May 1, 2016 (Vol. 36, No. 9)

Smaller, modern multiproduct biopharmaceutical production facilities require greater flexibility and higher throughput at lower cost, so many manufacturers are responding by implementing continuous processing and single­-use technologies. Continuous upstream perfusion processes can, however, create downstream bottlenecks in tank capacity for manufacturing plants designed for fed­batch systems or with limited footprints.

Concentration using tangential flow filtration (TFF) helps to some extent but, because it involves recirculation of the product solution, it can be damaging to sensitive biotherapeutics. The significant limitations of conventional TFF can be overcome with new, in­line single­-pass TFF (SPTFF) disposable technology from Pall Life Sciences, and can be operated virtually anywhere within a given bioprocess.

Benefits of Continuous Processing with SPTFF

Transitioning from batch to continuous bioprocessing delivers process time savings and lower capital costs via smaller equipment, tanks and tubing sizes. In addition, some hold­-tanks between unit operations may be eliminated, and reduced system holdup volumes also improve product recoveries, which is particularly valuable for smaller footprint facilities.

SPTFF technology with inline concentration (ILC) from Pall eliminates the conventional TFF recirculation loop with a staged flow­path design, allowing concentrations of 2–30× to be achieved continuously in a single pass. The single­-pass pump reduces minimum working volume concerns and, when optimized, is ideal for the processing of fragile biomolecules, delivering:

  • Greater flow ratio control
  • 5–10× lower feed rate
  • 2–3× smaller lines sizes
  • Lower working volumes
  • Lower shear exposure
  • Product recoveries typically 99%+
  • No mixing or foaming issues
  • Easy coupling throughout the bioprocess

Pall has demonstrated that in­line concentration with SPTFF is a reliable, scalable technology suitable for R&D, process development, and pilot and production scales. In this context, SPTFF technology is not only an important addition to the process development toolbox for platform process evaluation, but also a crucial enabler of integrated, streamlined and continuous bioprocessing initiatives.

Simplifying Bioprocessing with SPTFF

A leading global biologics manufacturer saw ILC with disposable SPTFF technology as a potential solution for overcoming facility constraints and increasing the flexibility and throughput of operations. The company evaluated the performance of ILC modules in key downstream processing steps to determine its feasibility over a wide range of feed streams and at varying concentrations and process temperatures. Module sizing was also evaluated for process optimization at commercial scale.

Perfusion Recovery
Perfusion culture processes create downstream bottlenecks in tank capacity for fed­batch manufacturing processes. However, ILC of clarified culture fluid (CCF) allows the perfused volume to be collected in an existing tank and batch processed downstream without facility modifications. Processing at the reactor temperature (37°C) increased the convective transport and overall throughput by 30% volumetric concentration factors (VCF = 3.2×) compared with the results obtained under ambient conditions (21°C). The VCF was then further increased by applying additional backpressure to the ILC module.

Overall, it was determined that currently available ILC modules can process up to 2,000 L bioreactor volumes operated in continuous mode and provide a streamlined solution to facility fit constraints.

Pre­Capture Chromatography
For mAb processes, protein A chromatography is commonly used as a capture step. Due to the high cost of this resin, it is desirable to reduce the packed bed volume (PBV) and perform multiple cycles, thus exchanging raw material cost for operational cost. However, the lower volumes and higher titers obtained following ILC via SPTFF lead to shorter load times, which reduces both raw material and operational costs.

In one case, Pall’s customer determined that, after ILC, the increase in total processing time due to the use of a reduced column volume was shifted by as much as a factor of two. Overall, the PBV was decreased from 57 L (60 cm) to 31 L (45 cm), saving $416,000 in raw material costs. In addition, a 6× concentrated CCF loaded onto a protein A capture column exhibited no difference in eluate CHOP, DNA, percent yield or MAb purity compared with the control.

In-­Process Volume Reduction
Purification of biopharmaceuticals consists of multiple chromatography steps combined with additional filtration and treatment steps to remove and inactivate viruses. Linkage of these steps occasionally requires adjustments or dilutions that can pose facility fit constraints. The use of ILC can decrease pool volumes to enable increased disposables usage and to provide options for increasing the operability range of manufacturing plants.

Studies by the global biologics producer revealed that the feed concentration impacts the VCF and throughput obtained at a given operating pressure. In addition, the reproducibility and responsiveness of SPTFF systems to changing operating conditions were confirmed.

When used to concentrate a chromatography column effluent, it was found that less ILC area was needed, but that back­pressure from the ILC may impact column operation. When used prior to membrane chromatography, ILC decreased both the processing time and pool volume.

Ultrafiltration/Diafiltration (UF/DF)

UF/DF system design is critical for multiproduct facility fit. However, because a wide range of process titers and drug substance (DS) concentrations are processed in such a facility, it is difficult to design a universal UF/DF system.

As a result, some facilities employ two UF/DF systems (UF2) to achieve high DS concentrations (>150 g/L), which increases their costs and the process complexity. Pall’s customer found that replacing a UF2 system with an SPTFF ILC system reduced the required capital investment while providing lower holdup volumes and higher recoveries with reduced overconcentration targets and minimal pump passes. It was also determined that currently available ILC modules are suitable for processing 12,000 L reactor volumes up to 2.7 g/L titer in three hours.

Studies revealed that the use of SPTFF for ILC of biopharmaceutical solutions during commercial­scale downstream processing delivers:

  • 3–8× VCF for a wide range of feed streams and concentrations;
  • No difference in product quality (mAb purity, enzyme activity) under various conditions employed;
  • Support for perfusion process flows of 1–2 vessel volumes per day for reactor volumes from 100 L to 2,000 L;
  • The potential to save significant cost while maintaining acceptable processing times;
  • Improved chromatographic selectivity through the use of shallow gradients; and
  • a replacement for UF/DF, which can alleviate the working volume constraints of conventional systems to decrease overconcentration minimums, increase recoveries, and improve the operating ranges of existing systems.

These results confirm that SPTFF systems can enable biopharmaceutical manufacturers to achieve greater flexibility and higher throughput at lower cost, facilitating continuous bioprocessing and maximizing the operational use of facilities with smaller footprints.

Pall Life Sciences

Engin Ayturk, Ph.D. 
Senior R&D Manager 
Biopharm Applications
[email protected]
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