Biacore surface matrix effects on the binding kinetics and affinity of an antigen/antibody complex

To characterize a proprietary therapeutic monoclonal antibody (mAb) candidate, a rigorous biophysical study consisting of 53 Biacore and kinetic exclusion assay (KinExA) experiments was undertaken on the therapeutic mAb complexing with its target antigen. Unexpectedly, the observed binding kinetics depended on the chip used, suggesting that the negatively charged carboxyl groups on CM5, CM4, and C1 chips were adversely affecting the Biacore kinetic results. To study this hypothesis, Biacore solution-phase and KinExA equilibrium titrations, as well as KinExA kinetic measurements, were performed to establish accurate values for the affinity and kinetic rate constants of the binding reaction between antigen and mAb. The results revealed that as the negative charge on the biosensor surface decreased, the binding kinetics and K(D) approached the accurate binding parameters more closely when measured in solution. Two potential causes for the artifactual Biacore surface-based measurements are (i) steric hindrance of antigen binding arising from an interaction of the negatively charged carboxymethyldextran matrix with the mAb, which is a highly basic protein with a pI of 9.4, and (ii) an electrostatic repulsion between the negatively charged antigen and the carboxymethyldextran matrix. Importantly, simple diagnostic tests can be performed early in the measurement process to identify these types of matrix-mediated artifacts.     

Exploring the dynamic range of the kinetic exclusion assay in characterizing antigen-antibody interactions

Therapeutic antibodies are often engineered or selected to have high on-target binding affinities that can be challenging to determine precisely by most biophysical methods. Here, we explore the dynamic range of the kinetic exclusion assay (KinExA) by exploiting the interactions of an anti-DKK antibody with a panel of DKK antigens as a model system. By tailoring the KinExA to each studied antigen, we obtained apparent equilibrium dissociation constants (K(D) values) spanning six orders of magnitude, from approximately 100 fM to 100 nM. Using a previously calibrated antibody concentration and working in a suitable concentration range, we show that a single experiment can yield accurate and precise values for both the apparent K(D) and the apparent active concentration of the antigen, thereby increasing the information content of an assay and decreasing sample consumption. Orthogonal measurements obtained on Biacore and Octet label-free biosensor platforms further validated our KinExA-derived affinity and active concentration determinations. We obtained excellent agreement in the apparent affinities obtained across platforms and within the KinExA method irrespective of the assay orientation employed or the purity of the recombinant or native antigens.     

Confirmation of the validity of the current characterization of immunochemical reactions by kinetic exclusion assay

Prior observations that questioned the validity of kinetic exclusion assays were based on the mistaken assumption that the assays quantified the fraction of those antibody molecules that had unoccupied binding sites. Instead, the standard KinExA assay quantifies the fraction of total antibody binding sites that are unoccupied, regardless of the number of unoccupied sites on each antibody molecule. Although the standard KinExA analysis assumes that there is only a small probability of antibody-site capture by the affinity matrix, the results of numerical simulations demonstrate the reliability of dissociation constants obtained by the standard KinExA analysis for capture probabilities as high as 30%. This finding further strengthens the potential of kinetic exclusion assays as the procedure of choice for the rapid and accurate characterization of immunochemical reactions that forms part of screening processes in the search for therapeutic antibodies.     

Rapid affinity measurement of protein-protein interactions in a microfluidic platform

A rapid screening method has been developed to determine binding affinities for protein-ligand interactions using the Gyrolab workstation, a commercial microfluidic platform developed to accurately and precisely quantify proteins in solution. This method was particularly suited for assessing the high-affinity interactions that have become typical of therapeutic antibody-antigen systems. Five different commercially available antibodies that bind digoxin and a digoxin-bovine serum albumin (BSA) conjugate with high affinity were rigorously evaluated by this method and by the more conventional kinetic exclusion assay (KinExA) method. Binding parameter values obtained using Gyrolab were similar to those recovered from KinExA. However, the total experimental time for 20 binding affinity titrations, with each titration covering 12 data points in duplicate, took approximately 4h by the Gyrolab method, which reduced the experimental duration by more than 10-fold when compared with the KinExA method. This rapid binding analysis method has significant applications in the screening and affinity ranking selection of antibodies from a very large pool of candidates spanning a wide range of binding affinities from the low pM to μM range.     

