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.