A continuous integrated bioprocess available from the earliest stages of process development allows for an easier, more efficient and faster development and characterization of an integrated process as well as production of small‐scale drug candidates. The process presented in this article is a proof‐of‐concept of a continuous end‐to‐end monoclonal antibody production platform at a very small scale based on a 200 ml alternating tangential flow filtration perfusion bioreactor, integrated with the purification process with a model‐based design and control. The downstream process, consisting of a periodic twin‐column protein A capture, a virus inactivation, a CEX column and an AEX column, was compactly implemented in a single chromatography system, with a purification time of less than 4 hr. Monoclonal antibodies were produced for 17 days in a high cell density perfusion culture of CHO cells with titers up to 1.0 mg/ml. A digital twin of the downstream process was created by modelling all the chromatography steps. These models were used for real‐time decision making by the implementation of control strategies to automatize and optimize the operation of the process. A consistent glycosylation pattern of the purified product was ensured by the steady state operation of the process. Regarding the removal of impurities, at least a 4‐log reduction in the HCP levels was achieved. The recovery yield was up to 60%, and a maximum productivity of 0.8 mg/ml/day of purified product was obtained.

An optimization study of an integrated periodic counter-current chromatography (PCC) process in a monoclonal antibody (mAb) downstream process at lab scale, is presented in this paper. The optimization was based on a mechanistic model of the breakthrough curve in the protein-A capture step. Productivity and resin utilization were the objective functions, while yield during the loading of the capture column was set as a constraint. Different integration approaches were considered, and the effect of the feed concentration, yield and the protein-A resin was studied. The breakthrough curve and the length of the product recovery, which depended on the integration approach, determined the process scheduling. Several optimal Pareto solutions were obtained. At 0.5 mg mL-1 and 99% yield, a maximum productivity of 0.38 mg mL-1 min-1 with a resin utilization of 60% was obtained. On the other hand, the maximum resin utilization was 95% with a productivity of 0.10 mg mL-1 min-1. Due to the constraint of the process scheduling, a lower productivity could be achieved in the integration approaches with higher recovery time, which was more remarkable at higher concentrations. Therefore, it was shown that a holistic approach, where all the purification steps are considered in the process optimization, is needed to design a PCC in a downstream process.

In this study, we demonstrated the first, to our knowledge, integrated continuous bioprocess (ICB) designed for the production of acid‐sensitive monoclonal antibodies, prone to aggregate at low pH, on pilot scale. A high cell density perfusion culture, stably maintained at 100 × 10⁶ cells/ml, was integrated with the downstream process, consisting of a capture step with the recently developed Protein A ligand, Z_Ca; a solvent/detergent‐based virus inactivation; and two ion‐exchange chromatography steps. The use of a mild pH in the downstream process makes this ICB suitable for the purification of acid‐sensitive monoclonal antibodies. Integration and automation of the downstream process were achieved using the Orbit software, and the same equipment and control system were used in initial small‐scale trials and the pilot‐scale downstream process. High recovery yields of around 90% and a productivity close to 1 g purified antibody/L/day were achieved, with a stable glycosylation pattern and efficient removal of impurities, such as host cell proteins and DNA. Finally, negligible levels of antibody aggregates were detected owing to the mild conditions used throughout the process. The present work paves the way for future industrial‐scale integrated continuous biomanufacturing of all types of antibodies, regardless of acid stability.

Integrated continuous downstream processes with process analytical technology offer a promising opportunity to reduce production costs and increase process flexibility and adaptability. In this case study, an integrated continuous process was used to purify a recombinant protein on laboratory scale in a two‐system setup that can be used as a general downstream setup offering multiproduct and multipurpose manufacturing capabilities. The process consisted of continuous solvent/detergent virus inactivation followed by periodic countercurrent chromatography in the capture step, and a final chromatographic polishing step. A real‐time controller was implemented to ensure stable operation by adapting the downstream process to external changes. A concentration disturbance was introduced to test the controller. After the disturbance was applied, the product output recovered within 6 h, showing the effectiveness of the controller. In a comparison of the process with and without the controller, the product output per cycle increased by 27%, the resin utilization increased from 71.4% to 87.9%, and the specific buffer consumption was decreased by 21% with the controller, while maintaining a similar yield and purity as in the process without the disturbance. In addition, the integrated continuous process outperformed the batch process, increasing the productivity by 95% and the yield by 28%.