The transition to continuous biomanufacturing is considered the next step to reduce costs and improve process robustness in the biopharmaceutical industry, while also improving productivity and product quality. The platform production process for monoclonal antibodies (mAbs) is eligible for continuous processing to lower manufacturing costs due to patent expiration and subsequent growing competition. One of the critical quality attributes of interest during mAb purification is aggregate formation, with several processing parameters and environmental factors known to influence antibody aggregation. Therefore, a real-time measurement to monitor aggregate formation is crucial to have immediate feedback and process control and to achieve a continuous downstream processing. Miniaturized biosensors as an in-line process analytical technology tool could play a pivotal role to facilitate the transition to continuous manufacturing. In this review, miniaturization of already wellestablished methods to detect protein aggregation, such as dynamic light scattering, Raman spectroscopy and circular dichroism, will be extensively evaluated for the possibility of providing a real-time measurement of mAb aggregation. The method evaluation presented in this review shows which limitations of each analytical method still need to be addressed and provides application examples of each technique for mAb aggregate characterization. Additionally, challenges related to miniaturization are also addressed, such as the design of the microfluidic chip and the microfabrication material. The evaluation provided in this review shows why the development of microfluidic biosensors is considered the key for real-time measurement of mAb aggregates and how it can contribute to the transition to a continuous processing.

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.

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.

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.