Integrated continuous biomanufacturing on pilot scale for acid-sensitive monoclonal antibodies

Abstract

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.

Keywords: Chinese Hamster Ovary cells; ZCa ligand; antibody aggregation; antibody manufacturing; continuous chromatography; integrated continuous bioprocess; perfusion culture.

Figure 1

Process overview: on pilot scale, a 30 L TFF perfusion bioreactor was linked to a PCC system for the Protein A capture via an intermediate hold‐up vessel. Further downstream, a solvent/detergent‐based VI and two polishing steps with a CEX and an AEX resin are shown. AEX, anion exchange; CEX, cation exchange; PCC, periodic counter‐current chromatography; TFF, tangential flow filtration.

Figure 2

Perfusion culture data of the 0.2 L (blue) and 30 L pilot‐scale (green) run: (a) VCD (circles) and viability (squares) for N‐1 culture and production culture. (b) Harvest flow rate (circles), perfusion (feed) flow rate (squares) and bleed rate (diamonds). (c) mAb concentration in the harvest flow. mAb, monoclonal antibody; VCD, viable cell density.

Figure 3

Steady‐state operation of the downstream process on pilot scale. (a) Capture step: UV signals from the three columns in the PCC operation. The breakthrough curves are expressed as percentages with respect to the feed concentration. The blue shaded peaks correspond to the product collected in the same VI bottle. (b) Polishing steps: UV signals from the two polishing steps. UV1 corresponds to the CEX column, and UV2 to the AEX column. The blue shaded area is the product loaded onto the CEX column, and the red shaded peak is the AEX eluate. AEX, anion exchange; CEX, cation exchange; PCC, periodic counter‐current chromatography; UV, ultraviolet.

Figure 4

Recovery yield for the different downstream steps (a) and productivities (b) in the pilot‐scale run. AEX, anion exchange; CEX, cation exchange; DSP, downstream process; USP, upstream process.

Figure 5

Quality and purity of the final product obtained in the pilot‐scale ICB throughout the entire run. The arrows mark the time point where the capture columns were switched. (a) The mAb glycosylation pattern indicating the percentage of the different glycoforms. (b) HCP log reduction values (LRV) demonstrating the removal of HCP after each purification step of the downstream process as compared to the initial harvest. (c) LRVs for DNA after capture and CEX at three‐time points throughout the process. LRVs for AEX is not included since the DNA levels were below the limit of detection. AEX, anion exchange; CEX, cation exchange; HCP, host cell proteins; ICB, integrated continuous bioprocess; mAb, monoclonal antibodies.