Peak decay analysis and biointeraction studies of immunoglobulin binding and dissociation on protein G affinity microcolumns.

Protein G can be a valuable binding agent for antibodies and immunoglobulins in methods such as immunosensors, chromatographic-based immunoassays, and immunoaffinity chromatography. This report used the method of peak decay analysis along with frontal analysis and zonal elution studies to characterize the binding, elution and regeneration properties of affinity microcolumns that contained immobilized protein G. Frontal analysis was employed with rabbit immunoglobulin G (IgG) to characterize the binding capacity of these affinity microcolumns. Zonal elution experiments looking at the retained peaks for small injections of labeled rabbit IgG were used to optimize the column regeneration conditions. Peak decay analysis was then used to look at the effects of flow rate and elution pH on the release of several types of IgG from the protein G microcolumns. This approach made it possible to obtain detailed information on the use and behavior of such columns, as could be used in future work to optimize the capture or analysis of IgG and antibodies by such devices. The same approach and tools that were used in this report could also be adapted for work with affinity columns that make use of other supports, binding agents or targets.

On-column entrapment of alpha1-acid glycoprotein for studies of drug-protein binding by high-performance affinity chromatography.

An on-column approach for protein entrapment was developed to immobilize alpha1-acid glycoprotein (AGP) for drug-protein binding studies based on high-performance affinity chromatography. Soluble AGP was physically entrapped by using microcolumns that contained hydrazide-activated porous silica and by employing mildly oxidized glycogen as a capping agent. Three on-column entrapment methods were evaluated and compared to a previous slurry-based entrapment method. The final selected method was used to prepare 1.0 cm × 2.1 mm I.D. affinity microcolumns that contained up to 21 (±4) µg AGP and that could be used over the course of more than 150 sample applications. Frontal analysis and zonal elution studies were performed on these affinity microcolumns to examine the binding of various drugs with the entrapped AGP. Site-selective competition studies were also conducted for these drugs. The results showed good agreement with previous observations for these drug-protein systems and with binding constants that have been reported in the literature. The entrapment method developed in this study should be useful for future work in the area of personalized medicine and in the high-throughput screening of drug interactions with AGP or other proteins. Graphical abstract On-column protein entrapment using a hydrazide-activated support and oxidized glycogen as a capping agent.

Use of protein G microcolumns in chromatographic immunoassays: A comparison of competitive binding formats.

Affinity microcolumns containing protein G were used as general platforms for creating chromatographic-based competitive binding immunoassays. Human serum albumin (HSA) was used as a model target for this work and HSA tagged with a near infrared fluorescent dye was utilized as the label. The protein G microcolumns were evaluated for use in several assay formats, including both solution-based and column-based competitive binding immunoassays and simultaneous or sequential injection formats. All of these methods were characterized by using the same amounts of labeled HSA and anti-HSA antibodies per sample, as chosen for the analysis of a protein target in the low-to-mid ng/mL range. The results were used to compare these formats in terms of their response, precision, limit of detection, and analysis time. All these methods gave detection limits in the range of 8-19ng/mL and precisions ranging from ±5% to ±10% when using an injection flow rate of 0.10mL/min. The column-based sequential injection immunoassay provided the best limit of detection and the greatest change in response at low target concentrations, while the solution-based simultaneous injection method had the broadest linear and dynamic ranges. These results provided valuable guidelines that can be employed to develop and extend the use of protein G microcolumns and these competitive binding formats to other protein biomarkers or biological agents of clinical or pharmaceutical interest.

Entrapment of alpha1-acid glycoprotein in high-performance affinity columns for drug-protein binding studies.

A slurry-based method was developed for the entrapment of alpha1-acid glycoprotein (AGP) for use in high-performance affinity chromatography to study drug interactions with this serum protein. Entrapment was achieved based on the physical containment of AGP in hydrazide-activated porous silica supports and by using mildly oxidized glycogen as a capping agent. The conditions needed for this process were examined and optimized. When this type of AGP column was used in binding studies, the association equilibrium constant (Ka) measured by frontal analysis at pH 7.4 and 37°C for carbamazepine with AGP was found to be 1.0 (±0.5)×10(5)M(-1), which agreed with a previously reported value of 1.0 (±0.1)×10(5)M(-1). Binding studies based on zonal elution were conducted for several other drugs with such columns, giving equilibrium constants that were consistent with literature values. An entrapped AGP column was also used in combination with a column containing entrapped HSA in a screening assay format to compare the binding of various drugs to AGP and HSA. These results also agreed with previous data that have been reported in literature for both of these proteins. The same entrapment method could be extended to other proteins and to the investigation of additional types of drug-protein interactions. Potential applications include the rapid quantitative analysis of biological interactions and the high-throughput screening of drug candidates for their binding to a given protein.

