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1 Press Release Presse-Information Information de presse Contact/Kontakt Dr. Kathrin Rübberdt Tel (0) 69 / Fax ++49 (0) 69 / presse@dechema.de May 2015 Trend Report No 13: Single-Use (Disposable) Systems in Biopharmaceutical Processing: Quo Vadis? Single-use systems (SUS) are now used in the majority of biopharmaceutical processes involving animal cell cultures. Single-use filters, plastic storage bags, single-use mixers and single-use bioreactors for upstream processing (USP) in preclinical and clinical sample production are the items most commonly used. However the biopharmaceutical industry is also making greater use of SUS for downstream processing (DSP), formulation and filling. Stirred-tank single-use bioreactors for microorganisms are one of the current development priorities. There is also a need for single-use equipment to process stem cells and T-cells. Single-use (disposable) systems are only intended to be used once. All parts that come into contact with the product are made of Food and Drug Administration (FDA) approved plastics such as polyethylene or polycarbonate. The systems are normally beta or gamma sterilized by the manufacturer so that they are ready to use. Acceptance of SUS in biopharmaceutical production has greatly increased over the past 15 years. This is particularly the case for all processes which involve animal cell cultures where there is a need for rapid development and efficient production of low to medium volume products such as therapeutic antibodies, hormones, enzymes and vaccines. It is probably safe to assume that SUS is used in 2/3 of new process designs for these applications. The trend can be explained by increased product titers (and the resulting decrease in the size of production bioreactors down to 1 m 3 or 2 m 3 ) as well as by the advantages of SUS compared to re-useable equipment made of glass or steel. Besides faster production throughput (elimination of the sterilization and cleaning procedures), increased flexibility (faster product changeover) and improved safety (minimum risk of product cross- 1

2 contamination) are also cited as reasons. In addition, SUS reduces the upfront investment cost of a production system by around 40%, and it takes less time to install the system and get it up and running. Single-use equipment is ubiquitous in production of pre-clinical and clinical samples, but it is used to a lesser extent in commercial biopharmaceutical production. Limitations affecting the single-use systems which are currently available on the market is cited as the reason why this is the case. Leachables and Extractables (L&E) are the main weakness of SUS Leachables and Extractables (L&E) are chemical substances which migrate from plastic under process conditions or in worst case scenarios and which degrade the product. The lack of procedures for standardized tests to detect L&E along with the absence of appropriate analysis protocols is the biggest weakness of SUS. The identification of bis(2,4- di-tert-butylphenyl)phosphate (bdtbpp) as a cytotoxic leachable for various Chinese Hamster Ovary (CHO) cell lines has not changed that. The results of investigations conducted by a team of Amgen specialists under the direction of Matthew Hammond did, however, accelerate development of more robust, optimized films (e.g. Aegis5-14 films from ThermoFisher Scientific or Flexsafe S80 from Sartorius Stedim Biotech) for storage, mixer and bioreactor bags. Recent studies based on a DECHEMA recommendation showed that the risk of cell growth inhibition can be excluded for Flexsafe S80 if the concentration of the antioxidant Irgafors 168 is minimized, and that applies not only to CHO cells. In addition to DECHEMA which is developing recommendations and guidelines for standardization of SUS in the German-speaking region based on the efforts of its Single-Use Technology in Biopharmaceutical Production Working Group, the organizations listed in Fig. 1 are also active. Besides the L&E problem, issues relating to integrity, supplier qualification, material and component exchangeability and process characterization of single-use bioreactors are also being addressed. 2

3 Figure 1: Organizations which support SUS implementation Single-use process platforms, complete single-use USP lines and initial single-use production plants Nevertheless, the advantages of SUS outweigh the disadvantages listed above if the right equipment is selected and it is used properly. Single-use systems now exist for all USP and DSP process steps. Suitable single-use equipment (mixers, transfer systems, dosing systems, filling needles, etc.) is even available for formulation and filling. Complete singleuse production plants such as WuXi PharmaTech in Shanghai are, however, still the exception (approx. 10%). Hybrid production plants (approx. 75%) are the dominant form. Single-use filters, plastic storage bags, single-use mixers and single-use bioreactors are used together with equipment made of stainless steel. Process platforms (e.g. for media production, fermentation, biomass separation, virus separation and inactivation, formulation and filling) designed by the SUS market leaders (GE Healthcare, Pall Life Sciences, Merck MilliPore, Sartorius Stedim Biotech and ThermoScientific) are widely used at these plants. The fact that this application space remains a bottleneck for volumes > 500 L (single-use centrifugation limitations, single-use protein A chromatography, lack of sensor systems) despite the progress that has been made in single-use DSP explains the trend towards production facilities with USP which is completely SUS based (see Fig. 2). A prime example of this approach is Shire s manufacturing facility with a 2000 L bioreactor in Lexington, Massachusetts. Velaglucerase Alfa, which has been approved by the European Medicines Agency (EMA) and the FDA to treat Gaucher disease, is the first commercial 3

