysis. MS data demonstrated the conservation of the pseudonative form of IgGs and allowed identification of the separated variants. Native FcRn affinity LC-ESI-MS was performed on a therapeutic mAb undergoing various oxidation stress. Native MS detection was used to determine the sample oxidation level. Lower retention was observed for mAbs oxidized variants compared to their intact counterparts indicating decreased affinities for the receptor. This methodology proved to be suitable to identify and quantify post-translational modifications at native protein level in order to correlate their influence on the binding to the FcRn receptor. Native FcRn affinity LC-ESI-MS can tremendously reduce the time required to assess the biological relevance of the IgG microheterogeneities thus providing valuable information for biopharmaceutical research and development.

Application of coupled affinity-sizing chromatography for the detection of proteolyzed HSA-tagged proteins

Coupled affinity liquid chromatography and size exclusion chromatography (ALC–SEC) is a technique that has been shown to successfully report product quality of proteins during cell expression and prior to the commencement of downstream processing chromatography steps. This method was applied to monitoring the degradation and subsequent partial remediation of a HSA-tagged protein which showed proteolysis, allowing for rapid cell line development to address this product quality dilemma. This paper outlines the novel application of this method for measuring and addressing protease-induced proteolysis.

Analytical FcRn affinity chromatography for functional characterization of monoclonal antibodies

The neonatal Fc receptor (FcRn) is important for the metabolic fate of IgG antibodies in vivo. Analysis of the interaction between FcRn and IgG in vitro might provide insight into the structural and functional integrity of therapeutic IgG that may affect pharmacokinetics (PK) in vivo. We developed a standardized pH gradient FcRn affinity liquid chromatography method with conditions closely resembling the physiological mechanism of interaction between IgG and FcRn. This method allows the separation of molecular IgG isoforms, degradation products and engineered molecules based on their affinity to FcRn. Human FcRn was immobilized on the column and a linear pH gradient from pH 5.5 to 8.8 was applied. FcRn chromatography was used in comparison to surface plasmon resonance to characterize different monoclonal IgG preparations, e.g., oxidized or aggregated species. Wild-type and engineered IgGs were compared in vitro by FcRn chromatography and in vivo by PK studies in huFcRn transgenic mice. Analytical FcRn chromatography allows differentiation of IgG samples and variants by peak pattern and retention time profile. The method can distinguish: 1) IgGs with different Fabs, 2) oxidized from native IgG, 3) aggregates from monomer and 4) antibodies with mutations in the Fc part from wild-type IgGs. Changes in the FcRn chromatographic behavior of mutant IgGs relative to the wild-type IgG correlate to changes in the PK profile in the FcRn transgenic mice. These results demonstrate that FcRn affinity chromatography is a useful new method for the assessment of IgG integrity.

Detection, Identification, and Quantification of Selenoproteins in a Candidate Human Plasma Standard Reference Material

To understand the effect of Se supplementation on health, it is critical to accurately assess the Se status in the human body by measuring reliable biomarkers. The preferred biomarkers of the Se status are selenoprotein P (SelP) and glutathione peroxidase 3 (GPx3) along with selenoalbumin (SeAlb), but there is still a real need for reference methods and reference materials to validate their measurements. Therefore, this work presents a systematic approach to provide quality control data in selenoprotein measurements. This approach combines online isotope dilution affinity liquid chromatography (LC) coupled to inductively coupled plasma mass spectrometry (ICPMS), laser ablation ICPMS, and tandem mass spectrometry (MS/MS) to identify and quantify SelP, GPx3, and SeAlb in a human plasma reference material SRM 1950. Quantitative determinations of SelP, GPx3, and SeAlb were 50.2 ± 4.3, 23.6 ± 1.3, and 28.2 ± 2.6 ng g(-1) as Se, respectively. The subsequent identification of the selenoproteins included nine SelP peptides, including two selenopeptides and nine GPx3 peptides, while albumin was identified with a protein coverage factor >95%. The structural elucidation of selenoproteins in the target Se affinity fractions in SRM 1950 provides information needed for method validation and quality control measurements of selenoproteins and therefore the selenium status in human plasma.