Chromatographic analysis of the effects of fatty acids and glycation on binding by probes for Sudlow sites I and II to human serum albumin.

The primary endogenous ligands of human serum albumin (HSA) are non-esterified fatty acids, with 0.1-2mol of fatty acids normally being bound to HSA. In type II diabetes, fatty acid levels in serum are often elevated, and the presence of high glucose results in an increase in the non-enzymatic glycation of HSA. High-performance affinity chromatography (HPAC) was used to examine the combined effects of glycation and the presence of long chain fatty acids on the binding of HSA with R-warfarin and l-tryptophan (i.e., probes for Sudlow sites I and II, the major sites for drugs on this protein). Zonal elution competition studies were used to examine the interactions of myristic acid, palmitic acid and stearic acid with these probes on HSA. It was found that all these fatty acids had direct competition with R-warfarin at Sudlow site I of normal HSA and glycated HSA, with the glycated HSA typically having stronger binding for the fatty acids at this site. At Sudlow site II, direct competition was observed for all the fatty acids with l-tryptophan when using normal HSA, while glycated HSA gave no competition or positive allosteric interactions between these fatty acids and l-tryptophan. These data indicated that glycation can alter the interactions of drugs and fatty acids at specific binding sites on HSA. The results of this study should lead to a better understanding of how these interactions may change during diabetes and demonstrate how HPAC can be used to examine drug/solute-protein interactions in complex systems.

Analysis of drug-protein interactions by high-performance affinity chromatography: interactions of sulfonylurea drugs with normal and glycated human serum albumin.

High-performance affinity chromatography (HPAC) is a type of liquid chromatography that has seen growing use as a tool for the study of drug-protein interactions. This report describes how HPAC can be used to provide information on the number of binding sites, equilibrium constants, and changes in binding that can occur during drug-protein interactions. This approach will be illustrated through recent data that have been obtained by HPAC for the binding of sulfonylurea drugs and other solutes to the protein human serum albumin (HSA), and especially to forms of this protein that have been modified by non-enzymatic glycation. The theory and use of both frontal analysis and zonal elution competition studies in such work will be discussed. Various practical aspects of these experiments will be presented, as well as factors to consider in the extension of these methods to other drugs and proteins or additional types of biological interactions.

Development of microcolumn-based one-site immunometric assays for protein biomarkers.

One-site immunometric assays that utilize affinity microcolumns were developed and evaluated for the analysis of protein biomarkers. This approach used labeled antibodies that were monitored through on-line fluorescence or near-infrared (NIR) fluorescence detection. Human serum albumin (HSA) was utilized as a model target protein for this approach. In these assays, a fixed amount of labeled anti-HSA antibodies was mixed with samples or standards containing HSA, followed by the injection of this mixture onto an HSA microcolumn to remove excess antibodies and detect the non-retained labeled antibodies that were bound to HSA from the sample. The affinity microcolumns were 2.1mm i.d. ×5mm and contained 8-9nmol of immobilized HSA. These microcolumns were used from 0.10 to 1.0mL/min and gave results within 35s to 2.8min of sample injection. Limits of detection down to 0.10-0.28ng/mL (1.5-4.2pM) or 25-30pg/mL (0.38-0.45pM) were achieved when using fluorescein or a NIR fluorescent dye as the label, with an assay precision of ±0.1-4.2%. Several parameters were examined during the optimization of these assays, and general guidelines and procedures were developed for the extension of this approach for use with other types of affinity microcolumns and protein biomarkers.

Studies of drug interactions with glycated human serum albumin by high-performance affinity chromatography.

Diabetes is a health condition associated with elevated levels of glucose in the bloodstream and affects 366 million people worldwide. Type II diabetes is often treated with sulfonylurea drugs, which are known to bind tightly in blood to the transport protein human serum albumin (HSA). One consequence of the elevated levels of glucose in diabetes is the non-enzymatic glycation of proteins such as HSA. Several areas of HSA are now known to be affected by glycation-related modifications, which may in turn affect the binding of sulfonylurea drugs and other solutes to this protein. This review discusses some recent studies that have examined these changes in drug-protein binding by employing high-performance affinity chromatography (HPAC). A description of the theoretical and experimental techniques that were used in these studies is given. The information on drug interactions with glycated HSA, as obtained through this method, is also summarized. In addition, the potential advantages of this approach in the areas of biointeraction analysis and personalized medicine are considered.