4 biopharmaceutical to be produced at a production facility which has a complete single-use USP line. However without scalable single-use biotechnology and the associated peripheral equipment, this type of manufacturing facility would not exist. Figure 2: SUS for USP (with the kind permission of Sartorius Stedim Biotech AG) New generation of single-use bioreactors Table 1 contains a list of commercially available single-use bioreactors ranging from benchtop to cubic meter scale. The list is not intended to be exhaustive. Automated singleuse mini bioreactors for milliliter-scale cultivation such as the ambr15 and ambr250 (Sartorius Stedim Biotech, formerly TAP Systems), the BioBLU 0.3 systems (Eppendorf), the Pall Micro-24 bioreactor system (Pall Life Sciences), the BioLector (mp2-labs), the series H ZRP bioreactors from Zellwerk and the PBS Biotech MagDrive mini bioreactors were not included. Single-use bioreactors used for tissue engineering were omitted as well. Bioreactors from a variety of suppliers are listed. They can be differentiated by energy input mechanism, size, cultivation container construction material, sensors and control unit. They are used both for cultivation of cells which grow in suspension and for adherent cells. Singleuse bioreactors with mechanical or pneumatic drive are suitable for suspension cells. Adherent cells on the other hand are cultivated either on micro carriers in the same single- 4

5 use bioreactors, in rotating bed bioreactors with mechanical drive or single-use hollow fiber bioreactors with hydraulic drive or in parallel plate bioreactors and fixed bed bioreactors. Most users prefer stirred-tank or wave single-use bioreactors with mechanical drive. Wave reactors are used (with some exceptions) primarily for cell expansion, whereas bioreactions normally take place in stirred-tank bioreactors. Over the past three years, a number of wave and stirred-tank single-use reactors have been redesigned to simplify handling and expand the application horizon beyond animal cell cultures (see Table 1). The development of stirred-tank single-use reactors for microbial processing is worth mentioning. The sensors, controllers and monitoring & control software used on single-use bioreactors have been revised and upgraded, guaranteeing better data acquisition and process monitoring. Development work continues on single-use bioreactors and a number of mergers have taken place (e.g. Xcellerex/GE Healthcare, Eppendorf/New Brunswick/DASGIP and ATMI/Pall Life Sciences), so some of the bioreactor names have changed. 5

6 Table 1: List of commercially available instrumented single-use bioreactors (liter-scale) Reactor Category Reactor Name Working Volume [L] Used successfully 1 for cultivation of Manufacturer mechanical drive, wave action AppliFlex animal and plant cell cultures, algae 2 Applikon CELL-tainer (20 L, 200 L) microorganisms Cell tainer Biotech animal cell cultures CellTumbler (various Systems 50) animal cell cultures CerCell SmartRocker Bioreactor animal and plant cell cultures Finesse Wave Bioreactor System (System 20/50 and System 500/1000) animal and plant cell cultures GE Healthcare ReadyToProcess WAVE Xuri Cellbag bioreactor (System W5 and W25) human cells (e.g. cells and stem cells) Allegro XRS animal cell cultures, microorganisms and T-cells Pall Life Sciences BIOSTAT RM bioreactor (System 20/50 and System 200) animal and plant cell cultures, algae 2 and microorganisms Sartorius Stedim Biotech n.a. not available, 1 application notes and/or papers exist, 2 version with LED illumination, 3 announced, 4 cubic plastic bag, 5 paddle stirrer, 6 Fibra- Cel carrier, 7 polyester microfibers, 8 working volume fixed bed 6

7 Table 1 continued Reactor Category Reactor Name Working Volume [L] Used successfully 1 for cultivation of Manufacturer mechanical drive, stirred-tank, rigid tank CellVessel 21 and 23 Series animal cell cultures CerCell CellVessel 21 and 23 Series microorganisms BactoVessel 25 Series 2-75 CelliGen BLU benchtop stirred-tank reactor (BioBLU 1c, 5c, 14c, 50c) CelliGen BLU benchtop stirred-tank reactor (BioBLU 1f) DASGIP Parallel Bioreactor (BioBlock with BioBLU 1c) DASGIP Parallel Bioreactor (BioBlock with BioBLU 1f) Mobius CellReady 3 L bioreactor animal cell cultures and human cells (stem cells) microorganisms animal cell cultures and human cells (stem cells) microorganisms animal cell cultures and human cells (stem cells) Eppendorf Merck Millipore UniVessel SU animal and plant cell cultures, human cells (stem cells) and microorganisms Sartorius Stedim Biotech SmartVessel 3 single-use bioreactor animal and plant cell cultures Finesse n.a. not available, 1 application notes and/or papers exist, 2 version with LED illumination, 3 announced, 4 cubic plastic bag, 5 paddle, 6 Fibra-Cel carrier, 7 polyester microfibers, 8 working volume fixed bed 7