Kinetic exclusion assay of monoclonal antibody affinity to the membrane protein Roundabout 1 displayed on baculovirus

The reliable assessment of monoclonal antibody (mAb) affinity against membrane proteins in vivo is a major issue in the development of cancer therapeutics. We describe here a simple and highly sensitive method for the evaluation of mAbs against membrane proteins by means of a kinetic exclusion assay (KinExA) in combination with our previously developed membrane protein display system using budded baculovirus (BV). In our BV display system, the membrane proteins are displayed on the viral surface in their native form. The BVs on which the liver cancer antigen Roundabout 1 (Robo1) was displayed were adsorbed onto magnetic beads without fixative (BV beads). The dissociation constant (K_d, ∼10⁻¹¹ M) that was measured on the Robo1 expressed BV beads correlated well with the value from a whole cell assay (the coefficient of determination, R² = 0.998) but not with the value for the soluble extracellular domains of Robo1 (R² = 0.834). These results suggest that the BV–KinExA method described here provides a suitably accurate K_d evaluation of mAbs against proteins on the cell surface.     

Allowance for antibody bivalence in the determination of association rate constants by kinetic exclusion assay

This investigation completes the amendment of theoretical expressions for the characterization of antigen–antibody interactions by kinetic exclusion assay—an endeavor that has been marred by inadequate allowance for the consequences of antibody bivalence in its uptake by the affinity matrix (immobilized antigen) that is used to ascertain the fraction of free antibody sites in a solution with defined total concentrations of antigen and antibody. A simple illustration of reacted site probability considerations in action confirms that the square root of the fluorescence response ratio, R_Ag/R_o, needs to be taken in order to determine the fraction of unoccupied antibody sites, which is the parameter employed to describe the kinetics of antigen uptake in the mixture of antigen and antibody with defined initial composition. The approximately 2-fold underestimation of the association rate constant (k_a) that emanates from the usual practice of omitting the square root factor gives rise to a corresponding overestimate of the equilibrium dissociation constant (K_d)—a situation that is also encountered in the thermodynamic characterization of antigen–antibody interactions by kinetic exclusion assay.     

Interpolation method for accurate affinity ranking of arrayed ligand–analyte interactions

The values of the affinity constants (k_d, k_a, and K_D) that are determined by label-free interaction analysis methods are affected by the ligand density. This article outlines a surface plasmon resonance (SPR) imaging method that yields high-throughput globally fitted affinity ranking values using a 96-plex array. A kinetic titration experiment without a regeneration step has been applied for various coupled antibodies binding to a single antigen. Globally fitted rate (k_d and k_a) and dissociation equilibrium (K_D) constants for various ligand densities and analyte concentrations are exponentially interpolated to the K_D at R_max = 100 RU response level (K_D^R100).     

Direct comparison of binding equilibrium, thermodynamic, and rate constants determined by surface- and solution-based biophysical methods

The binding interactions of small molecules with carbonic anhydrase II were used as model systems to compare the reaction constants determined from surface- and solution-based biophysical methods. Interaction data were collected for two arylsulfonamide compounds, 4-carboxybenzenesulfonamide (CBS) and 5-dimethyl-amino-1-naphthalene-sulfonamide (DNSA), binding to the enzyme using surface plasmon resonance, isothermal titration calorimetry, and stopped-flow fluorescence. We demonstrate that when the surface plasmon resonance biosensor experiments are performed with care, the equilibrium, thermodynamic, and kinetic constants determined from this surface-based technique match those acquired in solution. These results validate the use of biosensor technology to collect reliable data on small molecules binding to immobilized macromolecular targets. Binding kinetics were shown to provide more detailed information about complex formation than equilibrium constants alone. For example, although carbonic anhydrase II bound DNSA with twofold higher affinity than CBS, kinetic analysis revealed that CBS had a fourfold slower dissociation rate. Analysis of the binding and transition state thermodynamics also revealed significant differences in the enthalpy and entropy of complex formation. The lack of labeling requirements, high information content, and high throughput of surface plasmon resonance biosensors will make this technology an important tool for characterizing the interactions of small molecules with enzymes and receptors.     