Determination of Immunoglobulin G in Bovine Colostrum and Milk Powders, and in Dietary Supplements of Bovine Origin by Protein G Affinity Liquid Chromatography: Collaborative Study

An AOAC collaborative study was conducted to evaluate an affinity LC procedure for measuring immunoglobulin G (IgG) in selected dairy powders. The powders were extracted with 0.15 M sodium chloride solution and the pH was adjusted to 4.6 to precipitate caseins, which would otherwise lead to an overestimation of IgG. The analyte was then bound to a commercially available Protein G affinity cartridge and selectively eluted with a glycine buffer at pH 2.5. Detection was at 280 nm and quantification was made against a calibration curve prepared from bovine serum IgG. The samples analyzed included the likely matrixes for which this assay will find commercial use, namely, high- and low-protein-content colostrum powders, tablets containing colostrum powder, and some IgG-containing dairy powders; milk protein isolate, whey protein concentrate, and skim milk powder. Eleven laboratories provided data for the study and assayed blind duplicates of six materials. The repeatability RSD values ranged from 2.1 to 4.2% and the reproducibility RSD values ranged from 6.4 to 18.5%. The Protein G method with casein removal has adequate reproducibility for measuring IgG in colostrum-derived powders that are traded on the basis of IgG content as a colostral marker.

Heparin-mimetic sulfated peptides with modulated affinities for heparin-binding peptides and growth factors

Heterogeneity in the composition and in the polydispersity of heparin has motivated the development of homogeneous heparin mimics, and peptides of appropriate sequence and chemical function have therefore recently emerged as potential replacements for heparin in selected applications. Here, we report the assessment of the binding affinities of multiple sulfated peptides (SPs) for a set of heparin-binding peptides (HBPs) and for vascular endothelial growth factor isoform 165 (VEGF165); these binding partners have application in the selective immobilization of proteins and in hydrogel formation through non-covalent interactions. Sulfated peptides were produced via solid-phase methods, and their affinity for the HBPs and VEGF165 was assessed via affinity liquid chromatography (ALC), surface plasmon resonance (SPR), and in selected cases, isothermal titration calorimetry (ITC). The shortest peptide, SP a, showed the highest affinity binding of HBPs and VEGF165 in both ALC and SPR measurements, with slight exceptions. Of the investigated HBPs, a peptide based on the heparin-binding domain of human platelet factor 4 showed greatest binding affinities toward all of the SPs, consistent with its stronger binding to heparin. The affinity between SP a and PF4 ZIP was indicated via SPR ( K D = 5.27 μM) and confirmed via ITC ( K D = 8.09 μM). The binding by SP a of both VEGF and HBPs suggests its use as a binding partner to multiple species, and the use of these interactions in assembly of materials. Given that the peptide sequences can be varied to control binding affinity and selectivity, opportunities are also suggested for the production of a wider array of matrices with selective binding and release properties useful for biomaterials applications.

Study of dynamic adsorption behavior of large-size protein-bearing particles

The subject of this paper is an investigation of the peculiarities of dynamic adsorption behavior of nanoparticles. For this purpose, virus-mimicking synthetic particles bearing different proteins at their outer surface were specially constructed using two approaches, e.g. the cross-linking of proteins and modification of polystyrene microsphere surface by proteins. Two chromatographic modes, namely ion-exchange and affinity liquid chromatography on ultra-short monolithic columns [Convective Interaction Media (CIM) DEAE and CIM QA disks] have been used as a tool for dynamic adsorption experiments. Such parameters as maximum adsorption capacity and its dependence on applied flow rate were established and compared with those obtained for individual proteins. Similarly to individual proteins, it was shown that the maximum of adsorption capacity was not changed at different flow rates. In addition, the permeability of porous space of used monolithic sorbents appeared to be sufficient for efficient separation of large particles and quite similar to the well-studied process applied for individual proteins.

Affinity Liquid Chromatography Method for the Quantification of Immunoglobulin G in Bovine Colostrum Powders

An affinity liquid chromatography (LC) method for the determination of bovine immunoglobulin G (IgG), using protein G coupled to an agarose support, was modified to permit the quantification of IgG in colostrum-based powders. Sample preparation included pH adjustment to 4.6 to precipitate casein and denatured whey protein. The method was applied to a range of colostrum powders and was compared with the alternative independent methods of surface plasmon resonance immunoassay, radial immunodiffusion, and reversed-phase LC. The method was rapid, and performance parameters included a working range of 10-150 microg IgG and precision relative standard deviation values of <10%.

