October 1, 2011 (Vol. 31, No. 17)

Regine Eibl, Ph.D.
Dieter Eibl, Ph.D. Ph.D. Zurich University of Applied Sciences, School of Life Sciences and Facility Management, Institute of Chemistry and Biotechnology

Tracking the Evolution and Development of an Increasingly Used Technology

Mammalian cells are most commonly used for the production of biopharmaceuticals. Over the past 25 years the focus has mainly been on protein-based therapeutic hormones, enzymes, antibodies, and vaccines.

Early mammalian cell culture systems have been increasingly replaced by high-productivity CHO and human-designed cell lines. The majority grow as suspended cells in serum-free, protein-free, or even chemically defined culture media and currently deliver up to 10 times higher product titers. The result has been a shrinkage in both batch sizes and bioreactor volumes during upstream processing.

As a result of a growing understanding of mammalian cellular processes at the molecular level, the number of potential product candidates for clinical research has been rising steadily.


Regine Eibl, Ph.D.

Thus, the ability to utilize a range of different bioprocesses at varying scales has become necessary. In addition, the rising cost of healthcare has led to the need for shortened development and production times for biotherapeutics and to increased production capacities. Use of single-use systems, whose beginnings date back nearly 60 years to transfusion medicine, have made these advances possible.


Dieter Eibl, Ph.D.

Slow Beginnings

In 1953 Fenwal’s invention of the plastic blood bag paved the way for single-use technology. For routine work in cell culture labs, plastic flasks, dishes, and 96-well plates (which have been commercially available since the 1960s) have increasingly replaced their counterparts made of glass. This changeover has contributed to a reduction in cleaning and sterilization times as well as to lower contamination rates.

Another important milestone in the history of single-use, the development of the first hollow-fiber reactor, was achieved by Knazek and his team in the early 1970s.

By carrying out perfusion culture operations with hollow-fiber membranes bundled in a single-use cartridge, mammalian cells can grow to high cell densities in in vivo like conditions. This ability has revolutionized the in vitro production of small amounts of hybridoma-derived antibodies.

In the mid ’70s, Nunc and Bioferon (now part of Rentschler Biotechnologie) started to manufacture cell factories from polystyrene. In the 1990s these multitray systems were shown to be capable of producing bioproducts (e.g., vaccines) with anchorage-dependent cells (while replacing roller bottles in GMP production).


Platform products that bundle fundamental process steps and are based on standardized single-use modules are increasingly popular.

Single-Use Bioreactors

The 1990s and 2000s saw a great increase in single-use technology for upstream production. In addition to the first single-use bags for storage and transport of buffers and media from HyClone (today a unit of Thermo Fisher Scientific), two-compartment membrane bioreactors and the first wave-mixed bag bioreactor system for mammalian cell cultures, the Wave Bioreactor 20, entered the market. The product was developed by Wave Biotech U.S, which was later acquired by GE Healthcare, and Wave Biotech Switzerland, which is now part of Sartorius Stedim Biotech.

Despite their lack of in-line sensors, single-use bioreactors prevailed in cell-expansion projects, screening experiments, and preclinical sample production at lab scale. Studies documented the advantages of wave-induced motion, which was initially viewed with skepticism, in surface-aerated bags manufactured from multilayer films. Positives included lower shear stress acting on cells, the opportunity for direct inoculation with cells pooled from T flasks (omitting intermediate cultivation steps), removal of need to add antifoam, and cost savings of 30–50%.

The Wave Bioreactor 20 and its successful application beyond cell expansion was a driver for the rapid further development of single-use technology in the 2000s.

In addition to aseptic connection devices, new sampling and transfer systems, single-use membrane filters and bioprocess containers, as well as stirred single-use mixers (e.g., LevTech’s Magnetic Mixer), have found their way into the development and commercial manufacture of small- and mid-volume protein therapeutics.

Since 2005 stirred single-use bioreactors have been available from HyClone and Xcellerex (XDR™ Disposable Stirred Tank Bioreactor) in addition to further wave-mixed systems (Tsunami Bioreactor and the AppliFlex from Applikon) for mammalian cells. In addition, all single-use bioreactors operating with bags have been available with optical single-use or standard sensors for in-process control of pH and DO.

Second-Generation Products

Between 2006 and 2008 the second generation of wave-mixed single-use bioreactors entered the market. They established a new gold standard for seed inoculum and seed-train production in mammalian cell semi-commercial and commercial production processes.

Nevertheless, stirred bag bioreactors (already available up to 2 m3 culture volume in 2008) remain popular. To this group also belongs ATMI Life Science’s Nucleo Bioreactor, which has a cubical bag shape and a tumbling impeller, and is used in vaccine and antibody productions.

Available engineering and biological data indicate that comparable product quantities and quality are achievable with single-use wave-mixed and stirred-bag bioreactors. In addition, a number of studies demonstrate that leachables and extractables have more impact downstream than upstream.

