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Dr. Kathrin Rübberdt
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e-Mail: 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 m3 or 2 m3) 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
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(2,4di-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
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
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
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
Table 1: List of commercially available instrumented single-use bioreactors (liter-scale)
Reactor Category
Reactor Name
Working
Volume [L]
Used successfully1 for cultivation of
Manufacturer
mechanical drive, wave action
AppliFlex
0.5-25
animal and plant cell cultures, algae2
Applikon
CELL-tainer (20 L, 200 L)
0.015-150
microorganisms
Cell tainer Biotech
0.015-200
animal cell cultures
CellTumbler (various
Systems 50)
0.5-10
animal cell cultures
CerCell
SmartRocker Bioreactor
0.5-25
animal and plant cell cultures
Finesse
Wave Bioreactor System
(System 20/50 and System
500/1000)
0.2-500
animal and plant cell cultures
GE Healthcare
ReadyToProcess WAVE25
0.2-25
Xuri Cellbag bioreactor
(System W5 and W25)
0.2-25
human cells (e.g. cells and stem cells)
Allegro XRS 20
0.1-20
animal cell cultures, microorganisms
and T-cells
Pall Life Sciences
BIOSTAT RM bioreactor
(System 20/50 and System
200)
0.1-100
animal and plant cell cultures, algae2
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 FibraCel carrier, 7 polyester microfibers, 8 working volume fixed bed
6
Table 1 continued
Reactor Category
mechanical drive, stirred-tank,
rigid tank
Reactor Name
Working
Volume [L]
Used successfully1 for cultivation of
Manufacturer
CellVessel 21 and 23 Series
0.250-75
animal cell cultures
CerCell
CellVessel 21 and 23 Series
0.25-1
microorganisms
BactoVessel 25 Series
2-75
CelliGen BLU benchtop
stirred-tank reactor (BioBLU
1c, 5c, 14c, 50c)
0.25-40
animal cell cultures and human cells
(stem cells)
CelliGen BLU benchtop
stirred-tank reactor (BioBLU
1f)
0.25-1.25
microorganisms
DASGIP Parallel Bioreactor
(BioBlock with BioBLU 1c)
0.25-1.25
animal cell cultures and human cells
(stem cells)
DASGIP Parallel Bioreactor
(BioBlock with BioBLU 1f)
Eppendorf
microorganisms
Mobius CellReady 3 L
bioreactor
1-2.4
animal cell cultures and human cells
(stem cells)
Merck Millipore
UniVessel SU
0.6-2
animal and plant cell cultures, human
cells (stem cells) and microorganisms
Sartorius Stedim
Biotech
SmartVessel3 single-use
bioreactor
0.5-2.2
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
Table 1 continued
Reactor Category
mechanical drive, stirred-tank,
plastic bag
Reactor Name
Working
Volume [L]
Used successfully1 for cultivation of
Manufacturer
Xcellerex XDR (10 L, 50 L,
200 L, 500 L, 1000 L, 2000
L)
4.5-2000
animal cell cultures
GE Healthcare
Xcellerex XDR-50 MO
Fermentor
10-50
microorganisms
Mobius CellReady (50 L,
200 L, 1000 L3, 2000 L3)
10-20003
animal cell cultures
Merck Millipore
Allegro STR (200 L, 1000
L3, 2000 L3)
60-20003
animal cell cultures
Pall Life Sciences
PadReactor (25 L, 50 L,
125 L, 250 L, 600 L, 1200
L)4, 5
8-960
animal cell cultures
BIOSTAT STR (50 L, 200 L,
500 L, 1000 L, 2000 L3)
12.5-20003
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)
25-2000
animal and plant cell cultures
ThermoScientific
HyPerforma S.U.F (30 L,
300 L)
6-300
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
Table 1 continued
Reactor Category
Reactor Name
Working
Volume [L]
Used successfully1 for cultivation of
Manufacturer
OrbShake bioreactor (SB10X, SB-50X, SB-200X,
SB-2500X)
1-2500
animal and plant cell cultures,
microorganisms
Adolf Kühner
Current Bioreactor (5 L, 50
L, 150 L, 300 L)
0.5-300
animal cell cultures
Hangzhou Amprotein
Bioengineering
mechanical drive, rotating
Vertical Wheel bioreactor
MagDrive (PBS3MAG,
PBS15 MAG)
1.8-15
human cells (stem cells)
PBS Biotech
pneumatic drive
CEllMaker Felix3
n.a.
animal cell cultures
Cellexus
CEllMaker Regular and
CEllMaker Plus (8 L, 50 L)
1-50
microorganisms and algae
Vertical Wheel bioreactor
AirDrive (PBS 3 Air, PBS 15
AIR, PBS 80 AIR, PBS 500
AIR)
0.6-500
animal cell cultures
PBS Biotech
Quantum Cell Expansion
absolute cell
culture
surface area:
2.1 m2
adherent animal cell cultures and
human cells (stem cells and T-cells)
Terumo BCT
mechanical drive, orbital shaken
hydraulic drive, hollow fiber
bioreactor
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
Table 1 continued
Reactor Category
Reactor Name
Working
Volume [L]
Used successfully1 for cultivation of
Manufacturer
hydraulic drive, parallel plate
bioreactor
Xpansion Multiplate
Bioreactor
absolute cell
culture
surface area:
-0.06-12.2 m2
animal cell cultures and human cells
(stem cells)
Pall Life Sciences
Celligen BLU fixed bed
bioreactor (5 L, BioBLU 5p)6
3.758
adherent animal cell cultures and
human cells (stem cells)
Eppendorf
iCELLis7
0.04-258
animal cell cultures and human cells
(stem cells)
Pall Life Sciences
(10 - 200 plates)
hydraulic drive, fixed bed
bioreactor
(nano, 500)
1
n.a. not available, 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
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 25  60486 Frankfurt am Main  Germany  T + 49(0)69 75 64-0  F +49(0)69 75 64-201  presse@dechema.de
www.dechema.de
New DSP process designs: clarification, capture, polishing
However the high cell densities (in the region of 108 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 m3 and 2 m3. 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 25  60486 Frankfurt am Main  Germany  T + 49(0)69 75 64-0  F +49(0)69 75 64-201  presse@dechema.de
www.dechema.de
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 25  60486 Frankfurt am Main  Germany  T + 49(0)69 75 64-0  F +49(0)69 75 64-201  presse@dechema.de
www.dechema.de
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
www.achema.de)
14 / 14
DECHEMA Gesellschaft für Chemische Technik und Biotechnologie e.V.
Theodor-Heuss-Allee 25  60486 Frankfurt am Main  Germany  T + 49(0)69 75 64-0  F +49(0)69 75 64-201  presse@dechema.de
www.dechema.de