Scintillation proximity assay (SPA) as a new approach to determine a ligand’s kinetic profile. A case in point for the adenosine A<Subscript>1</Subscript> receptor

Scintillation proximity assay (SPA) is a radio-isotopic technology format used to measure a wide range of biological interactions, including drug-target binding affinity studies. The assay is homogeneous in nature, as it relies on a “mix and measure” format. It does not involve a filtration step to separate bound from free ligand as is the case in a traditional receptor-binding assay. For G protein-coupled receptors (GPCRs), it has been shown that optimal binding kinetics, next to a high affinity of a ligand, can result in more desirable pharmacological profiles. However, traditional techniques to assess kinetic parameters tend to be cumbersome and laborious. We thus aimed to evaluate whether SPA can be an alternative platform for real-time receptor-binding kinetic measurements on GPCRs. To do so, we first validated the SPA technology for equilibrium binding studies on a prototypic class A GPCR, the human adenosine A₁ receptor (hA₁R). Differently to classic kinetic studies, the SPA technology allowed us to study binding kinetic processes almost real time, which is impossible in the filtration assay. To demonstrate the reliability of this technology for kinetic purposes, we performed the so-called competition association experiments. The association and dissociation rate constants (k _on and k _off) of unlabeled hA₁R ligands were reliably and quickly determined and agreed very well with the same parameters from a traditional filtration assay performed simultaneously. In conclusion, SPA is a very promising technique to determine the kinetic profile of the drug-target interaction. Its robustness and potential for high-throughput may render this technology a preferred choice for further kinetic studies.     

The strengths and limitations of using biolayer interferometry to monitor equilibrium titrations of biomolecules

Every method used to quantify biomolecular interactions has its own strengths and limitations. To quantify protein‐DNA binding affinities, nitrocellulose filter binding assays with ³²P‐labeled DNA quantify K_d values from 10^?12 to 10^?8 M but have several technical limitations. Here, we considered the suitability of biolayer interferometry (BLI), which monitors association and dissociation of a soluble macromolecule to an immobilized species; the ratio k_off/k_on determines K_d. However, for lactose repressor protein (LacI) and an engineered repressor protein (“LLhF”) binding immobilized DNA, complicated kinetic curves precluded this analysis. Thus, we determined whether the amplitude of the BLI signal at equilibrium related linearly to the fraction of protein bound to DNA. A key question was the effective concentration of immobilized DNA. Equilibrium titration experiments with DNA concentrations below K_d (equilibrium binding regime) must be analyzed differently than those with DNA near or above K_d (stoichiometric binding regime). For ForteBio streptavidin tips, the most frequent effective DNA concentration was ~2 × 10^?9 M. Although variation occurred among different lots of sensor tips, binding events with K_d ≥ 10^?8 M should reliably be in the equilibrium binding regime. We also observed effects from multi‐valent interactions: Tetrameric LacI bound two immobilized DNAs whereas dimeric LLhF did not. We next used BLI to quantify the amount of inducer sugars required to allosterically diminish protein‐DNA binding and to assess the affinity of fructose‐1‐kinase for the DNA‐LLhF complex. Overall, when experimental design corresponded with appropriate data interpretation, BLI was convenient and reliable for monitoring equilibrium titrations and thereby quantifying a variety of binding interactions.     

Development and Application of a High Throughput Protein Unfolding Kinetic Assay

The kinetics of folding and unfolding underlie protein stability and quantification of these rates provides important insights into the folding process. Here, we present a simple high throughput protein unfolding kinetic assay using a plate reader that is applicable to the studies of the majority of 2-state folding proteins. We validate the assay by measuring kinetic unfolding data for the SH3 (Src Homology 3) domain from Actin Binding Protein 1 (AbpSH3) and its stabilized mutants. The results of our approach are in excellent agreement with published values. We further combine our kinetic assay with a plate reader equilibrium assay, to obtain indirect estimates of folding rates and use these approaches to characterize an AbpSH3-peptide hybrid. Our high throughput protein unfolding kinetic assays allow accurate screening of libraries of mutants by providing both kinetic and equilibrium measurements and provide a means for in-depth ϕ-value analyses.     