During the mid stages of single-use systems development, users showed interest in scalable stirred-bag bioreactors and in stirred single-use benchtop systems. The challenge was met with the launch of the Biostat® CultiBag STR and the UniVessel SU® (Sartorius Stedim Biotech), as well as the Mobius® CellReady (EMD Millipore) and CelliGEN® BLU (New Brunswick Scientific). Instead of polypropylene bags, the benchtop systems came with rigid polycarbonate vessels.

The finding that mammalian cells are more sensitive to orbital shaking than previously assumed gained further recognition as this technology was developed for mL-range and scale-up applications. Orbital shaking single-use bioreactors, e.g., M24 microbioreactor (MicroReactor Technologies), BioLector (m2p-labs), the Current Bioreactor (AmProtein), and the OrbShake Bioreactor (Sartorius Stedim Biotech, Kühner), currently form the largest group behind stirred and wave-mixed single-use bioreactors.


Single-use systems originated nearly 60 years ago in transfusion medicine.

Downstream Bottlenecks

The increase in single-use technology for upstream processing paralleled the development of single-use systems for downstream processing and fill/finish operations as well. The first centrifuge (Sorvall’s Centritech Cell II), different pumps (peristaltic, syringe, and diaphragm), isolators, a freeze-thaw system (Sartorius Stedim Biotech’s Celsius®-Pak), mixers up to 5,000 L in culture volume, and filling systems are relatively recent single-use additions.

Moreover, new single-use systems for large-scale purification and polishing have been introduced. These include scalable tangential flow and depth filtration systems from different suppliers (e.g., EMD Millipore, SciLog, TangenX, GE Healthcare, and 3M Purification) as well as pre-packed chromatography columns.

Single-use systems for downstream processing have not yet reached the same level of importance as those for upstream work. In downstream processing, chromatography is particularly problematic—there being a demand for larger chromatography columns due to enhanced protein loading. To date, 20 L single-use columns remain the maximum size for affinity purification.

Furthermore, to improve downstream processing efficiency, attempts have been made to increase the number of chromatography cycles (simulated moving bed chromatography) and to use single-use membrane adsorbers, e.g., Sartobind (Sartorium Stedim Biotech), ChromaSorb (Millipore).

Consequently, it comes as no surprise that hybrid bioprocessing facilities are commonly used. There are no single-use facilities completely equipped with single-use systems at large scale.

Platform concepts that bundle fundamental process steps and are based on standardized single-use modules—FlexAct® (Sartorius Stedim Biotech), Mobius® (EMD Millipore), ReadyToProcess™ (GE Healthcare), and FlexFactory (Xcellerex)—are gaining favor.

Next Ten Years

Single-use technology for the production of protein therapeutics is not expected to continue to grow as rapidly. Standardization activities, however, for single-use systems should be carried out and efforts made to overcome the existing limitations (pressure, flow rates, mixing, temperature control, in-process monitoring, oxygen/CO2 stripping rates, leachables/extractables).

Sensors for relevant process parameters as well as improved equipment for depth-, ultra-, and diafiltration, chromatography, and filling at large scale are already under way.

If all these changes are implemented, we will be a step closer to a complete single-use production facility and to the concept of a “single-use factory in a box.”

The extent to which 4 or 5 m3 single-use bioreactors are necessary remains questionable. For most new protein therapeutics (vaccines, personalized antibodies) 1 and 2 m3 sizes are sufficient. The difficulty lies not in the development of a 4 or 5 m3 single-use bioreactor but in its handling and storage.

New-generation biotherapeutics (based on autologous or allogeneic human stem cells or T cells) will largely determine the further development of single-use technology. About 40 cell-therapy products are currently on the market and more than 200 are at the clinical-research stage. For the manufacture of cell-therapy products to be commercially successful, innovative equipment and new technologies are required.

Due to the production demands of cell therapeutics, single-use systems are essential. The manufacture of cell therapeutics is focused on the generation of bioactive cells that are directly infused into a patient. For cell-based therapeutics it is assumed that culture volumes in upstream processing are smaller (0.01 to 1 m3) than those for protein-based products.

GMP-compliant platform solutions with single-use bioreactors that allow efficient expansion and/or differentiation of adherent and suspension cells are also needed.

Open centrifugation tubes and blood-processing equipment, which have to date been used in already certified processes, are not suitable for the downstream processing of large amounts of cell culture broth. Solutions, which include automation of the filling process and cryopreservation at large scale, are required.

Finally, single-use systems for nonmammalian cell applications (insect, microbial, plant cell-derived) to generate niche products are on the horizon. Single-use systems are also expected to become important in areas with high cleaning and safety demands in industrial biotechnology.

Regine Eibl, Ph.D. ([email protected]), and Dieter Eibl, Ph.D. ([email protected]), are lecturers in cell cultivation techniques and biochemical engineering at the Zurich University of Applied Sciences in Switzerland.

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