Microfluidic Platform for Assessment of Therapeutic Proteins Using Molecular Charge Modulation Enhanced Electrokinetic Concentration Assays

Abstract

Therapeutic proteins (TPs) are critical in modern medicine, yet shortage of TPs in disaster situations and remote areas remains a worldwide challenge. Manufacturing and real-time release of TPs on demand at the point-of-care is considered the key to this issue, which requires reliable and rapid analytics techniques for quality assurance. Herein we report a microfluidic platform that could be implemented in-line and at the point-of-care for real-time decision-making about the quality of a TP. The in vivo efficacy and duration of efficacy of TPs were assessed by the equilibrium and kinetics of TP and TP receptor (TPR) binding, using electrokinetic concentration (EC) and molecular charge modulation (MCM). EC can simultaneously concentrate and separate bound and unbound species in an assay based on electrical mobility, allowing for the quantification of binding. MCM enables the application of EC to arbitrary TPs by enhancing the mobility differences between TPs, TPRs, and TP-TPR complexes. This technology is homogeneous and overcomes many practical challenges of conventional heterogeneous assays. We developed various formats of assays for equilibrium and kinetic analysis and rapid determination of degradation of TPs, obtaining results comparable to state-of-the-art technologies with significantly less time (<1 h) and simpler setup. Finally, we demonstrated that the results of MCM-EC based assays correlated well with those from mass spectrometry and cell-based assay, which are the industrial standards for quality testing of TPs.

Electropreconcentration‐induced local pH change

“…It would be more attractive when the electropreconcentration is integrated with other analytical methods such as capillary electrophoresis and electrospray ionization mass spectrometry. Recently, the electropreconcentration has been applied for quantifying therapeutic proteins , detecting kinase activities , analysing enzyme‐linked immunosorbent assay (ELISA) sensitivity , and quantifying enzyme reaction rate . However, the effective charge of a protein is directly related with the buffer pH .…”

Recent advances in protein analysis by capillary and microchip electrophoresis

“…141 In a previous report, 142 the authors stated that electrokinetic concentration could simultaneously preconcentrate and separate bound and unbound species in aptamer-protein binding assay. However, the usefulness of sole electrokinetic concentration for TP and TP receptor (TPR) binding assays is quite limited.…”

Force fields of charged particles in micro-nanofluidic preconcentration systems

“…Features that are not explainable in 1D, namely the protrusions in focused band and the mechanism behind the low enrichment factor and instability of focused band of large particles, are clarified. Although 2D results are not expected to match the experimental data directly, qualitative mechanisms and physical insights obtained here may help researchers better understand, design and optimize ICP-based preconcentrators for mixed species, 29,30 as well as devices for desalination, 31–33 separation, 25,34 reaction, 35–37 micro-nano-fluidic mixing, 38,39 and other related applications 12,40 …”