8 Table 1 continued Reactor Category Reactor Name Working Volume [L] Used successfully 1 for cultivation of Manufacturer mechanical drive, stirred-tank, plastic bag Xcellerex XDR (10 L, 50 L, 200 L, 500 L, 1000 L, 2000 L) Xcellerex XDR-50 MO Fermentor Mobius CellReady (50 L, 200 L, 1000 L 3, 2000 L 3 ) Allegro STR (200 L, 1000 L 3, 2000 L 3 ) PadReactor (25 L, 50 L, 125 L, 250 L, 600 L, 1200 L) 4, animal cell cultures GE Healthcare microorganisms animal cell cultures Merck Millipore animal cell cultures Pall Life Sciences animal cell cultures BIOSTAT STR (50 L, 200 L, 500 L, 1000 L, 2000 L 3 ) animal cell cultures, human cells (stem cells) and microorganisms Sartorius Stedim Biotech HyPerforma S.U.B (50 L, 100 L, 250 L, 500 L, 1000 L, 2000 L) HyPerforma S.U.F (30 L, 300 L) animal and plant cell cultures ThermoScientific microorganisms n.a. not available, 1 application notes and/or papers exist, 2 version with LED illumination, 3 announced, 4 cubic plastic bag, 5 paddle, 6 Fibra-Cel carrier, 7 polyester microfibers, 8 working volume fixed bed 8

9 Table 1 continued Reactor Category Reactor Name Working Volume [L] Used successfully 1 for cultivation of Manufacturer mechanical drive, orbital shaken OrbShake bioreactor (SB- 10X, SB-50X, SB-200X, SB-2500X) animal and plant cell cultures, microorganisms Adolf Kühner mechanical drive, rotating Current Bioreactor (5 L, 50 L, 150 L, 300 L) Vertical Wheel bioreactor MagDrive (PBS3 MAG, PBS15 MAG ) animal cell cultures Hangzhou Amprotein Bioengineering human cells (stem cells) PBS Biotech pneumatic drive CEllMaker Felix 3 n.a. animal cell cultures Cellexus CEllMaker Regular and CEllMaker Plus (8 L, 50 L) Vertical Wheel bioreactor AirDrive (PBS 3 Air, PBS 15 AIR, PBS 80 AIR, PBS 500 AIR) 1-50 microorganisms and algae animal cell cultures PBS Biotech hydraulic drive, hollow fiber bioreactor Quantum Cell Expansion absolute cell culture surface area: 2.1 m 2 adherent animal cell cultures and human cells (stem cells and T-cells) Terumo BCT n.a. not available, 1 application notes and/or papers exist, 2 version with LED illumination, 3 announced, 4 cubic plastic bag, 5 paddle, 6 Fibra-Cel carrier, 7 polyester microfibers, 8 working volume fixed bed 9

10 Table 1 continued Reactor Category Reactor Name Working Volume [L] Used successfully 1 for cultivation of Manufacturer hydraulic drive, parallel plate bioreactor Xpansion Multiplate Bioreactor ( plates) absolute cell culture surface area: m 2 animal cell cultures and human cells (stem cells) Pall Life Sciences hydraulic drive, fixed bed bioreactor Celligen BLU fixed bed adherent animal cell cultures and bioreactor (5 L, BioBLU 5p) 6 human cells (stem cells) Eppendorf icellis 7 (nano, 500) animal cell cultures and human cells (stem cells) Pall Life Sciences n.a. not available, 1 application notes and/or papers exist, 2 version with LED illumination, 3 announced, 4 cubic plastic bag, 5 paddle, 6 Fibra-Cel carrier, 7 polyester microfibers, 8 working volume fixed bed 10

