Ultrafast Affinity Extraction Research Articles

Determination of binding constants by ultrafast affinity extraction: Theoretical and experimental studies of optimum conditions for analysis

Ultrafast affinity extraction (UAE) is a form of microscale affinity HPLC that can be employed to quickly measure equilibrium constants for solute-binding agent interactions in solution. This study used chromatographic and equilibrium theory with universal plots to examine the general conditions that are needed in UAE to obtain accurate, precise, and robust measurements of equilibrium constants for such interactions. The predicted results were compared to those obtained by UAE in studies that examined the binding of various drugs with two transport proteins: human serum albumin and α1-acid glycoprotein. The most precise and robust conditions for these binding studies occurred for systems with intermediate values for their equilibrium free fraction for the solute (F0 ≈ 0.20–0.80). These trends showed good agreement with those seen in prior studies using UAE. It was further determined how the apparent free fraction of a solute was related to the dissociation rate of this solute, the time allowed for solute dissociation during UAE, and the equilibrium free fraction for the solute. These results also agreed with experimental results, as obtained for the binding of warfarin and gliclazide with human serum albumin. The final section examined how a change in the apparent free fraction, as caused by solute dissociation, affected the accuracy of an equilibrium constant that was measured by UAE. In addition, theoretical plots were generated to allow the selection of conditions for UAE that provided a given level of accuracy during the measurement of an equilibrium constant. The equations created and trends identified for UAE were general ones that can be extended in future work to other solutes and binding agents.

Analysis of the binding of warfarin to glyoxal- and methylglyoxal-modified human serum albumin by ultrafast affinity extraction

Ultrafast affinity extraction (UAE) and affinity microcolumns containing immobilized human serum albumin (HSA) were employed to evaluate the effect of advanced stage glycation on HSA and its binding to warfarin, a common site-specific probe for Sudlow site I of this protein. The modification of HSA by glyoxal (GO) and methylglyoxal (MGO) was considered, where GO and MGO are known to be important in the formation of many types of advanced glycation end products. Free drug fractions were measured by UAE for warfarin in solutions containing normal HSA or HSA that had been modified by GO or MGO at levels seen in serum during diabetes. The free fractions measured with the GO-modified HSA gave association equilibrium constants that ranged from 2.42–2.63 × 105 M−1 at pH 7.4 and 37 °C. These values were not significantly different from a value of 2.33 (±0.15) × 105 M−1 that was determined by the same method for warfarin with normal HSA. Similar studies using MGO-modified HSA gave association equilibrium constants for warfarin in the range of 3.07–3.31 × 105 M−1, which were 1.32- to 1.42-fold higher than the value seen for normal HSA (differences that were significant at the 95% confidence level). These results will be valuable in future binding studies based on affinity chromatography or other methods that employ warfarin as a probe to examine drug interactions at Sudlow site I of HSA and modified forms of this protein. This work also illustrates how UAE can be used, with analysis times of only minutes, to detect and measure small changes in the binding by drugs with unmodified or modified forms of a soluble binding agent or protein.

Evaluation of microcolumn stability in ultrafast affinity extraction for binding and rate studies

Ultrafast affinity extraction (UAE) has recently been developed and employed for measuring non-bound (or free) fractions and binding or rate constants for drugs and other targets with soluble binding agents such as serum proteins. This study examined the long-term stability of 10 mm × 2.1 mm i.d. affinity microcolumns when used in UAE at both low and high flow rates (e.g., 0.5 and 3.5 mL/min) over an extended series of injections. This stability was investigated by using immobilized human serum albumin (HSA) and samples containing the drug warfarin with or without soluble HSA as a model system. The free warfarin fractions measured at 0.5 mL/min in the presence of soluble HSA were stable up to 150 injections and changed by <10% at 3.5 mL/min. The association equilibrium constant for warfarin with HSA that was estimated by UAE at 3.5 mL/min had no significant change over 50 injections and a change of only ∼18–22% over 100–150 injections. The dissociation rate constant for warfarin from HSA was found by combining UAE results at 0.5 and 3.5 mL/min and employing a new two-point approach, with no significant changes in this value being seen even after 200 injections. The effects of extended microcolumn use on the retention time, peak width, and peak asymmetry for warfarin, and on the backpressure of the microcolumn, were also considered. These results indicated that UAE and HSA microcolumns could be used to provide consistent values for free solute fractions, binding constants, and rate constants over a large series of injections. These results should be useful in future work by providing guidelines for the assessment, further development, and use of UAE in characterizing interactions involving other drugs and binding agents in solution-based samples.

