Design of a microfluidic mixer channel: First steps into creating a fluorescent dye‐based biosensor for mAb aggregate detection

Abstract

A major challenge in the transition to continuous biomanufacturing is the lack of process analytical technology (PAT) tools which are able to collect real-time information on the process and elicit a response to facilitate control. One of the critical quality attributes (CQAs) of interest during monoclonal antibodies production is aggregate formation. The development of a real-time PAT tool to monitor aggregate formation is then crucial to have immediate feedback and process control. Miniaturized sensors placed after each unit operation can be a powerful solution to speed up an analytical measurement due to their characteristic short reaction time. In this work, a micromixer structure capable of mixing two streams is presented, to be employed in the detection of mAb aggregates using fluorescent dyes. Computational fluid dynamics (CFD) simulations were used to compare the mixing performance of a series of the proposed designs. A final design of a zigzag microchannel with 45 • angle was reached and this structure was subsequently fabricated and experimentally validated with colour dyes and, later, with a FITC-IgG molecule. The designed zigzag micromixer presents a mixing index of around 90%, obtained in less than 30 seconds. Therefore, a micromixer channel capable of a fast and efficient mixing is hereby demonstrated, to be used as a real-time PAT tool for a fluorescence based detection of protein aggregation.

Real‐time detection of mAb aggregates in an integrated downstream process

“…The zigzag micromixer (100 μm high × 100 μm wide × 17.2 mm long) presents two inlets and one outlet (Figure 1). More information on the dimensions and characteristics of the micromixer channel are found in São Pedro et al (2022). In terms of the structure fabrication, the designed mold was ordered from INESC Microsystems and Nanotechnologies.…”

“…The micromixer provides around 90% of mixing efficiency when both streams are simultaneously pumped into the structure at 1 µL min −1 (São Pedro et al, 2022). Due to the ÄKTA's inherent pump limitations for lower flows, a flow rate of 3 µL min −1 (0.003 mL min −1 ) was used, which still provided a high mixing efficiency, of around 85%, determined according to São Pedro et al (2023).…”

“…Since these dyes provide an immediate and straightforward measurement of aggregation, a FD‐based microfluidic biosensor for aggregate detection was designed and developed. A zigzag micromixer, represented in Figure 1, is capable of effectively mixing two different streams within 30 s (São Pedro et al, 2022). The micromixer is comprised of two inlets and one outlet, where the mixing occurs due to the 45° zigzag design, with a total of 30 mixing units.…”