A global benchmark study using affinity-based biosensors.

To explore the variability in biosensor studies, 150 participants from 20 countries were given the same protein samples and asked to determine kinetic rate constants for the interaction. We chose a protein system that was amenable to analysis using different biosensor platforms as well as by users of different expertise levels. The two proteins (a 50-kDa Fab and a 60-kDa glutathione S-transferase [GST] antigen) form a relatively high-affinity complex, so participants needed to optimize several experimental parameters, including ligand immobilization and regeneration conditions as well as analyte concentrations and injection/dissociation times. Although most participants collected binding responses that could be fit to yield kinetic parameters, the quality of a few data sets could have been improved by optimizing the assay design. Once these outliers were removed, the average reported affinity across the remaining panel of participants was 620 pM with a standard deviation of 980 pM. These results demonstrate that when this biosensor assay was designed and executed appropriately, the reported rate constants were consistent, and independent of which protein was immobilized and which biosensor was used.

Comparative analyses of a small molecule/enzyme interaction by multiple users of Biacore technology.

To gauge the experimental variability associated with Biacore analysis, 36 different investigators analyzed a small molecule/enzyme interaction under similar conditions. Acetazolamide (222 g/mol) binding to carbonic anhydrase II (CAII; 30000 Da) was chosen as a model system. Both reagents were stable and their interaction posed a challenge to measure because of the low molecular weight of the analyte and the fast association rate constant. Each investigator created three different density surfaces of CAII and analyzed an identical dilution series of acetazolamide (ranging from 4.1 to 1000 nM). The greatest variability in the results was observed during the enzyme immobilization step since each investigator provided their own surface activating reagents. Variability in the quality of the acetazolamide binding responses was likely a product of how well the investigators' instruments had been maintained. To determine the reaction kinetics, the responses from the different density surfaces were fit globally to a 1:1 interaction model that included a term for mass transport. The averaged association and dissociation rate constants were 3.1+/-1.6 x 10(6)M(-1)s(-1) and 6.7+/-2.5 x 10(-2)s(-1), respectively, which corresponded to an average equilibrium dissociation constant (K(D) of 2.6+/-1.4 x 10(-8)M. The results provide a benchmark of variability in interpreting binding constants from the biosensor and highlight keys areas that should be considered when analyzing small molecule interactions.

Kinetic exclusion assay technology: characterization of molecular interactions.

Characterization of intermolecular interactions in terms of affinity, binding kinetics, stoichiometry, specificity, and thermodynamics can facilitate the selection of lead compounds in the discovery and development of protein therapeutics. KinExA (Sapidyne Instruments, Inc., Boise, ID) is a relatively new technology that is gaining use in characterizing molecular interactions, particularly with respect to antibody therapeutics. KinExA offers a platform that allows the measurement of true equilibrium binding affinity and kinetics using unmodified molecules in solution phase. This is accomplished by using a solid-phase immobilized molecule to probe for free concentration of one interaction component after allowing sufficient time to reach equilibrium (affinity measurements), or under pre-equilibrium conditions (kinetics). In this review, the theory behind KinExA technology is discussed, and examples of applying this technology to antibody characterization are provided. Finally, a comparison among KinExA, Biacore (surface plasmon resonance), and isothermal titration calorimetry is presented, and potential future improvements and applications of KinExA are discussed.

Glycosylation of erythropoietin affects receptor binding kinetics: role of electrostatic interactions.

Erythropoietin (EPO) is a cytokine produced by the kidney whose function is to stimulate red blood cell production in the bone marrow. Previously, it was shown that the affinity of EPO for its receptor, EPOR, is inversely related to the sialylation of EPO carbohydrate. To better understand the properties of EPO that modulate its receptor affinity, various glycoforms were analyzed using surface plasmon resonance. The system used has been well characterized and is based on previous reports employing an EPOR-Fc chimera captured on a Protein A surface. Using three variants of EPO containing different levels of sialylation, we determined that sialic acid decreased the association rate constant (k(on)) about 3-fold. Furthermore, glycosylated EPO had a 20-fold slower k(on) than nonglycosylated EPO, indicating that the core carbohydrate also negatively impacted k(on). The effect of electrostatic forces on EPO binding was studied by measuring binding kinetics in varying NaCl concentrations. Increasing NaCl concentration resulted in a slower k(on) while having little impact on k(off), suggesting that long-range electrostatic interactions are primarily important in determining the rate of association between EPO and EPOR. Furthermore, the glycosylation content (i.e., nonglycosylated vs glycosylated, sialylated vs desialylated) affected the overall sensitivities of k(on) to [NaCl], indicating that sialic acid and the glycan itself each impact the overall effect of these electrostatic forces.