Advances in chromatography in the study of drug-plasma protein interactions

小分子药物进入人体血液循环系统后与人血清白蛋白(HSA)、α1 -酸性糖蛋白(AGP)等血浆蛋白存在广泛的相互作用,这些相互作用深刻影响药物在体内的分布及其与靶标蛋白的结合,进而影响药物效应的发挥。深入探究药物与血浆蛋白间的相互作用对于候选药物的成药性优化、新药研发、联合用药的风险评控等意义重大。而发展高效、灵敏、准确的分析检测方法是开展药物-血浆蛋白相互作用研究的关键。近年来,色谱技术由于其高通量、高分离性能、高灵敏度等特点在该领域得到了广泛的应用,包括测定血浆蛋白翻译后修饰对药物结合的影响,多种药物的竞争性结合等。其中,高效亲和色谱(HPAC)和毛细管电泳(CE)应用最为广泛,能够通过多种分析方法获取结合常数、结合位点数、解离速率常数等相互作用信息。该文着重综述了HPAC和CE在药物-血浆蛋白相互作用研究中的常用策略及最新研究进展,包括HPAC中常用的前沿色谱法、竞争洗脱法、超快亲和提取法、峰值分析法和峰衰减分析法,以及CE中常用的亲和毛细管电泳法(ACE)和毛细管电泳前沿分析法(CE-FA)等。最后,该文还对当前色谱方法存在的不足进行了总结,并对色谱技术在药物-血浆蛋白相互作用研究领域的应用前景和发展方向进行了展望。

Characterization of drug binding with alpha1-acid glycoprotein in clinical samples using ultrafast affinity extraction

Many drugs bind to serum transport proteins, which can affect both drug distribution and activity in the body. α1-Acid glycoprotein (AGP) is a key transport protein for basic and neutral drugs. Both elevated levels and altered glycosylation patterns of AGP have been seen in clinical conditions such as systemic lupus erythematosus (SLE). This study developed, optimized, and used the method of ultrafast affinity extraction (UAE) to examine whether these changes in AGP are associated with changes in the binding by some drugs to this transport protein. This approach used affinity microcolumns to capture and measure, in serum, the free fractions of several drugs known to bind AGP. These measurements were made with pooled normal control serum and serum samples from individuals with SLE. Immunoaffinity chromatography was used to obtain the content of AGP and HSA in these samples, and CE was used to examine the glycoform pattern for AGP in each serum sample. The free drug fractions measured for normal control serum ranged from 3.5 to 29.1%, in agreement with the results of ultrafiltration, and provided binding constants of ~105–106 M−1 for the given drugs with AGP at 37⁰C. Analysis of a screening set of SLE serum samples by UAE gave decreased free fractions (relative change, 12-55%) vs normal serum when spiked with the same types and amounts of drugs. These changes were related in some cases to AGP content, with some SLE samples having AGP levels 1.3- to 2.1-fold above the upper end of the normal range. In other cases, the changes in free fractions appeared to be linked to alterations in the glycoforms and binding constants of AGP, with some affinities differing by 1.2- to 1.5-fold vs normal AGP. This approach can be employed with other solute-protein systems and to investigate binding by other drugs or transport proteins directly in clinical samples.

Kinetic Analysis by Affinity Chromatography.

Important information on chemical processes in living systems can be obtained by the rates at which these biological interactions occur. This review will discuss several techniques based on traditional and high-performance affinity chromatography that may be used to examine the kinetics of biological reactions. These methods include band-broadening measurements, techniques for peak fitting, split-peak analysis, peak decay studies, and ultrafast affinity extraction. The general principles and theory of each method, as applied to the determination of rate constants, will be discussed. The applications of each approach, along with its advantages and limitations, will also be considered.