Studies of metabolite-protein interactions: a review.

The study of metabolomics can provide valuable information about biochemical pathways and processes at the molecular level. There have been many reports that have examined the structure, identity and concentrations of metabolites in biological systems. However, the binding of metabolites with proteins is also of growing interest. This review examines past reports that have looked at the binding of various types of metabolites with proteins. An overview of the techniques that have been used to characterize and study metabolite-protein binding is first provided. This is followed by examples of studies that have investigated the binding of hormones, fatty acids, drugs or other xenobiotics, and their metabolites with transport proteins and receptors. These examples include reports that have considered the structure of the resulting solute-protein complexes, the nature of the binding sites, the strength of these interactions, the variations in these interactions with solute structure, and the kinetics of these reactions. The possible effects of metabolic diseases on these processes, including the impact of alterations in the structure and function of proteins, are also considered.

Effects of Fatty Acids and Glycation on Drug Interactions with Human Serum Albumin.

The presence of elevated glucose concentrations in diabetes is a metabolic change that leads to an increase in the amount of non-enzymatic glycation that occurs for serum proteins. One protein that is affected by this process is the main serum protein, human serum albumin (HSA), which is also an important carrier agent for many drugs and fatty acids in the circulatory system. Sulfonylureas drugs, used to treat type 2 diabetes, are known to have significant binding to HSA. This study employed ultrafiltration and high-performance affinity chromatography to examine the effects of HSA glycation on the interactions of several sulfonylurea drugs (i.e., acetohexamide, tolbutamide and gliclazide) with fatty acids, whose concentrations in serum are also affected by diabetes. Similar overall changes in binding were noted for these drugs with normal HSA or glycated HSA and in the presence of the fatty acids. For most of the tested drugs, the addition of physiological levels of the fatty acids to normal HSA and glycated HSA produced weaker binding. At low fatty acid concentrations, many of these systems followed a direct competition model while others involved a mixed-mode interaction. In some cases, there was a change in the interaction mechanism between normal HSA and glycated HSA, as seen with linoleic acid. Systems with only direct competition also gave notable changes in the affinities of fatty acids at their sites of drug competition when comparing normal HSA and glycated HSA. This research demonstrated the importance of considering how changes in the concentrations and types of metabolites (e.g., in this case, glucose and fatty acids) can alter the function of a protein such as HSA and its ability to interact with drugs or other agents.

Review: Glycation of human serum albumin.

Glycation involves the non-enzymatic addition of reducing sugars and/or their reactive degradation products to amine groups on proteins. This process is promoted by the presence of elevated blood glucose concentrations in diabetes and occurs with various proteins that include human serum albumin (HSA). This review examines work that has been conducted in the study and analysis of glycated HSA. The general structure and properties of HSA are discussed, along with the reactions that can lead to modification of this protein during glycation. The use of glycated HSA as a short-to-intermediate term marker for glycemic control in diabetes is examined, and approaches that have been utilized for measuring glycated HSA are summarized. Structural studies of glycated HSA are reviewed, as acquired for both in vivo and in vitro glycated HSA, along with data that have been obtained on the rate and thermodynamics of HSA glycation. In addition, this review considers various studies that have investigated the effects of glycation on the binding of HSA with drugs, fatty acids and other solutes and the potential clinical significance of these effects.

Use of entrapment and high-performance affinity chromatography to compare the binding of drugs and site-specific probes with normal and glycated human serum albumin.

Protein entrapment and high-performance affinity chromatography were used with zonal elution to examine the changes in binding that occurred for site-specific probes and various sulfonylurea drugs with normal and glycated forms of human serum albumin (HSA). Samples of this protein in a soluble form were physically entrapped within porous silica particles by using glycogen-capped hydrazide-activated silica; these supports were then placed into 1.0 cm × 2.1 mm inner diameter columns. Initial zonal elution studies were performed using (R)-warfarin and L-tryptophan as probes for Sudlow sites I and II (i.e., the major drug binding sites of HSA), giving quantitative measures of binding affinities in good agreement with literature values. It was also found for solutes with multisite binding to the same proteins, such as many sulfonylurea drugs, that this method could be used to estimate the global affinity of the solute for the entrapped protein. This entrapment and zonal approach provided retention information with precisions of ±0.1-3.3% (± one standard deviation) and elution within 0.50-3.00 min for solutes with binding affinities of 1 × 10(4)-3 × 10(5) M(-1). Each entrapped-protein column was used for many binding studies, which decreased the cost and amount of protein needed per injection (e.g., the equivalent of only 125-145 pmol of immobilized HSA or glycated HSA per injection over 60 sample application cycles). This method can be adapted for use with other proteins and solutes and should be valuable in high-throughput screening or quantitative studies of drug-protein binding or related biointeractions.

