Therapeutic monoclonal antibodies (mAb) are used for the treatment of numerous serious diseases, which have led to an increasing demand over the last decades. Increased cell density and mAb titer of the cultivation broth lead to great challenges for the subsequent clarification and capture operations in the downstream process. As an alternative approach to the conventional downstream process, a selective mAb extraction via an aqueous two-phase system (ATPS) directly from the cultivation broth of a mAb producing industrial relevant chinese hamster ovary (CHO) cell line was investigated. An efficient purification of the mAb was accomplished by the ATPS composition. The phase separation was realized by a newly developed membrane based phase separator. Moreover, a complete cell removal was integrated into this process by the used membrane. A selectivity between both phases was achieved by membrane modification. Yields up to 93% in the light phase and removal of process related impurities were obtained after aqueous two-phase extraction (ATPE). Phase separation performance as well as contact angles on the membrane were characterized for different ATPS. ATPE directly from the cultivation broth in combination with the new membrane based phase separation led to a mAb yield of 78% with a simultaneous reduction of deoxyribonucleic acid (DNA) and host cell protein (HCP) load.

Safety-critical systems-on-chip currently undergo extensive fault-effect analyses. To meet the safety requirements of ISO 26262, most frequently fault-injection campaigns are per- formed. Due to the exponentially growing fault-verification space, faster simulation possibilities than enabled by register transfer (RT) and gate-level (GL) models are under investigation. Fault injection on virtual prototypes (VPs) is one measure to speed up simulation. However, VPs require the injection of complex abstract faults to observe the same effects of, for example, single- bit fault injection into GL models. As a consequence, VPs often suffer from injection of incorrect faults (i.e., faults whose effects cannot be reproduced on the RT or gate levels). Therefore, we developed an efficient approach to verify or falsify failures detected with VP fault simulation. As a result, incorrect faults are discovered early in the development phase helping to improve the design of accurate safety mechanisms. Moreover, the exclusion of incorrect faults from fault-effect analyses further improves the accuracy and efficiency of fault-injection campaigns. The benefit of the presented method has been validated using a medium-size controller design.

Downstream processing is the bottleneck in the continuous manufacturing of monoclonal antibodies (mAbs). To overcome throughput limitations, two different continuous processes with a novel convective diffusive protein A membrane adsorber (MA) were investigated: the rapid cycling parallel multi‐column chromatography (RC‐PMCC) process and the rapid cycling simulated moving bed (RC‐BioSMB) process. First, breakthrough curve experiments were performed to investigate the influence of the flow rate on the mAb dynamic binding capacity and to calculate the duration of the loading steps. In addition, customized control software was developed for an automated MA exchange in case of pressure increase due to membrane fouling to enable robust, uninterrupted, and continuous processing. Both processes were performed for 4 days with 0.61 g L⁻¹ mAb‐containing filtrate and process performance, product purity, productivity, and buffer consumption were compared. The mAb was recovered with a yield of approximately 90% and productivities of 1010 g L⁻¹ d⁻¹ (RC‐PMCC) and 574 g L⁻¹ d⁻¹ (RC‐BioSMB). At the same time, high removal of process‐related impurities was achieved with both processes, whereas the buffer consumption was lower for the RC‐BioSMB process. Finally, the attainable productivity for perfusion bioreactors of different sizes with suitable MA sizes was calculated to demonstrate the potential to operate both processes on a manufacturing scale with bioreactor volumes of up to 2000 L.

The interest in extracellular vesicles (EVs) has been increased in recent years due to their potential application in diagnosis and therapy of severe diseases. The versatile fields of application due to the numerous possible cargos and the targeted delivery system make them a promising biopharmaceutical product. However, their broad size range as well as varied surface protein content result in challenges for the purification, characterization, and quantification. In this study a novel method, based on high‐resolution flow cytometry, was examined for the enumeration of EVs in purified as well as crude process samples. In addition to quantification, samples were characterized by dynamic light scattering, zeta potential measurement, and analytical size exclusion chromatography. It has been demonstrated that EVs were successfully enumerated with the novel method, offering great benefits for development and monitoring of EV processes.

The semi and fully continuous production of monoclonal antibodies (mAbs) has been gaining traction as a lower cost, and efficient production of mAbs to broaden patient access. To be truly flexible and adaptive to process demands, the industry has lacked sufficient advanced control strategies. The variation of the upstream product concentration typically cannot be handled by the downstream capture step, which is configured for a constant feed concentration and fixed binding capacity. This inflexibility leads to losses of efficiency and product yield.This study shows that these challenges can be overcome by a novel advanced control strategy concept that includes dynamic control throughout a perfusion bioreactor, with cell retention by alternating tangential flow, integrated with simulated moving bed (SMB) multi‐column chromatography. The automation workflow and advanced control strategy were implemented through the use of a visual programming development environment. This enabled dynamic flow control across the upstream and downstream process integrated with a dynamic column loading of the SMB. A sensor prototype, based on continuous biolayer interferometry measurements was applied to detect mAb breakthrough within the last column flow‐through to manage column switching. This novel approach provided higher specificity and lower background signal compared to commonly used spectroscopy methods, resulting in an optimized resin utilization while simultaneously avoiding product loss. The dynamic loading was found to provide a twofold increase of the mAb concentration in the eluate compared to a conservative approach with a predefined recipe with similar impurity removal. This concept shows that advanced control strategies can lead to significant process efficiency and yield improvement.

Harmonizing unit operations in the downstream process of monoclonal antibodies (mAbs) has a high potential to overcome throughput limitations and reduce manufacturing costs. This study proposes a streamlined clarification and capture (S‐CC) process concept for the continuous processing of cell broth harvested from a connected bioreactor. The process was realized with a fluidized bed centrifuge connected to depth and sterile filters, a surge tank, and a multi‐column chromatography (MCC) unit. The MCC unit was operated in the rapid cycling simulated moving bed (RC‐BioSMB) mode with five convective diffusive membrane adsorbers (MAs). A control strategy and the surge tank were used to adjust the loading flow rate of the MCC unit. The mAb was recovered with a total process yield of 90%, with high removal of the process‐related impurities HCP (2.1 LRV) and DNA (2.9 LRV). Moreover, the S‐CC process productivity of 4.2 g h⁻¹ was up to 5.3 times higher than for comparable, hypothetical batch MA processes. In addition, the buffer consumption of the capture step could be reduced from 2.0 L g⁻¹ in batch mode to 1.2 L g⁻¹ in the RC‐BioSMB mode. These results demonstrate the high potential of streamlined interconnected unit operations to improve the overall mAb downstream process performance.