Binding studies based on ultrafast affinity extraction and single- or two-column systems: Interactions of second- and third-generation sulfonylurea drugs with normal or glycated human serum albumin

Ultrafast affinity extraction was evaluated and used with microcolumns containing human serum albumin (HSA) to measure the global affinity constants and dissociation rate constants for several second- and third-generation sulfonylurea drugs with solution-phase normal HSA or glycated HSA. Glibenclamide, glimepiride and glipizide were used as model drugs for this work. Both single- and two-column systems were considered for the analysis of global affinities for the model drugs. These methods were optimized with respect to the flow rates, column sizes and sample residence times that were employed with each drug for ultrafast affinity extraction. Data acquired with single-column systems were further utilized to estimate the dissociation rate constants for normal HSA and glycated HSA with the given drugs. The binding constants obtained by the single- and two-column systems showed good agreement with each other and with values obtained from the literature. Use of a single-column system indicated that levels of glycation found in controlled or advanced diabetes resulted in a 18–44% decrease in the overall binding strength of the model drugs with HSA. Although the two-column system allowed work with smaller free drug fractions and clinically-relevant drug/protein concentrations, the single-column system required less protein, provided better precision, and was easier to use in binding studies.

Characterization of solution-phase drug-protein interactions by ultrafast affinity extraction.

A number of tools based on high-performance affinity separations have been developed for studying drug-protein interactions. An example of one recent approach is ultrafast affinity extraction. This method has been employed to examine the free (or non-bound) fractions of drugs and other solutes in simple or complex samples that contain soluble binding agents. These free fractions have also been used to determine the binding constants and rate constants for the interactions of drugs with these soluble agents. This report describes the general principles of ultrafast affinity extraction and the experimental conditions under which it can be used to characterize such interactions. This method will be illustrated by utilizing data that have been obtained when using this approach to measure the binding and dissociation of various drugs with the serum transport proteins human serum albumin and alpha1-acid glycoprotein. A number of practical factors will be discussed that should be considered in the design and optimization of this approach for use with single-column or multi-column systems. Techniques will also be described for analyzing the resulting data for the determination of free fractions, rate constants and binding constants. In addition, the extension of this method to complex samples, such as clinical specimens, will be considered.

Chromatographic studies of drug interactions with alpha1-acid glycoprotein by ultrafast affinity extraction and peak profiling

Interactions with serum proteins such as alpha1-acid glycoprotein (AGP) can have a significant effect on the behavior and pharmacokinetics of drugs. Ultrafast affinity extraction and peak profiling were used with AGP microcolumns to examine these processes for several model drugs (i.e., chlorpromazine, disopyramide, imipramine, lidocaine, propranolol and verapamil). The association equilibrium constants measured for these drugs with soluble AGP by ultrafast affinity extraction were in the general range of 104–106M−1 at pH 7.4 and 37°C and gave good agreement with literature values. Some of these values were dependent on the relative drug and protein concentrations that were present when using a single-site binding model; these results suggested a more complex mixed-mode interaction was actually present, which was also then used to analyze the data. The apparent dissociation rate constants that were obtained by ultrafast affinity extraction when using a single-site model varied from 0.14 to 7.0s−1 and were dependent on the relative drug and protein concentrations. Lower apparent dissociation rate constants were obtained by this approach as the relative amount of drug versus protein was decreased, with the results approaching those measured by peak profiling at low drug concentrations. This information should be useful in better understanding how these and other drugs interact with AGP in the circulation. In addition, the chromatographic approaches that were optimized and used in this report to examine these systems can be adapted for the analysis of other solute-protein interactions of biomedical interest.

Analysis of stereoselective drug interactions with serum proteins by high-performance affinity chromatography: A historical perspective

The interactions of drugs with serum proteins are often stereoselective and can affect the distribution, activity, toxicity and rate of excretion of these drugs in the body. A number of approaches based on affinity chromatography, and particularly high-performance affinity chromatography (HPAC), have been used as tools to study these interactions. This review describes the general principles of affinity chromatography and HPAC as related to their use in drug binding studies. The types of serum agents that have been examined with these methods are also discussed, including human serum albumin, α1-acid glycoprotein, and lipoproteins. This is followed by a description of the various formats based on affinity chromatography and HPAC that have been used to investigate drug interactions with serum proteins and the historical development for each of these formats. Specific techniques that are discussed include zonal elution, frontal analysis, and kinetic methods such as those that make use of band-broadening measurements, peak decay analysis, or ultrafast affinity extraction.