Simple and sensitive bacterial quantification by a flow-based kinetic exclusion fluorescence immunoassay

A flow-based immunoassay system utilizing secondary-antibody coated microbeads and Cy5-secondary antibody for signal production was successfully developed to quantitate target bacteria with a kinetic exclusion assay (KinExA 3000 Instrument). It directly measured the concentration of unliganded antibody separated from the equilibrated mixture of antibody and bacteria through a 0.2 microm polyethersulfone membrane, enabling it to quantify the concentration of bacteria. The novel method demonstrated the qualities of rapidness, sensitivity, high accuracy and reproducibility, and ease to perform. Detection of Pseudomonas aeruginosa and Staphylococcus aureus was accomplished with low detection limits of 4.10 x 10(6) and 5.20 x l0(4)cells/mL, respectively, with an assay time of less than 15 min. The working ranges for quantification were 4.10 x l0(6) to 1.64 x l0(10)cells/mL for P. aeruginosa, and 5.20 x l0(4) to 1.04 x l0(9)cells/mL for S. aureus. It yielded an assay with at least 10-fold greater sensitivity than ELISA and could correctly assess the concentration of predominant bacterium spiked in the mixture of P. aeruginosa and S. aureus. With this reliable platform, the average amount of antibody bound by one cell in the maximum capability could be further provided: (1.6-2.5) x l0(5) antibodies for one P. aeruginosa cell and (2.2-2.7) x l0(8) antibodies for one S. aureus cell. The KinExA system is flexible to determine different kinds of bacteria conveniently by using anti-mouse IgG as the same immobilizing agent. However, a higher specificity of the antibodies to the target bacteria will be required for the use of this system with higher detection sensitivity.     

The influence of exogenous peptide on β2–microglobulin exchange in the HLA complex: analysis in real-time

 We used an optical biosensor to determine the relative binding affinity of peptides to purified HLA class I molecules. In this assay we monitor β₂–microglobulin (β₂m) exchange within the HLA-A2 molecule, whereby native β₂m in the complex is replaced by β₂m immobilized at the surface of the biosensor. Quantitative kinetic measurements permit us to obtain association rate (k_ass), dissociation rate (k_diss) and affinity constants (K_A) for the β₂m exchange reaction, alone, (control) and in the presence of exogenous peptide. We tested a panel of six peptides which had been designed and synthesized with an HLA-A2 binding motif, and had also been tested by the T2-cell binding assay, along with control peptides. The biosensor results demonstrate that exogenous peptide influences the dynamics of β₂m exchange in a sequence-specific manner. Five of six peptides increased the association rate, decreased the dissociation rate, and significantly increased the affinity (K_A=1.55–1.88×10⁹ M^–1) of HLA-A2 for immobilized β₂m compared with the control (K_A =1.14±0.04×10⁹ M^–1), demonstrating stabilization of the complex. One peptide was unable to stabilize the complex, as also shown in the T2 binding assay. However, analysis of peptide sequences demonstrated that the HLA-A2 secondary motif as well as primary motif residues are required for HLA-A2 stabilization. Further experiments demonstrated that β₂m exchange alone cannot stabilize the HLA class I complex at the cell surface until a peptide of sufficient binding affinity is bound. Hence kinetics equal to or below the control values in our biosensor assay probably represent an unstable complex in vivo. Unlike other methods described for the analysis of peptide stabilization, this approach is significantly faster, provides full kinetic analysis, and is simpler, since it requires no labeling of peptides. Furthermore, this may have important implications in the assessment of peptide vaccines. Received: 9 October 1997 / Revised: 20 January 1998     

Characterizing high-affinity antigen/antibody complexes by kinetic- and equilibrium-based methods

Two biophysical methods, Biacore and KinExA, were used to kinetically and thermodynamically characterize high-affinity antigen/antibody complexes. Three to five independent experiments were performed on each platform with three different antigen/antibody complexes possessing nanomolar to picomolar equilibrium dissociation constants. By monitoring the dissociation phase on Biacore for 4 h, we were able to measure dissociation rate constants (kd) on the order of 1 x 10(-5)s(-1). To characterize high-affinity interactions by KinExA, samples needed to be equilibrated for up to 35 h to reach equilibrium. In the end, we show that similar kinetic rate constants and affinities were determined with both solution-phase and solid-phase methodologies. These results help further validate both interaction technologies and illustrate their suitability for characterizing extremely high-affinity interactions.     