Development of affinity microcolumns for drug-protein binding studies in personalized medicine: interactions of sulfonylurea drugs with in vivo glycated human serum albumin.

This report used high-performance affinity microcolumns to examine the changes in binding by sulfonylurea drugs to in vivo glycated HSA that had been isolated from individual patients with diabetes. An immunoextraction approach was developed to isolate HSA and glycated HSA from clinical samples, using only 20 µL of plasma or serum and 6-12 nmol of protein to prepare each affinity microcolumn. It was found that the affinity microcolumns could be used in either frontal analysis or zonal elution studies, which typically required only 4-8 min per run. The microcolumns had good stability and allowed data to be obtained for multiple drugs and experimental conditions over hundreds of sample application cycles. Both the overall binding, as measured by frontal analysis, and site-specific interactions, as examined by zonal elution, showed good agreement with previous data that had been obtained for in vitro glycated HSA with similar levels of modification. It was also possible to directly compare the changes in site-specific binding that occurred between sulfonylurea drugs or as the level of HSA glycation was varied. This method is not limited to clinical samples of glycated HSA but could be adapted for work with other modified proteins of interest in personalized medicine.

Analysis of drug interactions with modified proteins by high-performance affinity chromatography: binding of glibenclamide to normal and glycated human serum albumin.

High-performance affinity chromatography (HPAC) was used to examine the changes in binding that occur for the sulfonylurea drug glibenclamide with human serum albumin (HSA) at various stages of glycation for HSA. Frontal analysis on columns containing normal HSA or glycated HSA indicated glibenclamide was interacting through both high affinity sites (association equilibrium constant, K(a), 1.4-1.9 × 10(6)M(-1) at pH 7.4 and 37 °C) and lower affinity sites (K(a), 4.4-7.2 × 10(4)M(-1)). Competition studies were used to examine the effect of glycation at specific binding sites of HSA. An increase in affinity of 1.7- to 1.9-fold was seen at Sudlow site I with moderate to high levels of glycation. An even larger increase of 4.3- to 6.0-fold in affinity was noted at Sudlow site II for all of the tested samples of glycated HSA. A slight decrease in affinity may have occurred at the digitoxin site, but this change was not significant for any individual glycated HSA sample. These results illustrate how HPAC can be used as tool for examining the interactions of relatively non-polar drugs like glibenclamide with modified proteins and should lead to a more complete understanding of how glycation can alter the binding of drugs in blood.

Pharmaceutical and biomedical applications of affinity chromatography: recent trends and developments.

Affinity chromatography is a separation technique that has become increasingly important in work with biological samples and pharmaceutical agents. This method is based on the use of a biologically related agent as a stationary phase to selectively retain analytes or to study biological interactions. This review discusses the basic principles behind affinity chromatography and examines recent developments that have occurred in the use of this method for biomedical and pharmaceutical analysis. Techniques based on traditional affinity supports are discussed, but an emphasis is placed on methods in which affinity columns are used as part of HPLC systems or in combination with other analytical methods. General formats for affinity chromatography that are considered include step elution schemes, weak affinity chromatography, affinity extraction and affinity depletion. Specific separation techniques that are examined include lectin affinity chromatography, boronate affinity chromatography, immunoaffinity chromatography, and immobilized metal ion affinity chromatography. Approaches for the study of biological interactions by affinity chromatography are also presented, such as the measurement of equilibrium constants, rate constants, or competition and displacement effects. In addition, related developments in the use of immobilized enzyme reactors, molecularly imprinted polymers, dye ligands and aptamers are briefly considered.

High-performance affinity chromatography and the analysis of drug interactions with modified proteins: binding of gliclazide with glycated human serum albumin.