Analysis of free drug fractions in serum by ultrafast affinity extraction and two-dimensional affinity chromatography using α1-acid glycoprotein microcolumns

In the circulatory system, many drugs are reversibly bound to serum proteins such as human serum albumin (HSA) and alpha1-acid glycoprotein (AGP), resulting in both free and protein-bound fractions for these drugs. This report examined the use of microcolumns containing immobilized AGP for the measurement of free drug fractions by ultrafast affinity extraction and a two-dimensional affinity system. Several drugs known to bind AGP were used as models to develop and evaluate this approach. Factors considered during the creation of this method included the retention of the drugs on the microcolumns, the injection flow rate, the microcolumn size, and the times at which a second AGP column was placed on-line with the microcolumn. The final system had residence times of only 110–830ms during sample passage through the AGP microcolumns and allowed free drug fractions to be determined within 10–20min when using only 3–10μL of sample per injection. This method was used to measure the free fractions of the model drugs at typical therapeutic levels in serum, giving good agreement with the results obtained by ultrafiltration. This approach was also used to estimate the binding constants for each drug with AGP in serum, even for drugs that had significant interactions with both AGP and HSA in such samples. These results indicated that AGP microcolumns could be used with ultrafast affinity extraction to measure free drug fractions in a label-free manner and to study the binding of drugs with AGP in complex samples such as serum.

High-Performance Affinity Chromatography: Applications in Drug-Protein Binding Studies and Personalized Medicine.

The binding of drugs with proteins and other agents in serum is of interest in personalized medicine because this process can affect the dosage and action of drugs. The extent of this binding may also vary with a given disease state. These interactions may involve serum proteins, such as human serum albumin or α1-acid glycoprotein, or other agents, such as lipoproteins. High-performance affinity chromatography (HPAC) is a tool that has received increasing interest as a means for studying these interactions. This review discusses the general principles of HPAC and the various approaches that have been used in this technique to examine drug-protein binding and in work related to personalized medicine. These approaches include frontal analysis and zonal elution, as well as peak decay analysis, ultrafast affinity extraction, and chromatographic immunoassays. The operation of each method is described and examples of applications for these techniques are provided. The type of information that can be obtained by these methods is also discussed, as related to the analysis of drug-protein binding and the study of clinical or pharmaceutical samples.

Analysis of Hormone-Protein Binding in Solution by Ultrafast Affinity Extraction: Interactions of Testosterone with Human Serum Albumin and Sex Hormone Binding Globulin.

Ultrafast affinity extraction was used to study hormone-protein interactions in solution, using testosterone and its transport proteins human serum albumin (HSA) and sex hormone binding globulin (SHBG) as models. Both single column and two-dimensional systems based on HSA microcolumns were utilized to measure the free fraction of testosterone in hormone/protein mixtures at equilibrium or that were allowed to dissociate for various lengths of time. These data were used to determine the association equilibrium constants (Ka) or global affinities (nKa') and dissociation rate constants (kd) for testosterone with soluble HSA and SHBG. This method was also used to measure simultaneously the free fraction of testosterone and its equilibrium constants with both these proteins in physiological mixtures of these agents. The kd and Ka values obtained for HSA were 2.1-2.2 s(-1) and 3.2-3.5 × 10(4) M(-1) at pH 7.4 and 37 °C. The corresponding constants for SHBG were 0.053-0.058 s(-1) and 0.7-1.2 × 10(9) M(-1). All of these results gave good agreement with literature values, indicating that this approach could provide information on a wide range of rate constants and binding strengths for hormone-protein interactions in solution and at clinically relevant concentrations. The same method could be extended to alternative hormone-protein systems or other solutes and binding agents.

Development of enhanced capacity affinity microcolumns by using a hybrid of protein cross-linking/modification and immobilization

A hybrid method was examined for increasing the binding capacity and activity of protein-based affinity columns by using a combination of protein cross-linking/modification and covalent immobilization. Various applications of this approach in the study of drug–protein interactions and in use with affinity microcolumns were considered. Human serum albumin (HSA) was utilized as a model protein for this work. Bismaleimidohexane (BMH, a homobifunctional maleimide) was used to modify and/or cross-link HSA through the single free sulfhydryl group that is present on this protein. Up to a 75–113% increase in protein content was obtained when comparing affinity supports that were prepared with BMH versus reference supports that were made by using only covalent immobilization. Several drugs that are known to bind HSA (e.g., warfarin, verapamil and carbamazepine) were further found to have a significant increase in retention on HSA microcolumns that were treated with BMH (i.e., a 70–100% increase in protein-based retention). These BMH-treated HSA microcolumns were used in chiral separations and in ultrafast affinity extraction to measure free drug fractions in drug/protein mixtures, with the latter method giving association equilibrium constants that had good agreement with literature values. In addition, it was found that the reversible binding of HSA with ethacrynic acid, an agent that can combine irreversibly with the free sulfhydryl group on this protein, could be examined by using the BMH-treated HSA microcolumns. The same hybrid immobilization method could be extended to other proteins or alternative applications that may require protein-based affinity columns with enhanced binding capacities and activities.