Affinity liquid chromatography and capillary electrophoresis of seminal plasma proteins

Interactions of boar, bull, and human seminal plasma proteins with heparin and phosphorylcholine were studied by affinity LC using heparin immobilized to a Toyopearl support. A step gradient elution from 0.15 to 1.50 M NaCl was employed to elute the seminal plasma proteins. Relative amounts of the heparin-binding fraction of seminal plasma proteins (H+) in seminal plasma of three species were determined. Further on, the fraction of seminal plasma proteins interacting with phosphorylcholine-binding proteins (P+) was evaluated. P+ proteins were not found in human seminal plasma and their highest amount was present in bull seminal plasma. A CE method was developed for separation of seminal plasma proteins. Various capillaries and separation conditions were tested; the best resolution was obtained in a bare-silica capillary, with a micellar system consisting of a 0.02 M borate buffer and 0.05 M SDS pH 10.0. The optimized conditions were applied to the identification of the components in boar plasma.

Temporal quantitation of mutant Kit tyrosine kinase signaling attenuated by a novel thiophene kinase inhibitor OSI-930

OSI-930, a potent thiophene inhibitor of the Kit, KDR, and platelet-derived growth factor receptor tyrosine kinases, was used to selectively inhibit tyrosine phosphorylation downstream of juxtamembrane mutant Kit in the mast cell leukemia line HMC-1. Inhibition of Kit kinase activity resulted in a rapid dephosphorylation of Kit and inhibition of the downstream signaling pathways. Attenuation of Ras-Raf-Erk (phospho-Erk, phospho-p38), phosphatidyl inositol-3' kinase (phospho-p85, phospho-Akt, phospho-S6), and signal transducers and activators of transcription signaling pathways (phospho-STAT3/5/6) were measured by affinity liquid chromatography tandem mass spectrometry, by immunoblot, and by tissue microarrays of fixed cell pellets. To more globally define additional components of Kit signaling temporally altered by kinase inhibition, a novel multiplex quantitative isobaric peptide labeling approach was used. This approach allowed clustering of proteins by temporal expression patterns. Kit kinase, which dephosphorylates rapidly upon kinase inhibition, was shown to regulate both Shp-1 and BDP-1 tyrosine phosphatases and the phosphatase-interacting protein PSTPIP2. Interactions with SH2 domain adapters [growth factor receptor binding protein 2 (Grb2), Cbl, Slp-76] and SH3 domain adapters (HS1, cortactin, CD2BP3) were attenuated by inhibition of Kit kinase activity. Functional crosstalk between Kit and the non-receptor tyrosine kinases Fes/Fps, Fer, Btk, and Syk was observed. Inhibition of Kit modulated phosphorylation-dependent interactions with pathways controlling focal adhesion (paxillin, leupaxin, p130CAS, FAK1, the Src family kinase Lyn, Wasp, Fhl-3, G25K, Ack-1, Nap1, SH3P12/ponsin) and septin-actin complexes (NEDD5, cdc11, actin). The combined use of isobaric protein quantitation and expression clustering, immunoblot, and tissue microarray strategies allowed temporal measurement signaling pathways modulated by mutant Kit inhibition in a model of mast cell leukemia.

Preparation and testing of stationary phases and modified capillaries for affinity chromatography and affinity capillary electrophoresis of pepsin

Three stationary phases have been prepared for affinity liquid chromatography isolation and separation of porcine and human pepsin. The phases contain 3,5-diiodo- l-tyrosine (DIT) bound to the supports HEMA BIO VS, HEMA BIO E and EPOXY TOYOPEARL. These phases have been tested on a model sample of porcine pepsin A and applied to human pepsin. Fractions have been collected and the chymase activity determined in selected analyses. For affinity CE, capillaries have been prepared by modifying the wall with 3-aminopropyltriethoxysilane, followed either by direct binding of DIT, or by binding l-tyrosine that was subsequently iodated. The dissociation constant K d has been determined for the pepsin–DIT complex from the changes in the electrophoretic mobilities.