Lectin bio-layer interferometry for assessing product quality of Fc- glycosylated immunoglobulin G

Glycosylation, as the most prominent posttranslational modification, is recognized as an important quality attribute of monoclonal antibodies affected by various bioprocess parameters and cellular physiology. A method of lectin-based bio-layer interferometry (LBLI) to relatively rank galactosylation and fucosylation levels was developed. For this purpose, Fc-glycosylated immunoglobulin G (IgG) was recombinantly produced with varying bioprocess conditions in 15 L bioreactor and accumulated IgG was harvested. The reliability, the robustness and the applicability of LBLI to different samples has been proven. Data obtained from LC–MS analysis served as reference and were compared to the LBLI results. The introduced method is based on non-fluidic bio-layer interferometry (BLI), which becomes recently a standard tool for determining biomolecular interactions in a label-free, real-time and high-throughput manner. For the intended purpose, biotinylated lectins were immobilized on disposable optical fiber streptavidin (SA) biosensor tips. Aleuria aurantia lectin (AAL) was used to detect the core fucose and Ricinus communis agglutinin 120 (RCA120) to determine galactosylation levels. In our case study it could be shown that fucosylation was not affected by variations in glucose feed concentration and cultivation temperature. However, the galactosylation could be correlated with the ratio of mean specific productivity (q_P) and ammonium (q_NH4+) but was unrelated to the ratio of mean q_P and the specific glucose consumption (q_gluc). This presented method strengthens the applicability of the BLI platform, which already enables measurement of several product related characteristics, such as product quantity as well as kinetic rates (k_d,k_on) and affinity constants (k_D) analysis.     

The accuracy of the rapid equilibrium assumption in steady-state enzyme kinetics in the case of a multipath arbitrary enzyme mechanism with a number of equilibrium segments

A quantitative evaluation of the accuracy of the rapid-equilibrium assumption in steady-state enzyme kinetics was obtained for a multipath arbitrary enzyme mechanism with a number of equilibrium segments. Explicit expressions for estimating the contribution of any equilibrium segment to the accuracy of the rapid-equilibrium assumption were obtained. This allowed us to determine the accuracy of the rapid-equilibrium assumption (Δ) in general: 1 + Δ = (1 + Δ₁)(1 + Δ₂)... (1 + Δ_k), where Δ₁, Δ₂,..., Δ_k is the contribution of each individual equilibrium segment. The accuracy depends only on the structure and properties of equilibrium segments, which have been accounted for in the rapid-equilibrium assumption, but it is independent of the number of paths in the mechanism of the enzymatic reaction and on the structure and properties of the remaining part (steady-state) of the kinetic scheme.     

精氨酸代谢调控蛋白ArgR调控嗜热链球菌胞外多糖合成

[目的] 研究精氨酸代谢调控蛋白ArgR对嗜热链球菌胞外多糖（EPS）合成的调控作用。[方法] 利用大肠杆菌异源表达嗜热链球菌ArgR蛋白,通过尿素变性-复性和Ni²⁺亲和层析纯化。采用凝胶电泳迁移（EMSA）和生物膜层干涉（BLI）分析ArgR和eps基因簇中P_epsA启动子的相互作用和动力学信息。构建过表达和弱化argR基因菌株,利用苯酚-硫酸法测定其合成EPS差异。[结果] 大肠杆菌异源表达的ArgR为包涵体,使用尿素变性-复性纯化可获得2.95 mg/mL可溶性蛋白;EMSA和BLI结果显示ArgR和启动子P_epsA有特异性结合,且结合因解离水平低而稳定;过表达argR基因可显著降低嗜热链球菌EPS合成,而弱化argR基因则提高EPS合成。[结论] 本研究表明ArgR能特异性结合嗜热链球菌eps基因簇启动子,并负调控EPS生物合成。     

