Introduction

Continuous manufacturing processes have gained significant interest from biopharmaceutical companies in recent years due to their potential to provide large productivity increases over traditional batch production modes (Konstantinov & Cooney, 2014; Pollock, 2017; Arnold 2018). These productivity increases allow a company to produce more biopharmaceutical product from an existing manufacturing facility or build new facilities with significantly reduced footprint compared to traditional designs.
Progress on upstream continuous processes has been widely published and presented at industry conferences. Companies have reported titers for perfusion processes reaching 2.3 g/L per day and beyond at scales over 500L (Coffman, 2021). This higher cell culture productivity results in a purification bottleneck for traditional batch purification processes. Perfusion cell culture volumes are typically on the order of 1.5 cell culture vessel volume per day over 30 days and have viable cell densities (VCD) and titers that vary over 10X across this duration. A traditional 4 x 12,000L facility can now be replaced with a 5 x 2,000L facility that outputs the same product mass, but at a cost of over 6X more cell culture volume that must be forward processed (Arnold, 2018). Purification facilities are not equipped with vessels to store the entire batch volume and therefore, at a minimum, must process the material through the purification unit operations in multiple sub-batches.
Continuous purification unit operations have matured in recent years and technologies now allow a full end-to-end continuous process without the need for large hold vessels (Pezzini, 2018). These technologies eliminate the challenge previously presented by large perfusion harvest volumes. In a continuous purification process, each unit operation in the process is connected in series and operated simultaneously. Since the material is continuously forward processed from one step to the next, there is no need to hold large intermediate volumes in tanks. Furthermore, the purification process keeps pace with the perfusion bioreactor and the full batch of drug substance is purified within 12 hours of completion of the cell culture operation (Coolbaugh, 2021).
Productivity improvements gained from continuous purification extend beyond those achieved with perfusion cell culture processes (Klutz, 2015). Regardless of cell culture method, continuous purification offers benefits of reduced equipment size and footprint, reduced capital and consumable costs, and decreased run duration. Facility footprint is significantly reduced by elimination of product hold tanks and the need for utilities to clean and sterilize stainless steel piping systems. Low flow rates allow for smaller piping diameters that can be provided by single use tubing instead of stainless-steel piping. Advanced automation allows processes to operate all steps 24/7 without intervention compared to one step performed per shift, greatly improving utilization of existing facility and personnel resources. Smaller chromatography columns are cycled continuously throughout a run, reaching more than 35 cycles compared to 3 to 5 cycles on a larger column for a traditional batch process. As a result, resin utilization is maximized and resin costs are reduced (Otez, 2020). Although the capture chromatography step sees the most consumable cost reduction in a continuous purification process, labor and time savings for an end-to-end continuous process are notable (Mahal, 2021).
End-to-end continuous purification processes have been published previously by our group and others (Godawat, 2015; Pezzini, 2018, Coolbaugh, 2021). They include processes up to 25 days in duration and a demonstration of bioburden control. Even so, significant opportunities for process maturity exist for certain unit operations, equipment hardware and automation. Here, we present the latest generation of continuous purification processes with an off-the-shelf Pilot PAK continuous purification system (PAK BioSolutions, Virginia, US) that includes single-use GMP flow kits, advanced control strategies, and supervisory control and data acquisition (SCADA).
In this work, we demonstrate a 14-day continuous monoclonal antibody (mAb) purification process for a 50L perfusion bioreactor. Bioburden was controlled within specifications for the duration of the process. A single PAK automated system with four flow kits installed was used to perform the following steps; harvested perfusion material was processed through a Protein A capture chromatography step, a low pH virus inactivation step, a depth filter, a 0.2µm sterile filter and an anion exchange membrane. The process performed did not require a cation exchange chromatography step to meet process impurity specifications. Ultrafiltration/ diafiltration operations were performed separately after breaking down and setting up the same PAK system in a UFDF setup.
As demonstrated in this work, continuous cell culture and purification processes have advanced beyond proof-of-concept studies. The significant advancements in off-the-shelf equipment now enable routine operation of continuous purification processes in pilot and GMP facilities. This equipment reduces footprint requirements for an end-to-end process by 75% and offers a level of automation that reduces hands on time by 90% compared to batch. Furthermore, we present low pH viral clearance data required to implement a continuous purification process for a GMP facility.