Analysis of free drug fractions by ultrafast affinity extraction: Interactions of sulfonylurea drugs with normal or glycated human serum albumin

Ultrafast affinity extraction and a multi-dimensional affinity system were developed for measuring free drug fractions at therapeutic levels. This approach was used to compare the free fractions and global affinity constants of several sulfonylurea drugs in the presence of normal human serum albumin (HSA) or glycated forms of this protein, as are produced during diabetes. Affinity microcolumns containing immobilized HSA were first used to extract the free drug fractions in injected drug/protein mixtures. As the retained drug eluted from the HSA microcolumn, it was passed through a second HSA column for further separation and measurement. Items that were considered during the optimization of this approach included the column sizes and flow rates that were used, and the time at which the second column was placed on-line with the HSA microcolumn. This method required only 1.0μL of a sample per injection and was able to measure free drug fractions as small as 0.09–2.58% with an absolute precision of ±0.02–0.5%. The results that were obtained indicated that glycation can affect the free fractions of sulfonylurea drugs at typical therapeutic levels and that the size of this effect varies with the level of HSA glycation. Global affinity constants that were estimated from these free drug fractions gave good agreement with those predicted from previous binding studies or determined through a reference method. The same approach could be utilized with other drugs and proteins or modified binding agents of clinical or pharmaceutical interest.

Determination of rate constants and equilibrium constants for solution-phase drug-protein interactions by ultrafast affinity extraction.

A method was created on the basis of ultrafast affinity extraction to determine both the dissociation rate constants and equilibrium constants for drug–protein interactions in solution. Human serum albumin (HSA), an important binding agent for many drugs in blood, was used as both a model soluble protein and as an immobilized binding agent in affinity microcolumns for the analysis of free drug fractions. Several drugs were examined that are known to bind to HSA. Various conditions to optimize in the use of ultrafast affinity extraction for equilibrium and kinetic studies were considered, and several approaches for these measurements were examined. The dissociation rate constants obtained for soluble HSA with each drug gave good agreement with previous rate constants reported for the same drugs or other solutes with comparable affinities for HSA. The equilibrium constants that were determined also showed good agreement with the literature. The results demonstrated that ultrafast affinity extraction could be used as a rapid approach to provide information on both the kinetics and thermodynamics of a drug–protein interaction in solution. This approach could be extended to other systems and should be valuable for high-throughput drug screening or biointeraction studies.

Analysis of biomolecular interactions using affinity microcolumns: A review

Affinity chromatography has become an important tool for characterizing biomolecular interactions. The use of affinity microcolumns, which contain immobilized binding agents and have volumes in the mid-to-low microliter range, has received particular attention in recent years. Potential advantages of affinity microcolumns include the many analysis and detection formats that can be used with these columns, as well as the need for only small amounts of supports and immobilized binding agents. This review examines how affinity microcolumns have been used to examine biomolecular interactions. Both capillary-based microcolumns and short microcolumns are considered. The use of affinity microcolumns with zonal elution and frontal analysis methods are discussed. The techniques of peak decay analysis, ultrafast affinity extraction, split-peak analysis, and band-broadening studies are also explored. The principles of these methods are examined and various applications are provided to illustrate the use of these methods with affinity microcolumns. It is shown how these techniques can be utilized to provide information on the binding strength and kinetics of an interaction, as well as on the number and types of binding sites. It is further demonstrated how information on competition or displacement effects can be obtained by these methods.

Analysis of free fractions for chiral drugs using ultrafast extraction and multi-dimensional high-performance affinity chromatography

A multi-dimensional chromatographic approach was developed to measure the free fractions of drug enantiomers in samples that also contained a binding protein or serum. This method, which combined ultrafast affinity extraction with a chiral stationary phase, was demonstrated using the drug warfarin and the protein human serum albumin.