11 Few new single-use bioreactors have been introduced recently, with Pall s Allegro STR 200 and the Vertical Wheel MagDrive bioreactors from PBS Biotech being two of the exceptions. A number of process developers and Contract Manufacturing Organizations (CMOs) see tangential flow filtration (TFF) as an effective tool for increasing the time-space yield of single-use bioreactors used for cultivation of animal cells or for overcoming the problem of limited scalability. They use stirred-tank and wave single-use bioreactors in combination with crossflow microfiltration systems such as the re-usable Refine Alternating Tangential Flow (ATF) module (Refine's biotechnology business is now part of Repligen) or single-use hollow fiber module systems from other suppliers (e.g. GE Healthcare, Spectrum Laboratories). With these types of equipment configurations, producers can run continuous perfusion, repeated fed batch, concentrated fed batch and concentrated perfusion processes supported by new single-use peripherals such as connectors, membrane valves and pumps (see Fig. 3). Figure 3: Levitronix PuraLev maglev single-use pump series with controller (with kind permission of Levitronix AG) 11 / 14 DECHEMA Gesellschaft für Chemische Technik und Biotechnologie e.v. Theodor-Heuss-Allee Frankfurt am Main Germany T + 49(0) F +49(0) presse@dechema.de

12 New DSP process designs: clarification, capture, polishing However the high cell densities (in the region of 10 8 cells per milliliter) and tenfold increase in product titer place increasing demands on DSP. This also creates opportunities to introduce innovative DSP process designs which minimize cost. Single-use body feed filtration modules such as the Sartoclear Dynamics from Sartorius Stedim Biotech (diatomaceous earth filtration) or the FILTROX FILTRODISC BIO SD modules can be used for efficient production-scale clarification of culture broth and they are a viable alternative to single-use centrifugation. 3M used the clarification steps as the starting point for development of the 3M Emphaze AEX Hybrid Purifier (a multifunctional single-use purifier) to reduce the level of contamination that needs to be removed in the expensive downstream chromatographic purification steps. Some users are already using continuous DSP processing. ASI recently introduced a single-use heat exchanger for these applications. In addition to fully-automated TFF modules, the leading SUS suppliers also offer TFF modules for diafiltration and virus filtration as well as single-use chromatography systems and various size membrane adsorbers. However, some biopharmaceutical producers and CMOs prefer pre-packaged disposable chromatographic systems which can be used multiple times. To mention a couple of examples, the Repligen Open Platform Specified (OPUS) columns come prepackaged with resin. They are used to clarify culture broths from single-use bioreactors ranging in size between 1 m 3 and 2 m 3. BIA Separations supplies chromatography columns made of monolithic materials. Summary and outlook Single-use systems are an accepted technology in all areas of biopharmaceutical processing which involve animal cell cultures (Fig. 4). Researchers are making a concerted effort to overcome the existing limitations (standardization, enhanced sensor technology, improved automation solutions and single-use chromatography on a large scale). Market analyses indicate that SUS will continue to grow on a sustained basis by at least 15% a year. Increased use for commercial production and microbial processing is also expected. Besides new construction of flexible, modular facilities for the production of biotherapeutics and biosimilars, re-usable equipment at existing plants is being replaced by SUS. 12 / 14 DECHEMA Gesellschaft für Chemische Technik und Biotechnologie e.v. Theodor-Heuss-Allee Frankfurt am Main Germany T + 49(0) F +49(0) presse@dechema.de

13 Based on the increasing level of success in cell, gene and immunotherapeutic applications, there will be greater demand for single-use equipment to process stem and T-cells (cell expansion, differentiation, harvesting and purification). It is not clear whether single-use equipment developed for permanent cell lines can be used in particular for induced pluripotent stem cells which are very sensitive to shear stress. Figure 4: Biopharmaceutical products which are potential candidates for SUS 13 / 14 DECHEMA Gesellschaft für Chemische Technik und Biotechnologie e.v. Theodor-Heuss-Allee Frankfurt am Main Germany T + 49(0) F +49(0) presse@dechema.de

14 Further reading (1) 6th Annual Survey of the Single-use Bioprocessing Market. Aspen Brook (2014). (2) 11th Annual Report and Survey of Biopharmaceutical Manufacturing Capacity and Production. BioPlan Associates (2014). (3) Technical Report No. 66. Application of Single-Use Systems in Pharmaceutical Manufacturing. PDA (2014). (4) Eibl, R. et al. Standardized cell culture test for the early identification of critical films for CHO cell lines and chemically defined culture media. DECHEMA Temporary Working Group on "Single-Use Technology in Biopharmaceutical Manufacturing" (2014). This ACHEMA Trend Report has been composed by Regine Eibl and Dieter Eibl, Zürcher Hochschule für Angewandte Wissenschaften, Departement N, in the name of the DECHEMA working group Single use technology in biopharmaceutical production. ACHEMA Trend Reports can be used for editorial purposes free of charge; the source has to be named (more details on 14 / 14 DECHEMA Gesellschaft für Chemische Technik und Biotechnologie e.v. Theodor-Heuss-Allee Frankfurt am Main Germany T + 49(0) F +49(0) presse@dechema.de