Efficient adeno-associated virus serotype 5 capture with affinity functionalized nanofiber adsorbents.

Adeno-associated viruses (AAVs) are one of the most promising tools for gene therapy applications. These vectors are purified using affinity and ion exchange chromatography, typically using packed beds of resin adsorbents. This leads to diffusion and pressure drop limitations that affect process productivity. Due to their high surface area and porosity, electrospun nanofiber adsorbents offer mass transfer and flow rate advantages over conventional chromatographic media. The present work investigated the use of affinity cellulose-based nanofiber adsorbents for adeno-associated virus serotype 5 (AAV5) capture, evaluating dynamic binding capacity, pressure drop, and AAV5 recovery at residence times (RT) less than 5 s. The dynamic binding capacity was found to be residence time-dependent, but nevertheless higher than 1.0 × 1014 TP mL-1 (RT = 1.6 s), with a pressure drop variation of 0.14 MPa obtained after loading more than 2,000 column volumes of clarified AAV5 feedstock. The single affinity chromatography purification step using these new affinity adsorbents resulted in 80% virus recovery, with the removal of impurities comparable to that of bead-based affinity adsorbents. The high binding capacity, virus recovery and reduced pressure drop observed at residence times in the sub-minute range can potentially eliminate the need for prior concentration steps, thereby reducing the overall number of unit operations, process time and costs.

Continuous Affinity Purification of Adeno-Associated Virus Using Periodic Counter-Current Chromatography.

Replacing batch unit operations of biopharmaceuticals by continuous manufacturing is a maturing concept, with periodic counter-current chromatography (PCC) favoured to replace batch chromatography. Continuous affinity capture of adeno-associated virus (AAV) using PCC has the potential to cope with the high doses required for AAV therapies thanks to its inherent high throughput. The implementation of continuous AAV affinity capture using a four-column PCC process is described herein. First, elution buffer screening was used to optimize virus recovery. Second, breakthrough curves were generated and described using a mechanistic model, which was later used to characterize the loading zone of the PCC. The experimental runs achieved a stable cyclic steady state yielding virus recoveries in line with the optimized batch process (>82%), with almost a three-fold improvement in productivity. The PCC affinity capture process developed here can bolster further improvements to process economics and manufacturing footprint, thereby contributing to the integrated continuous manufacturing concept.

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

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, ZCa ; 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.

Design of an integrated continuous downstream process for acid-sensitive monoclonal antibodies based on a calcium-dependent Protein A ligand.

Monoclonal antibodies (mAb) are used as therapeutics and for diagnostics of a variety of diseases, and novel antibodies are continuously being developed to find treatments for new diseases. Therefore, the manufacturing process must accommodate a range of mAb characteristics. Acid-sensitive mAbs can severely compromise product purity and yield in the purification process due to the potential formation of aggregates. To address this problem, we have developed an integrated downstream process for the purification of pH-sensitive mAbs at mild conditions. A calcium-dependent Protein A-based ligand, called ZCa, was used in the capture step in a 3-column periodic counter-current chromatography operation. The binding of ZCa to antibodies is regulated by calcium, meaning that acidic conditions are not needed to break the interaction and elute the antibodies. Further, the virus inactivation was achieved by a solvent/detergent method, where the pH could remain unchanged. The polishing steps included a cation and an anion exchange chromatography step, and screening of the capture and polishing steps was performed to allow for a seamless integration of the process steps. The process was implemented at laboratory scale for 9 days obtaining a high yield, and a consistently pure drug substance, including high reduction values of the host cell protein and DNA concentrations, as well as aggregate levels below the detection limit, which is attributed to the mild conditions used in the process.

Automated pH and conductivity conditioning using feedback control to support a two-step continuous purification process.

As discovery research organizations push more molecules and new modalities through their company pipelines, there comes a need to widen purification development and production bandwidth by increasing automation and throughput. Continuous processing technologies have the unique property of reducing manufacturing floor space and reducing costs. We can speed development and production by implementing automation and continuous process technologies early in discovery research. Here we describe an automated continuous instrument made up of an ÄKTA™ pcc for initial capture by protein A, an ÄKTA pure 150 retrofitted to automatically condition protein A eluate, and a second ÄKTA pure 150 built for flow-through anion exchange chromatography. The continuous instrument we have designed and built recirculates protein A eluate from the ÄKTA pcc in a closed loop while signals from the pH and conductivity meters direct addition of titrant for accurate and precise adjustments to the pH and salt concentration. The instrument is run without user intervention and can be used continuously for production or for development as a tool for screening running conditions on the anion exchange step.

Model-based design and control of a small-scale integrated continuous end-to-end mAb platform.

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.