Real-time label-free detection and kinetic analysis of Etanercept-Protein A interactions using quartz crystal microbalance.

A quartz crystal microbalance (QCM) was constructed to assess if such a biosensor has value as a complementary real-time label-free analysis platform for the biopharmaceutical industry. This was achieved through modifying QCM crystals with a low-fouling carboxymethyl-dextran layer bearing Protein A, and then injecting solutions containing Etanercept (i.e., Enbrel) into the QCM chambers. The kinetics of Enbrel - Protein A interactions was modeled using the Langmuir binding model and Enbrel concentrations between 0.75-300ngmL. The resulting equilibrium dissociation and association constants (K and K) were 5.06×10M and 1.98×10M, respectively. The association and dissociation rate constants (k and k) decreased substantially as Enbrel concentration, [C], increased, despite that the net binding rate, (k[C]+k), increased. The decrease in k and k was hypothesized to be a consequence of mass transport limitations. To verify this, QCM dissipation measurements were analyzed to provide insight on solution viscosity. As Enbrel concentration increased, the net change in dissipation, ΔD, became larger. An augmentation of ΔD is associated with a higher solution viscosity, which would result in an increase in mass transport limitations. Therefore, the decrease in k and k for increasing Enbrel concentration can be attributed to mass transport limitations. In conclusion, QCM is a valuable complementary real-time label-free biosensor analysis platform for the biopharmaceutical industry. Unlike the surface plasmon resonance (SPR) platform, QCM allows measuring dissipation, which can provide insight on how mass transport limitations impact interaction kinetics.