This study used high-performance affinity chromatography (HPAC) to examine the binding of gliclazide (i.e., a sulfonylurea drug used to treat diabetes) with the protein human serum albumin (HSA) at various stages of modification due to glycation. Frontal analysis conducted with small HPAC columns was first used to estimate the number of binding sites and association equilibrium constants (K(a)) for gliclazide with normal HSA and glycated HSA. Both normal and glycated HSA interacted with gliclazide according to a two-site model, with a class of high-affinity sites (average K(a), 7.1-10 × 10(4) M(-1)) and a group of lower-affinity sites (average K(a), 5.7-8.9 × 10(3) M(-1)) at pH 7.4 and 37 °C. Competition experiments indicated that Sudlow sites I and II of HSA were both involved in these interactions, with the K(a) values for gliclazide at these sites being 1.9 × 10(4) and 6.0 × 10(4) M(-1), respectively, for normal HSA. Two samples of glycated HSA had similar affinities to normal HSA for gliclazide at Sudlow site I, but one sample had a 1.9-fold increase in affinity at this site. All three glycated HSA samples differed from normal HSA in their affinity for gliclazide at Sudlow site II. This work illustrated how HPAC can be used to examine both the overall binding of a drug with normal or modified proteins and the site-specific changes that can occur in these interactions as a result of protein modification.

Characterization of drug interactions with serum proteins by using high-performance affinity chromatography.

The binding of drugs with serum proteins can affect the activity, distribution, rate of excretion, and toxicity of pharmaceutical agents in the body. One tool that can be used to quickly analyze and characterize these interactions is high-performance affinity chromatography (HPAC). This review shows how HPAC can be used to study drug-protein binding and describes the various applications of this approach when examining drug interactions with serum proteins. Methods for determining binding constants, characterizing binding sites, examining drug-drug interactions, and studying drug-protein dissociation rates will be discussed. Applications that illustrate the use of HPAC with serum binding agents such as human serum albumin, α(1)-acid glycoprotein, and lipoproteins will be presented. Recent developments will also be examined, such as new methods for immobilizing serum proteins in HPAC columns, the utilization of HPAC as a tool in personalized medicine, and HPAC methods for the high-throughput screening and characterization of drug-protein binding.

Binding of tolbutamide to glycated human serum albumin.

The presence of elevated levels of glucose in blood during diabetes can lead to the non-enzymatic glycation of serum proteins such as human serum albumin (HSA). This study examined the changes that occur in binding of the sulfonylurea drug tolbutamide to HSA as the level of glycation for this protein was increased. High-performance affinity chromatography was used in this work along with columns containing various preparations of in vitro glycated HSA. It was found in frontal analysis experiments that the binding of tolbutamide with all of the tested preparations of glycated HSA could be described by a two-site model involving both strong and weak affinity interactions. The association equilibrium constants (K(a)) for tolbutamide at its high affinity sites on glycated HSA were in the range of 0.8-1.2 x 105 M⁻¹ and increased by 1.4-fold in going from normal HSA to mildly glycated HSA. It was found through competition studies that tolbutamide was binding at both Sudlow sites I and II on the glycated HSA, in agreement with previous studies. The K(a) for tolbutamide at Sudlow site II increased by 1.1- to 1.4-fold in going from normal HSA to glycated HSA. At Sudlow site I, the K(a) for tolbutamide increased by 1.2- to 1.3-fold in going from normal HSA to the glycated HSA samples. This information demonstrates the effects that glycation can have on drug interactions on HSA and should provide a better quantitative understanding of how the protein binding of tolbutamide in serum may be affected for individuals with diabetes.

CHROMATOGRAPHIC ANALYSIS OF DRUG INTERACTIONS IN THE SERUM PROTEOME.

The binding of drugs with serum proteins and binding agents such as human serum albumin, α1-acid glycoprotein, and lipoproteins is an important process in determining the activity and fate of many pharmaceuticals in the body. A variety of techniques have been used to study drug interactions with serum proteins, but there is still a need for faster or better methods for such work. High-performance liquid chromatography (HPLC) is one tool that has been utilized in many formats for these types of measurements. Advantages of using HPLC for this application include its speed and precision, its ability to be automated, its good limits of detection, and its compatibility with a wide range of assay formats and detectors. This review will discuss various approaches in which HPLC can be employed for the study of drug-protein interactions. These techniques include the use of soluble proteins in zonal elution and frontal analysis methods or vacancy techniques such as the Hummel-Dreyer method. Zonal elution and frontal analysis methods that make use of immobilized proteins and high-performance affinity chromatography will also be presented. A variety of applications will be examined, ranging from the determination of free drug fractions to the measurement of the strength or rate of a drug-protein interaction. Newer developments that will be discussed include recent work in the creation of novel mathematical approaches for HPLC studies of drug-protein binding, the use of HPLC methods for the high-throughput screening of drug-protein binding, and the creation and use of affinity monoliths or affinity microcolumns for examining drug-protein systems.