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.
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.