A new dimension in single-use cell culture performance
The Allegro STR bioreactor family combines Pall’s bioprocess engineering expertise, cell culture know-how and our drive for quality into a series of single-use bioreactors that deliver consistent and scalable cell culture performance across the range. The Allegro STR bioreactor family has a direct bottom-driven impellor allowing power inputs up to 0.25 W/kg, resulting in high oxygen transfer rates and short mixing times.
From the outset of the design, the focus was on achieving the highest cell culture process assurance by introducing several easy and intuitive operation features:
CELL CULTURE SOLUTIONS
Pall has a comprehensive portfolio of advanced single-use solutions for cell culture applications
The single bottom-mounted impeller allows the biocontainer to be packed into a compact unit that can be placed directly into the hardware with minimal operator manipulation
Installation and inflation of the biocontainer is achieved in < 30 minutes through a guided sequence via the Human Machine Interface (HMI)
The low aspect ratio of the systems minimize the operations that have to be performed at height
Automatic biocontainer deflation speeds up turnaround time, minimizes operator activity and reduces the overall volume of waste for disposal
All product contact surfaces in the Allegro STR bioreactors are single-use components therefore the demands on maintenance, cleaning and cleaning validation are minimal. This makes single-use bioreactors the first choice for process development, CMO plants or multiproduct manufacturing facilities where maximum flexibility is required.
Extensive experiments were performed to characterize the bioreactor and gather data on mixing, mass transfer and cell culture performance. Additionally, all components of this bioreactor were rigorously tested, and several long term experiments were completed in order to ensure the integrity and compatibility of the materials with regards to cell culture demands. During the product development, numerous 28 to 45 day life tests were also performed under maximum operating conditions to prove the design of the single-use system: no leaks were observed during this testing. A smaller number of life tests were also performed for >100 days continuous operation with no leaks observed.
Mammalian suspension cell culture, using e.g. CHO or HEK293 cells
Insect cell culture, using e.g. Sf9 cells
Suspension cells in perfusion mode
Adherent cell growth on microcarriers, ex. VERO
Pall has conducted a range of calculations and experiments to characterize the STR bioreactors. Parameters such as mixing time, fluid velocity profiles and Kolmogoroff scale were assessed in Computational Fluid Dynamics studies (CFD). The mixing model was then verified in a tracer experiment. Other important parameters like kLa, dissolved CO2 removal and heating/cooling profile were also performed. The experimental setup and results are documented in separate application notes for each size of the Allegro STR bioreactor.
When filled with the maximum working volume, the Allegro STR bioreactors have an aspect ratio of approximately 1:1. This significantly reduces the overall height of the bioreactor compared to cylindrical versions of the same working volume. It is also much easier and safer for the operators to perform most installation steps without the use of ladders or platforms.
The filled biocontainer in the bioreactor tote forms a cube with rounded edges and corners. These edges act as natural baffles and increase the turbulence in the bioreactor. Additional baffles have been placed at three sides of the container to further increase that effect, especially at the high end of agitation speed. Pall has performed extensive studies using computational fluid dynamics (CFD) to show that there are no dead zones and 95% homogenization can be reached within less than 15 seconds (full impeller speed at up to 1000 L working volume).
Mixing profile on Allegro STR 1000 single-use bioreactor:
The bioreactor hardware provides the support for the biocontainer. It has been designed with a viewing window to observe the cell culture at any time. Heating is provided via a heating mat on the Allegro STR 200 bioreactor and with a water jacket on the Allegro STR 1000 bioreactor. The bioreactor hardware has integrated load cells to provide information on the liquid mass.
The controller uses a Siemens Programmable Logic Controller (PLC) and standard off-the-shelf components. The controller can connect to higher networks via OPC.
Operator interaction with the bioreactor is via a large HMI (15 in. color screen) which is supported on a moving arm offering a wide field of view and the flexibility to suit individual processes and operators of different heights. Industrial design guidelines were used to provide all the information in an intuitive and well-arranged format on screen.
Gas Flow Control
All biocontainers have the option to provide gas through the sparger, into the head space overlay and into a separate open pipe sparger for additional CO2 removal. The controller hosts 6 digital thermal mass flow controllers to control the gas flow. Four of them are calibrated to assure the proper mix of air, oxygen, nitrogen and carbon dioxide through the main sparger. Another is calibrated for air on the CO2 removal line. For the overlay gas, the flow rate of the 4-gas mix is also recorded by a digital mass flow meter. The gas mix is in this case created by pulsing up to 4 different gasses (air, oxygen, nitrogen and carbon dioxide) into the biocontainer.
The Allegro STR bioreactors are equipped with four integrated pumps for liquid dosing. These cover the flow ranges that are needed for the respective cascades (base, glucose, antifoam, etc.). For the addition of larger liquid volumes, e.g. during filling or inoculation, external pumps can be controlled via analog signals. All pumps, integrated and external, can be made part of a defined control cascade.
Liquid temperature within the biocontainer is measured using a PT 100 temperature sensor
Internal pressure within the biocontainer is measured using a single-use pressure sensor supplied with each single-use system
Level sensors in the hardware control the door interlocks and temperature control to ensure there is a minimum liquid volume prior to heating starting
Transmitters for conventional pH and DO sensors are integrated in the controller and redundant transmitters are available on selected models
Spare analog inputs (4-20 mA signal) can be used to connect external pumps, balances and sensors without modifications on the hardware
Single-use sensors for pH and DO are available on selected models.
