Active concentration measurements of recombinant biomolecules using biosensor technology

Abstract

Whereas the concentration of a biomolecule simply refers to the amount of chemical substance per unit of volume, its active concentration refers to a relational parameter that has meaning only with respect to the molecule's ability to interact specifically with one particular ligand. When proteins are studied in a biological context, it is the biologically active concentration that is relevant, and not the total concentration of correctly and incorrectly folded molecules. Using a biosensor instrument the concentration of active biomolecules in a preparation can be measured by injecting the preparation at different flow rates onto a sensor chip surface presenting a high concentration of a specific ligand. The method can be used under conditions of partial mass transport limitation and does not require a pre-established standard curve. When the method was used to measure the active concentration of several recombinant proteins it was found that the active concentration was much lower than the nominal concentration determined by conventional methods. The active concentration also depended on the ligand used in the binding assay, reflecting the fact that active concentration can only be defined with respect to one specific probe. Such discrepancies in concentration values, if undetected, may lead to erroneous conclusions regarding the properties and behaviour of recombinant proteins tested in different assays.

Phage display of human antibodies from a patient suffering from coeliac disease and selection of isotype‐specific scFv against gliadin

“…To clearly demonstrate the specificity of the selected scFv‐carrying phage for gliadin, surface plasmon resonance measurements were performed using the BIAcore system 19 . The relative binding capacity of different scFv‐displaying phage for α‐gliadin increased, on average, by 90 RUs during the association phase, and an average of 80 ± 5 RUs remained as the relative binding value after 100 seconds of elution, indicating a remarkable increase in the binding affinity.…”

“…Using the BIAcore system (Biacore, Uppsala, Sweden) the binding and specificity of the polyclonal phagemid particles, and of selected single clones, were measured 19 . For the immobilization of gliadin, the sensor chip CM‐5 was used.…”

Several regions of the repeat domain of theStaphylococcus capraeautolysin, AtlC, are involved in fibronectin binding

“…We first determined precisely the proportion of active recombinant protein molecules, i.e. able to bind Fn: these active concentration values are essential for the correct estimation of the binding parameters[7]. In the case of the denatured/renatured preparations, only 16% of AtlCR 1 R 2 R 3 , 17% of AtlCR 1 R 2 and 0.8% of AtlCR 3 molecules were functional, while this proportion increased to 31% for AtlCR 1 R 2 domains prepared in native conditions.…”

“…Fn was covalently linked, via its primary amine groups, to the carboxymethylated dextran matrix of a CM5 sensorchip, using a Biacore 2000 apparatus and the amine coupling kit (BIAcore, Uppsala, Sweden): a density of 1935 resonance units (approximately 2 ng mm −2 ) was thus attained. The active concentrations of the recombinant protein preparations were determined under conditions of partial mass transport limitation as previously described[7]. Briefly, two dilutions of each preparation (denatured/renatured AtlCR 1 R 2 R 3 , AtlCR 1 R 2 , and AtlCR 3 , and native AtlCR 1 R 2 ) were injected for 5 min across the CM5(Fn) surface at seven distinct flow rates ranging from 2 to 100 μl min −1 .…”

Limitations to the structure‐based design of HIV‐1 vaccine immunogens

“…It is indeed possible to predict in a fairly reliable manner the neutralizing capacity of antibodies induced by a vaccine immunogen because this capacity is correlated with the kinetic off‐rate constant of the antibody when it interacts with its antigen 158, 159. SPR biosensors allow the kinetics of antigen–antibody interactions to be measured with considerable precision and they should find many applications for predicting the efficacy of different vaccine regimens as well as for assessing the quality control of different batches of vaccines using suitable panels of Mabs for measuring the ‘active’ concentration of the vaccine material 160, 161…”