Antibody-based sensors for heavy metal ions

Competitive immunoassays for Cd(II), Co(II), Pb(II) and U(VI) were developed using identical reagents in two different assay formats, a competitive microwell format and an immunosensor format with the KinExA™ 3000. Four different monoclonal antibodies specific for complexes of EDTA–Cd(II), DTPA–Co(II), 2,9-dicarboxyl-1,10-phenanthroline–U(VI), or cyclohexyl–DTPA–Pb(II) were incubated with the appropriate soluble metal–chelate complex. In the microwell assay format, the immobilized version of the metal–chelate complex was present simultaneously in the assay mixture. In the KinExA format, the antibody was allowed to pre-equilibrate with the soluble metal-chelate complex, then the incubation mixture was rapidly passed through a microcolumn containing the immobilized metal-chelate complex. In all four assays, the KinExA format yielded an assay with 10–1000-fold greater sensitivity. The enhanced sensitivity of the KinExA format is most likely due to the differences in the affinity of the monoclonal antibodies for the soluble versus the immobilized metal–chelate complex. The KinExA 3000 instrument and the Cd(II)-specific antibody were used to construct a prototype assay that could correctly assess the concentration of cadmium spiked into a groundwater sample. Mean analytical recovery of added Cd(II) was 114.25±11.37%. The precision of the assay was satisfactory; coefficients of variation were 0.81–7.77% and 3.62–14.16% for within run and between run precision, respectively.     

Label‐free characterization of carbonic anhydrase—novel inhibitor interactions using surface plasmon resonance,isothermal titration calorimetry and fluorescence‐based thermal shift assays

This work describes the development of biophysical unbiased methods to study the interactions between new designed compounds and carbonic anhydrase II (CAII) enzyme. These methods have to permit both a screening of a series of sulfonamide derivatives and the identification of a lead compound after a thorough study of the most promising molecules. Interactions data were collected using surface plasmon resonance (SPR) and thermal shift assay (TSA). In the first step, experiments were performed with bovine CAII isoform and were extended to human CAII. Isothermal titration calorimetry (ITC) experiments were also conducted to obtain thermodynamics parameters necessary for the processing of the TSA data. Results obtained with this reference methodology demonstrate the effectiveness of SPR and TSA. K_D values obtained from SPR data were in perfect accordance with ITC. For TSA, despite the fact that the absolute values of K_D were quite different, the same affinity scale was obtained for all compounds. The binding affinities of the analytes studied vary by more than 50 orders of magnitude; for example, the K_D value determined by SPR were 6 ± 4 and 299 ± 25 nM for compounds 1 and 3, respectively. This paper discusses some of the theoretical and experimental aspects of the affinity‐based methods and evaluates the protein consumption to develop methods for the screening of further new compounds. The double interest of SPR, that is, for screening and for the quick thorough study of the interactions parameters (k_a, k_d, and K_D), leads us to choose this methodology for the study of new potential inhibitors. Copyright © 2013 John Wiley & Sons, Ltd.     

Oxygen transfer characteristics of miniaturized bioreactor systems

Since their introduction in 2001 miniaturized bioreactor systems have made great advances in function and performance. In this article the dissolved oxygen (DO) transfer performance of submilliliter microbioreactors, and 1–10 mL minibioreactors was examined. Microbioreactors have reached k_La values of 460 h^?1, and are offering instrumentation and some functionality comparable to production systems, but at high throughput screening volumes. Minibioreactors, aside from one 1,440 h^?1 k_La system, have not offered as high rates of DO transfer, but have demonstrated superior integration with automated fluid handling systems. Microbioreactors have been typically limited to studies with E. coli, while minibioreactors have offered greater versatility in this regard. Further, mathematical relationships confirming the applicability of k_La measurements across all scales have been derived, and alternatives to fluorescence lifetime DO sensors have been evaluated. Finally, the influence on reactor performance of oxygen uptake rate (OUR), and the possibility of its real‐time measurement have been explored. Biotechnol. Bioeng. 2013; 110: 1005–1019. © 2012 Wiley Periodicals, Inc.