<p>Studies of binding reactions are of highest importance in a vast number of areas of biomedicine and biotechnology. A demand for fast and accurate small-volume measurements grows stronger, partly due to the development of therapeutic antibodies. In this report, a novel method for studies of binding reactions of antibodies is described. The use of a microfluidic platform shows promising results in determination of affinity binding constants.</p><p>Affinities between 1E-09 and 1E-11 M have been determined for four TSH antibodies. Reproducibility tests give a CV below 10%, using different Gyrolab instruments and microfluidic CD:s. The method carries the advantages of using solution-based measurements of unmodified molecules. Also an initial proof-of-concept for measurement of binding reaction rate constants shows further usage of the method. The kinetic association rate constant has been determined to 2E+06 M-1s-1 for one antibody. The possibility of using this method for screening of antibody libraries is also discussed.</p>

Studies of binding reactions are of highest importance in a vast number of areas of biomedicine and biotechnology. A demand for fast and accurate small-volume measurements grows stronger, partly due to the development of therapeutic antibodies. In this report, a novel method for studies of binding reactions of antibodies is described. The use of a microfluidic platform shows promising results in determination of affinity binding constants. Affinities between 1E-09 and 1E-11 M have been determined for four TSH antibodies. Reproducibility tests give a CV below 10%, using different Gyrolab instruments and microfluidic CD:s. The method carries the advantages of using solution-based measurements of unmodified molecules. Also an initial proof-of-concept for measurement of binding reaction rate constants shows further usage of the method. The kinetic association rate constant has been determined to 2E+06 M-1s-1 for one antibody. The possibility of using this method for screening of antibody libraries is also discussed.

Même si les anticorps sont plus connus pour leur capacité à neutraliser les agents infectieux en se liant aux antigènes via leurs paratopes, leurs fragments cristallisables (Fc) sont aussi impliqués dans la signalisation des réponses immunitaires en se liant à des récepteurs spécifiques. Les interactions entre les récepteurs et les anticorps sont reconnues pour être affectées par la glycosylation des anticorps. Pour observer la cinétique de telles interactions biologiques, une des méthodes les plus utilisées est la spectroscopie de résonance des plasmons de surface (SPR). La synthèse et l’analyse, via SPR, d’anticorps en laboratoire sont un procédé long et exigeant si toutes les étapes sont réalisées manuellement. La possibilité d’effectuer le couplage d’un appareil SPR commercial à un bioréacteur pourrait être envisagée, mais le coût d’achat et d’opération d’un tel appareil SPR limiterait l’utilité d’un tel projet pour une possible utilisation à plus grande échelle. C’est pourquoi le couplage d’un appareil SPR de faible coût et d’un bioréacteur serait avantageux. Cela permettrait de superviser la synthèse des anticorps et leur affinité au récepteur en temps ‘’réel’’. Ce mémoire de maîtrise explorera le développement de deux des composantes nécessaires pour la réalisation de ce couplage entre le SPR et un bioréacteur, soit la chimie de surface pour passiver le capteur SPR et un algorithme d’optimisation par nuée de particules (Particles swarm Optimisation) évolutive. L’algorithme réalisera une corrélation du signal obtenue d’un instrument P4-SPR aux équations cinétiques décrivant les interactions entre les anticorps et leurs récepteurs dans le but d’obtenir les constantes cinétiques et thermodynamiques (Kd,kon,koff). De plus, ce mémoire présentera une étude qui a été réalisée afin de minimiser l’adsorption non spécifique des molécules composant le biocapteur et maximiser le signal de l’anticorps Trastuzumab (TZM), utilisé dans le couplage de l’instrument P4-SPR au bioréacteur, sur des monocouches de composition variée. / While antibodies are best known to help the neutralization of pathogens by binding to the antigens with their paratope, their crystallizable fragment region (Fc region) is also used to trigger immune response by binding to specific receptors. Interactions between receptors and antibodies are known to be affected by the glycosylation the antibodies. To observe the kinetic of those interactions, one of the favored method is surface plasmon resonance (SPR). However, a substantial time may have elapsed between synthesis of a modified antibody and its test in a SPR apparatus as the two are not coupled and oftentimes in different laboratories. The coupling of a SPR and a bioreactor would accelerate the process, but using a commercial instrument would limit it usefulness due to the high price and high cost of use of these SPR instruments. This is why the coupling of a low-cost SPR to a bioreactor is of interesting in the context of glycosylated antibody production. This could permit to monitor the synthesis of the antibody and it affinity to the target receptor in near real time. This masters’ thesis will show the development of two of the essential components, consisting in the surface chemistry to passivate the SPR chip and an algorithm using an evolving PSO (Particles Swarm Optimisation), to estimate kinetic and thermodynamics constants (Kd,kon,koff) by correlating the signal obtained of a P4-SPR instrument to the kinetic and thermodynamics equations describing the interactions between antibodies and their receptors. The thesis also presents the results of the tests while trying to minimize nonspecific adsorption of the molecules used for the biosensor on multiple self-assembled monolayers (SAM) and maximize signal of the antibody named Trastuzumab (TZM) and used in the coupling of the P4-SPR to the bioreactor.