Application of a fluorescent dye‐based microfluidic sensor for real‐time detection of mAb aggregates

Abstract

The lack of process analytical technologies able to provide real‐time information and process control over a biopharmaceutical process has long impaired the transition to continuous biomanufacturing. For the monoclonal antibody (mAb) production, aggregate formation is a major critical quality attribute (CQA) with several known process parameters (i.e., protein concentration and agitation) influencing this phenomenon. The development of a real‐time tool to monitor aggregate formation is then crucial to gain control and achieve a continuous processing. Due to an inherent short operation time, miniaturized biosensors placed after each step can be a powerful solution. In this work, the development of a fluorescent dye‐based microfluidic sensor for fast at‐line PAT is described, using fluorescent dyes to examine possible mAb size differences. A zigzag microchannel, which provides 90% of mixing efficiency under 30 s, coupled to an UV–Vis detector, and using four FDs, was studied and validated. With different generated mAb aggregation samples, the FDs Bis‐ANS and CCVJ were able to robustly detect from, at least, 2.5% to 10% of aggregation. The proposed FD‐based micromixer is then ultimately implemented and validated in a lab‐scale purification system, demonstrating the potential of a miniaturized biosensor to speed up CQAs measurement in a continuous process.

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

“…The function of each additional module is extensively described in Section 2.3.2. The microfluidic chip was operated at a flow rate of 3 µL min −1 (0.003 mL min −1 ), which provided a mixing efficiency of around 85% (São Pedro et al, 2023). Pump A and sample pump inject the sample and the FD into the micromixer, respectively.…”

“…Even though the size of these aggregates would still need to be assessed, 0.5% of aggregation on the final mAb formulation can be considerable acceptable (if the HMW species present are mainly reversible soluble aggregates). Therefore, Bis‐ANS can be an ideal choice to be employed in the mAb purification process since it only provides an increase of the UV signal at 520 nm for samples with around 2% of aggregation (São Pedro et al, 2023). Additionally, for the stripping of the AEX column, the micromixer was able to effectively detect aggregation, which was expected (Table 2).…”

“…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). With the incorporation of the two 50 mL superloops, this reduction of pump A's flow rate to 3 µL min −1 for aggregate detection in the micromixer could be achieved.…”

“…Regarding the wavelengths used in UV1 for aggregate detection, for the FD CCVJ, the selected excitation wavelength ( λ exc ) was of 435 nm and the emission wavelength ( λ em ) of 520 nm. For the FD Bis‐ANS, the selected λ exc was of 385 nm and the λ em of 520 nm (São Pedro et al, 2023).…”

“…This zigzag structure was applied to detect the presence of HMW species in a variety of mAb aggregation samples, induced by different induction factors (like temperature, freeze-thawing, or low pH incubation). Depending on the FD employed, the developed micromixer was able to robustly detect, at least, 2.5% of aggregation (São Pedro et al, 2023).…”