Integrated continuous manufacturing is entering the biopharmaceutical industry. The main drivers range from improved economics, manufacturing flexibility, and more consistent product quality. However, studies on fully integrated production platforms have been limited due to the higher degree of system complexity, limited process information, disturbance, and drift sensitivity, as well as difficulties in digital process integration. In this study, we present an automated end‐to‐end integrated process consisting of a perfusion bioreactor, CaptureSMB, virus inactivation (VI), and two polishing steps to produce an antibody from an instable cell line. A supervisory control and data acquisition (SCADA) system was developed, which digitally integrates unit operations and analyzers, collects and centrally stores all process data, and allows process‐wide monitoring and control. The integrated system consisting of bioreactor and capture step was operated initially for 4 days, after which the full end‐to‐end integrated run with no interruption lasted for 10 days. In response to decreasing cell‐specific productivity, the supervisory control adjusted the loading duration of the capture step to obtain high capacity utilization without yield loss and constant antibody quantity for subsequent operations. Moreover, the SCADA system coordinated VI neutralization and discharge to enable constant loading conditions on the polishing unit. Lastly, the polishing was sufficiently robust to cope with significantly increased aggregate levels induced on purpose during virus inactivation. It is demonstrated that despite significant process disturbances and drifts, a robust process design and the supervisory control enabled constant (optimum) process performance and consistent product quality.

Acetic acid can be generated through syngas fermentation, lignocellulosic biomass degradation, and organic waste anaerobic digestion. Microbial conversion of acetate into triacylglycerols for biofuel production has many advantages, including low-cost or even negative-cost feedstock and environmental benefits. The main issue stems from the dilute nature of acetate produced in such systems, which is costly to be processed on an industrial scale. To tackle this problem, we established an efficient bioprocess for converting dilute acetate into lipids, using the oleaginous yeast Yarrowia lipolytica in a semicontinuous system. The implemented design used low-strength acetic acid in both salt and acid forms as carbon substrate and a cross-filtration module for cell recycling. Feed controls for acetic acid and nitrogen based on metabolic models and online measurement of the respiratory quotient were used. The optimized process was able to sustain high-density cell culture using acetic acid of only 3% and achieved a lipid titer, yield, and productivity of 115 g/L, 0.16 g/g, and 0.8 g·L −1 ·h −1 , respectively. No carbon substrate was detected in the effluent stream, indicating complete utilization of acetate. These results represent a more than twofold increase in lipid production metrics compared with the current best-performing results using concentrated acetic acid as carbon feed.lipid production | acetate | tangential filtration | dynamic modeling | exhaust gas analysis

Chromatography is widely used in biotherapeutics manufacturing, and the corresponding underlying mechanisms are well understood. To enable process control and automation, spectroscopic techniques are very convenient as on-line sensors, but their application is often limited by their sensitivity. In this work, we investigate the implementation of Raman spectroscopy to monitor monoclonal antibody (mAb) breakthrough (BT) curves in chromatographic operations with a low titer harvest. A state estimation procedure is developed by combining information coming from a lumped kinetic model (LKM) and a Raman analyzer in the frame of an extended Kalman filter approach (EKF). A comparison with suitable experimental data shows that this approach allows for the obtainment of reliable estimates of antibody concentrations with reduced noise and increased robustness.

On‐line monitoring tools for downstream chromatographic processing (DSP) of biotherapeutics can enable fast actions to correct for disturbances in the upstream, gain process understanding, and eventually lead to process optimization. While UV/Vis spectroscopy is mostly assessing the protein's amino acid composition and the application of Fourier transform infrared spectroscopy is limited due to strong water interactions, Raman spectroscopy is able to assess the secondary and tertiary protein structure without significant water interactions. The aim of this work is to implement the Raman technology in DSP, by designing an in‐line flow cell with a reduced dead volume of 80 μL and a reflector to increase the signal intensity as well as developing a chemometric modeling path. In this context, measurement settings were adjusted and spectra were taken from different chromatographic breakthrough curves of IgG1 in harvest. The resulting models show a small average RMSEP of 0.12 mg/mL, on a broad calibration range from 0 to 2.82 mg/mL IgG1. This work highlights the benefits of model assisted Raman spectroscopy in chromatography with complex backgrounds, lays the fundamentals for in‐line monitoring of IgG1, and enables advanced control strategies. Moreover, the approach might be extended to further critical quality attributes like aggregates or could be transferred to other process steps.

Monoclonal antibodies (mAbs) and related recombinant proteins continue to gain importance in the treatment of a great variety of diseases. Despite significant advances, their manufacturing can still present challenges owing to their molecular complexity and stringent regulations with respect to product purity, stability, safety, and so forth. In this context, protein aggregates are of particular concern due to their immunogenic potential. During manufacturing, mAbs routinely undergo acidic treatment to inactivate viral contamination, which can lead to their aggregation and thereby to product loss. To better understand the underlying mechanism so as to propose strategies to mitigate the issue, we systematically investigated the denaturation and aggregation of two mAbs at low pH as well as after neutralization. We observed that at low pH and low ionic strength, mAb surface hydrophobicity increased whereas molecular size remained constant. After neutralization of acidic mAb solutions, the fraction of monomeric mAb started to decrease accompanied by an increase on average mAb size. This indicates that electrostatic repulsion prevents denatured mAb molecules from aggregation under acidic pH and low ionic strength, whereas neutralization reduces this repulsion and coagulation initiates. Limiting denaturation at low pH by D-sorbitol addition or temperature reduction effectively improved monomer recovery after neutralization. Our findings might be used to develop innovative viral inactivation procedures during mAb manufacturing that result in higher product yields. K E Y W O R D SANS fluorescence, downstream processing, monoclonal antibodies, protein aggregation, protein unfolding, viral inactivation

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Aggregates are amongst the most important product-related impurities to be removed during the downstream processing of antibodies due to their potential immunogenicity. Traditional operations use cation-exchange resins in bind-elute mode for their separation. However, frontal analysis is emerging as an alternative. In this study, a three-step process development for a membrane adsorber and a resin material is carried out, allowing the comparison between the stationary phases. Based on a screening study, optimal loading conditions are determined, which show that weak binding is favored on the membrane and strong binding on the resin. Transfer of these findings to breakthrough experiments shows that at 99% pool purity the yield is higher for the membrane, while the resin can be loaded twice as high, exceeding yields of 85%. For the investigated antibody and based on a given regeneration protocol, the productivity of the two phases is similar, ranging around 200 g/(L·h).Due to the higher loading, the resin requires about one-third less buffer than the membrane. Furthermore, the implementation of a wash step after loading allows to further increase yield by about 5%. In comparison to a generic bind-elute process, productivity and buffer consumption are improved by an order of magnitude.

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