title: Real-time label-free detection and kinetic analysis of Etanercept-Protein A interactions using quartz crystal microbalance.
abstract: 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-1. The resulting equilibrium dissociation and association constants (KDand KA) were 5.06×10-8M and 1.98×107M-1, respectively. The association and dissociation rate constants (konand koff) decreased substantially as Enbrel®concentration, [C], increased, despite that the net binding rate, (kon[C]+koff), increased. The decrease in konand koffwas 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 konand kofffor 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.
authors: Laboratoire de bio-ingénierie et de biophysique de l'Université de Sherbrooke, Department of Chemical and Biotechnological Engineering, Université de Sherbrooke, 2500, boulevard de l'Université, Sherbrooke, Québec J1K 2R1, Canada; Research Centre on Aging, Institut universitaire de gériatrie de Sherbrooke, 1036, rue Belvédère Sud, Sherbrooke, Québec J1H 4C4, Canada.
authors: Evan A
authors: EA
authors: Dubiel
authors: Laboratoire de bio-ingénierie et de biophysique de l'Université de Sherbrooke, Department of Chemical and Biotechnological Engineering, Université de Sherbrooke, 2500, boulevard de l'Université, Sherbrooke, Québec J1K 2R1, Canada.
authors: Bruno
authors: B
authors: Martin
authors: Laboratoire de bio-ingénierie et de biophysique de l'Université de Sherbrooke, Department of Chemical and Biotechnological Engineering, Université de Sherbrooke, 2500, boulevard de l'Université, Sherbrooke, Québec J1K 2R1, Canada; Research Centre on Aging, Institut universitaire de gériatrie de Sherbrooke, 1036, rue Belvédère Sud, Sherbrooke, Québec J1H 4C4, Canada.
authors: Sylvain
authors: S
authors: Vigier
authors: Laboratoire de bio-ingénierie et de biophysique de l'Université de Sherbrooke, Department of Chemical and Biotechnological Engineering, Université de Sherbrooke, 2500, boulevard de l'Université, Sherbrooke, Québec J1K 2R1, Canada; Research Centre on Aging, Institut universitaire de gériatrie de Sherbrooke, 1036, rue Belvédère Sud, Sherbrooke, Québec J1H 4C4, Canada. Electronic address: Patrick.Vermette@USherbrooke.ca.
authors: Patrick
authors: P
authors: Vermette
mesh_terms: D000327:Adsorption; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000737:chemistry / Q000302:isolation & purification*; D015374:Biosensing Techniques; D003911:Dextrans / Q000737:chemistry*; D004345:Drug Industry; D000068800:Etanercept / Q000737:chemistry / Q000302:isolation & purification*; D006801:Humans; D007700:Kinetics; D008970:Molecular Weight; D011485:Protein Binding; D058525:Quartz Crystal Microbalance Techniques / Q000295:instrumentation*; D012996:Solutions; D013205:Staphylococcal Protein A / Q000737:chemistry*; D014783:Viscosity
chemical_list: D000894:Anti-Inflammatory Agents, Non-Steroidal; D003911:Dextrans; D012996:Solutions; D013205:Staphylococcal Protein A; C014392:carboxymethyl dextran; D000068800:Etanercept
keywords: Biologics; Biopharmaceuticals; Biosensors; Interaction kinetics; Ligand-receptor interactions; Quartz crystal microbalance (QCM)