Mechanisms of Action of Calcium-Sensitizing Drugs

This review compares the mechanisms of action of the calcium-sensitizing agents levosimendan, pimobendan, MCI-154, and EMD 53998. By using purified human recombinant troponin-C (cTnC), the role of cTnC as a target protein for these compounds was investigated. Accordingly, the calcium-dependent binding to cTnC in a cTnC-high-performance liquid affinity chromatography (HPLAC) column and the stabilizing effects of the compounds on the calcium-induced conformation of dansylated cTnC were studied. Only levosimendan showed calcium-dependent action on cTnC. Of the studied compounds, levosimendan was the most potent calcium sensitizer in skinned fiber experiments. Furthermore, EMD 53998 and MCI-154, but not levosimendan and pimobendan, increased myosin ATPase activity, indicating that they may enhance the cycling rate of myosin-actin crossbridges. By analyzing the velocity (dT/dt) of isometric tension development in paced papillary muscles, it was shown that levosimendan probably enhances the association rate but decreases the dissociation rate of myosin-actin crossbridges. These effects occurred before the peak twitch tension was achieved. Therefore, levosimendan does not seem to affect the actual relaxation phase. The other calcium sensitizers, however, appear to act mainly by decreasing the dissociation rate of crossbridges. The weak calcium-sensitizing effect of pimobendan may be based on indirectly mediated increase in affinity of cTnC for calcium. MCI-154 might act in a similar way but, like EMD 53998, MCI-154 also acts on myosin-actin crossbridges. We suggest that levosimendan binds in a calcium-dependent manner to the N-terminal domain of cTnC, which magnifies the extent of the contraction produced by cTnC when it is calcium-activated.

Automated affinity liquid chromatography system for on-line isolation, separation, and quantitation of aflatoxins in methanol-water extracts of corn or peanuts

An automated liquid chromatography (LC) procedure has been developed for the determination of aflatoxins in corn and peanuts. The automated LC system consists of the following: two pumps, an autosampler, an isolation column (immunoaffinity or methacrylate copolymer), an automated switching valve, a C 18 column, an electrochemical cell, a fluorescence detector, and a data system. Filtered methanol: water (75:25) extracts of the test portions are injected into the LC system. With the switch in position 1, water is pumped through the autosampler and isolation column and then discarded. With the switch in position 2, the mobile phase consisting of methanol:acetonitrile:water (26:18:56), 1 mM KBr, and 1 mM HNO 3 is pumped through the isolation column, C 18 column, electrochemical cell, and detector

Coated silica and its behaviour in dye-affinity chromatography

Cellulose-coated silica as a support for the high-performance affinity liquid chromatography of proteins was prepared. Cibacron Blue 3G-A as an affinity ligand was grafted onto silica beads coated with cellulose in on or two steps. The prepared supports were used for the purification of lactate dehydrogenase from a crude bovine extract using solutions of potassium chloride, NADH and Cibacron Blue—dextran T 10 as eluents of the bound enzyme. The soprtion capacities of the dyed cellulose-coated silica for lactate dehydrogenase from bovine muscle were found to be much lower than those of Cibacron Blue—bead cellulose. The comparison of un-coated and coated silica supports showed that the non-specific interactions between the enzyme and the silica coated with cellulose were considerably reduced.

Selective effect of clonazepam and ( S)-uxepam on the binding of warfarin enantiomers to human serum albumin

Both clonazepam and ( S)-uxepam selectively increase the binding of ( S)-warfarin to human serum albumin. By liquid affinity chromatography, improved resolution of rac-warfarin was achieved.

Affinity chromatography of thrombin on modified polystyrene resins

Insoluble polystyrenes substituted with sulphonate and L-arginyl methyl ester (PAOM) present substituents mimicking the reactive binding site of antithrombin III. These materials have a specific affinity for thrombin. The binding of the enzyme is reversible and the eluted thrombin remains active. Consequently, these resins can be used as stationary phases in affinity liquid chromatography in order to purify thrombin with a high biological activity. The influence of different characteristics of such polymers (substitution ratio, average particle size, affinity constant, synthesis conditions) on the purification performance is studied. Human prothrombin complex concentrate is activated and applied onto the gel. A purified human thrombin of high specific activity is separated with a high recovery of biological activity of the enzyme.