December 1, 2010 (Vol. 30, No. 21)
Utilization of Disposable Fluid Flowpath Assemblies Can Deliver Optimal Performance
Tangential flow filtration (TFF) is an efficient separation technology that is widely used in the downstream purification of solutions containing molecules such as recombinant proteins (including mAbs), vaccines, or cellular debris. TFF has three primary applications in bioprocessing: clarification by microfiltration, concentration by ultrafiltration, and purification by diafiltration.
Important success criteria for all TFF processes are high product yield, high product quality, and high product purity. These success criteria are achieved, not just by the selection of appropriate filtration devices and operating parameters, but also by the implementation of well-designed process hardware.
Although the product processing time of a TFF step in a typical monoclonal antibody manufacturing operation is not excessive, the additional time required for installation, set-up, flushing, and cleaning of the TFF system and devices make it an ideal step to introduce disposable technology as a means of decreasing overall downstream processing time.
The introduction of single-use flowpath assemblies integrated with TFF membranes provides the opportunity to minimize set-up and cleaning time, decrease chances of cross-contamination from run to run, and capitalize on the opportunity for more flexible and rapid facility startup and change over.
The time and cost advantages of single-use systems over stainless-steel systems are well-documented; however, questions remain as to how the single-use system performs compared to historical processes. This study presents data demonstrating that a pre-engineered system for TFF utilizing completely single-use flowpaths can deliver optimal process performance in terms of yield, quality, and product purity through implementation of low working volume, excellent product recovery, effective mixing, and efficient removal of small molecules.
Mobius® FlexReady Solution for TFF
Millipore’s Mobius FlexReady Solution for TFF combines a process-ready hardware system with Flexware™ single-use assemblies designed for minimal working volume and high recovery of protein at high concentrations. The system is available with either a 10 L retentate recycle container utilizing Pellicon® cassettes of up to 0.5 m2 filtration area (TF-1), or a 50 L retentate recycle container using Pellicon cassettes of up to 2.5 m2 filtration area (TF-2). Pellicon cassettes were selected for their proven accuracy in scale-up and scale-down processes.
For both TF-1 and TF-2, the piping and instrument diagrams of the Flexware assemblies match the flowpaths of traditional stainless steel systems, with tubing diameters and materials of construction selected to enable the required flowrates and pressures of specific applications (Figure 1). The hardware and flowpaths were designed together to optimize layout and minimize excess tubing and connections. For this study, the TF-2 system was characterized over a range of process conditions.
Designing for High Product Yield
An optimized TFF system will ensure that all product can be recovered from the system after processing. One key to achieving high product yield is minimization of system volume, which increases the likelihood of enabling a buffer recirculation step for enhanced product recovery.
In addition, a system with low minimum working volume simplifies product recovery since a larger proportion of the final product pool ends up in the recycle tank and is easily recovered by pumping, and a lower proportion of the pool is distributed through the process lines and membranes. Finally, careful design of the process lines to ensure that they all slope toward a low-point product recovery point will enhance the volumetric recovery from the system.
Testing was performed on the Mobius FlexReady TF-2 system, with a model protein solution at concentrations ranging from 23 to 275 g/L and a starting pool volume of 2.5 L. A four-step product recovery procedure was implemented wherein 1) product was pumped from the tank to the low-point recovery port, 2) air or buffer was introduced at a high point in the process tubing to displace the product remaining in the membranes, membrane holder, and feed line into the product recovery container, 3) a small bolus of air or buffer was introduced at the high point to push any remaining product from the retentate line into the recycle bag, and 4) the feed pump was used again to move any last remaining product from the tank and feed line into the recovery vessel.
As shown in Figure 2, the Mobius FlexReady TF-2 System enables very high protein recovery over the full range of protein concentrations, using the buffer-displacement method. Additionally, this high recovery is achieved with minimal dilution of product (<10%), indicating excellent plug flow of the buffer through the tubing and membranes to the recovery port. Similar results are observed using the air blowdown method, which yields 98–100% recovery of product from solutions at 25–275 g/L without requiring any dilution of the product pool (data not shown).
Maintaining Product Quality
One way to maintain high product quality is by avoiding protein-damaging air-liquid interfaces, particularly during mixing in the recycle tank. In the Mobius FlexReady systems, a recycle bag that includes a vortex breaker enables low working volumes with no air entrainment or vortexing, while the inclusion of a bottom-mounted mixer enhances mixing and a retentate diverter plate inhibits turbulence and short-circuiting of the retentate stream as it re-enters the bag (data not shown). Air-product interaction is further reduced through a sub-surface retentate return to the recycle bag and careful sizing of the feed pump inlet tubing to avoid cavitation.
As shown in Figure 3, the Mobius FlexReady TF-2 system can operate efficiently over a wide range of volumes (0.5–40 L) at a range of mixing speeds (100–700 RPM) without generating turbulence, with larger volumes allowing higher RPM before turbulence is seen.
Optimizing Product Purity
Product purity is optimized through effective washing out of contaminants during diafiltration, as well as during any sanitization and flushing steps. The Mobius FlexReady TF-2 system was designed to minimize deadlegs and contamination points through the use of novel low dead volume T’s in the recycle path, and through avoidance of flex lines that are longer than necessary. As shown in Figure 4, this results in a system that can achieve >4 log clearance of small molecules as modeled by a 12-diavolume buffer exchange of 150 g/L NaCL solution against water using a 2.5 m2 Pellicon 2 30 kD cassette.
Implementation of process-ready hardware systems containing single-use processing assemblies permits rapid facility start-up as well as ongoing operational flexibility. This study presents data demonstrating that a preengineered system for TFF can deliver optimal process performance in terms of yield, quality, and product purity through implementation of low working volume, excellent product recovery, effective mixing, and efficient removal of small molecules.
The Mobius FlexReady TF-2 system enabled greater than 98% product recovery from even the most concentrated model protein feedstock tested, while minimizing turbulence during mixing and recirculation to maintain high product quality. Greater than 4 logs clearance of small molecules during buffer exchange delivers performance that is equivalent to traditional stainless steel TFF systems.
The data demonstrates that pre-engineered systems combining process-ready hardware systems with single-use, application-specific fluid flowpath assemblies are an effective means of realizing the full benefits of single-use technology.
Elizabeth Goodrich ([email protected]) is applications engineering manager at EMD Millipore.