Results and Discussion
This study successfully demonstrated a next-generation continuous
purification process, running for 14-days at a rate of 50L of cell
culture harvest material per day. Bioburden control was maintained for
the duration of the run and product quality results were within
acceptable limits. Advanced automation enabled tight control of the
process to meet specifications. Automation control strategies also
enabled break vessel volumes between 150 ml and 10L (20-fold or greater
reduction compared to batch). Facility footprint of the process was
reduced by 75% compared to the batch process, primarily due to the
reduction in vessel volumes and ability to perform all three process
steps in a single system. Setup time was to 1.5 days and was followed by
14 days of hands-free operation with the exception of daily sampling
(90% reduction in operator hours). These outcomes demonstrate the
robustness of this commercially available automation system for
continuous purification processes.
Bioburden Control
Most downstream operations for monoclonal antibody are not considered
aseptic and operate as low-bioburden. The guidance commonly used sets a
limit at 10 colony-forming units (CFU) per mL.
Bioburden control was demonstrated for the duration of the 14-day run.
Samples were taken daily from the harvest break vessel, protein A
product vessel, VI neutralized product vessel and AEX final product.
None of the sampling points tested positive for bioburden except, on day
7, when 1CFU/mL was observed for the protein A product vessel (below the
acceptable criteria of <10CFU/mL). This hit was attributed to
intentionally opening the protein A product vessel to fix an incorrectly
installed tubing assembly on the vessel. The bioburden objectives for
this process were clearly met. The 1CFU/mL sample from the protein A
elution product vessel was cleared by implementing a bioburden
mitigation strategy. This strategy consisted of six important steps: (1)
pausing the protein A step, (2) removing the contaminated vessel from
the system, (3) autoclaving a new vessel, (4) filtering material into
the new vessel under aseptic conditions, (5) integrating the new vessel
into the process and (6) flushing lines with sanitization buffer before
restarting the protein A step. No bioburden was detected after the
implementation of this strategy. Successful implementation of this
mitigation strategy demonstrates the ability to clear bioburden from an
ongoing process without significant process disruption, a necessity to
save a batch when operating a continuous operation.
Capture
Seventy protein A capture chromatography cycles were successfully run
during the 14-day process. The protein A capture step began
automatically once the harvest vessel reached a setpoint volume of 2L.
The capture step operated for the duration of the run.
The capture step was operated under a constant mass load principle,
targeting the resin capacity of 60g of mAb per L of resin: the load
volume onto the column changed as the titer varied throughout the run to
ensure that the grams of mAb loaded was the same for each cycle, as
illustrated in Figure 4. Load volume decreases as titers increase until
the middle of the run. Load volume then increases again as the titer
drops towards the end of the run. This strategy ensures a constant
concentration downstream of the protein A capture step, which greatly
simplifies the downstream process dynamics. The standard deviation for
the Protein A concentration over the duration of the run was 1.4 g/L.
With constant downstream concentration, the virus inactivation step does
not need to account for large mAb concentration changes that can affect
the titration profile. Additionally, polishing chromatography step
throughput can be calculated from volumetric throughput and the known
concentration. The capture step was triggered to stop once the harvest
vessel dropped to a low level due to lack of flow from the cell culture
feed.