Real-time detection of mAb aggregates in an integrated downstream process.

The implementation of continuous processing in the biopharmaceutical industry is hindered by the scarcity of process analytical technologies (PAT). To monitor and control a continuous process, PAT tools will be crucial to measure real-time product quality attributes such as protein aggregation. Miniaturizing these analytical techniques can increase measurement speed and enable faster decision-making. A fluorescent dye (FD)-based miniaturized sensor has previously been developed: a zigzag microchannel which mixes two streams under 30 s. Bis-ANS and CCVJ, two established FDs, were employed in this micromixer to detect aggregation of the biopharmaceutical monoclonal antibody (mAb). Both FDs were able to robustly detect aggregation levels starting at 2.5%. However, the real-time measurement provided by the microfluidic sensor still needs to be implemented and assessed in an integrated continuous downstream process. In this work, the micromixer is implemented in a lab-scale integrated system for the purification of mAbs, established in an ÄKTA™ unit. A viral inactivation and two polishing steps were reproduced, sending a sample of the product pool after each phase directly to the microfluidic sensor for aggregate detection. An additional UV sensor was connected after the micromixer and an increase in its signal would indicate that aggregates were present in the sample. The at-line miniaturized PAT tool provides a fast aggregation measurement, under 10 min, enabling better process understanding and control.

White paper on high-throughput process development for integrated continuous biomanufacturing.

Continuous manufacturing is an indicator of a maturing industry, as can be seen by the example of the petrochemical industry. Patent expiry promotes a price competition between manufacturing companies, and more efficient and cheaper processes are needed to achieve lower production costs. Over the last decade, continuous biomanufacturing has had significant breakthroughs, with regulatory agencies encouraging the industry to implement this processing mode. Process development is resource and time consuming and, although it is increasingly becoming less expensive and faster through high-throughput process development (HTPD) implementation, reliable HTPD technology for integrated and continuous biomanufacturing is still lacking and is considered to be an emerging field. Therefore, this paper aims to illustrate the major gaps in HTPD and to discuss the major needs and possible solutions to achieve an end-to-end Integrated Continuous Biomanufacturing, as discussed in the context of the 2019 Integrated Continuous Biomanufacturing conference. The current HTPD state-of-the-art for several unit operations is discussed, as well as the emerging technologies which will expedite a shift to continuous biomanufacturing.

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

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.

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

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.

Microfluidics as a high-throughput solution for chromatographic process development - The complexity of multimodal chromatography used as a proof of concept.

High-throughput technologies are fundamental to expedite the implementation of novel purification platforms. The possibility of performing process development within short periods of time while saving consumables and biological material are prime features for any high-throughput screening device. In this work, a microfluidic device is evaluated as high-throughput solution for a complete study of chromatographic operation conditions on ten different multimodal resins. The potential of this class of purification solutions is generally hindered by its complexity. Taking this into consideration, the microfluidic platform was herein applied and assessed as a tool for high-throughput applications. The commercially available multimodal ligands were studied for the binding of three antibody-based biomolecules (polyclonal mixture of whole antibodies, Fab and Fc fragments) at different pH and salt conditions, in a total of 450 experiments. The results obtained with the microfluidic device were comparable to a standard 96-well filtering microplate high-throughput tool. Additionally, five of the ten multimodal ligands tested were packed into a bench-scale column to perform a final validation of the microfluidic results obtained. All the data acquired in this work using different screening protocols corroborate each other, showing that microfluidic chromatography is a valuable tool for the fast implementation of a new purification step, particularly, if the goal is to narrow the downstream possibilities by being a first point of decision.

Minimizing the Influence of Fluorescent Tags on IgG Partition in PEG-Salt Aqueous Two-Phase Systems for Rapid Screening Applications.

Aqueous two-phase extraction (ATPE) has been showing significant potential in the biopharmaceutical industry, allowing the selective separation of high-value proteins directly from unclarified cell culture supernatants. In this context, effective high-throughput screening tools are critical to perform a rapid empirical optimization of operating conditions. In particular, microfluidic ATPE screening devices, coupled with fluorescence microscopy to continuously monitor the partition of fluorophore-labeled proteins, have been recently demonstrated to provide short diffusion distances and rapid partition, using minimal reagent volumes. Nevertheless, the currently overlooked influence of the labeling procedure on partition must be carefully evaluated to validate the extrapolation of results to the unlabeled molecule. Here, three fluorophores with different global charge and reactivity selected to label immunoglobulin G (IgG) at degrees of labeling (DoL) ranging from 0.5 to 7.6. Labeling with BODIPY FL maleimide (DoL = 0.5), combined with tris(2-carboxyethyl) phosphine (TCEP) to generate free thiol groups, is the most promising strategy to minimize the influence of the fluorophore on partition. In particular, the partition coefficient (Kp ) measured in polyethylene glycol (PEG) 3350-phosphate systems with and without the addition of NaCl using microtubes (batch) or microfluidic devices (continuous) is comparable to those quantified for the native protein.