Electronic Records (21 CFR 11)
The Allegro STR bioreactor controller offers all the technical features that are required to run the system compatible with 21 CFR 11. An administrator can set up users and define user rights and passwords. The passwords safety complies with the requirement in 21 CFR 11 for electronic signatures. The electronic records are saved within the system and options for backup to external systems are available. The controller also offers an OPC interface to fully control the operations on the bioreactor from an external SCADA system.
The biocontainer is made of the transparent, low extractable Allegro film as all other Allegro single-use systems, and which has been well characterized for extractables and leachables. The biocontainers are 100% integrity tested during manufacture. All ports and connections are labelled with colored number tags, providing guidance on how to set up and connect the biocontainer to the hardware. Each system is provided with multiple liquid addition lines and all are fitted with Kleenpak® sterile connectors as standard.
In many manufacturers’ current single-use bioreactor designs, there is risk of damage to the bioreactor bag during transportation and installation. It is therefore very important that the biocontainer is properly packaged and the interaction of the operator with the equipment is well defined. Protection of the single-use bioreactor was considered from the outset of the Allegro STR bioreactor development.
For each size of the Allegro STR biocontainers, Pall has designed specific protective packaging. Some components of the packaging are integral to the single-use system and provide support for the connectors, tubing and filters in the single-use system, thus reducing the risk of damage to the film during transport. On the larger systems, the packaging material provides handles and guides to easily move and position the single-use systems during unpacking and installation.
Impeller and Sparger Assembly
The Allegro STR bioreactors provide mixing via a bottom mounted pitched blade ‘elephant ear’ impeller. This shape has been selected from many candidates for good performance in mammalian cell culture. The impeller is directly connected to the motor, which allows excellent power transfer of approximately 0.25 W/kg into the liquid. A dynamic seal arrangement at the bottom of the biocontainer assures leak-free operation up to the claimed service life.
The ring sparger and the CO2 removal line are mounted directly under the impeller. This supports the distribution of the gas bubbles in the liquid and increases their residence time in the bioreactor. The sparger lines are fitted with sterilizing grade Pall Emflon II gas filters.
When filled with the maximum working volume, the Allegro STR bioreactors have an aspect ratio of approximately 1:1. This reduces the overall height of the bioreactor significantly compared to cylindrical version of the same working volume. It is also much easier and safer for the operators to perform many of the necessary installation steps on the ground level.
Pall has the standard biocontainer equipped with all liquid and gas connections for the most typical processes. Most operations, including perfusion, can be set up with the standard biocontainer. All ports are fitted with aseptic connections. Process liquids, including media and inoculation, can easily and safely be transferred into the bioreactor.
Pall can assist in customizing the bioreactor systems. This includes small changes on the biocontainer up to integrating the bioreactor with all surrounding unit operations into a fully automated process.
Documentation and Certificates
The Allegro STR bioreactors are delivered with complete documentation that qualifies the system. All units are tested prior to shipment and witnessed Factory Acceptance Testing (FAT) is available on request.
The biocontainers are delivered with all the batch specific certificates and calibration information for the included single-use sensors, where applicable.
Pall also can assist with hardware qualification, and standard Installation Qualification (IQ), Operational Qualification (OQ), Factory Acceptance Test (FAT) and Site Acceptance Test (SAT) test protocols are available.
Pall maintains a very stringent approach to quality of purchased and manufactured components. The Allegro bioreactor systems are designed and built to well recognized industry standards. The Allegro STR system hardware:
Good Automated Manufacturing Practice (GAMP) current version
CFR21 Part 11 for electronic records
Electromagnetic Compatibility (EMC) to EN 61326-1: 2013 and FCC CFR 47 Part 15B: 2013
Safety requirements for electrical equipment for measurement, control and laboratory use to IEC 61010-1:2010 (Third Edition), IEC 61010-2-010:2003 (Second Edition) and IEC 61010-3-051:2003 (Second Edition)
CE Marking to European Directives 20014/30/EU (2004/108/ EC extant until April 2016) and 2014/35/EU (2006/95/EC extant until April 2016)
The product is made in a manufacturing facility where the Quality Management System is ISO9001: 2008 certified
All electrical and electronic components used in the build of the hardware have been certified as compliant to the standard: Restriction of Hazardous Substances (RoHS) Directive 2011/65/EU
Waste Electrical and Electronic Equipment (WEEE) to EC Directive 2012/19/EU
The Allegro STR single-use biocontainers:
The product is made in a manufacturing facility where the Quality Management System is ISO13485 & ISO9001 certified
100% of STR biocontainers are checked for correct components, configuration, welding standard & dimensional correctness. A visual inspection confirms there are no embedded particles in the film or any loose particles inside or outside of the biocontainer
100% of STR biocontainers and seal housings undergo a pressure leak test during manufacturing
Samples from each biocontainer manufacturing batch undergo a tensile test of the film weld area & a microscope inspection of the weld area to ensure the quality of the welded seal
Fluid Path Endotoxins: Periodically, rinse effluents from representative samples of Allegro systems are tested for endotoxins in accordance with USP <85> Bacterial Endotoxins Test using Limulus Amoebocyte Lysate (LAL) reagent. Fluid path rinses meet the internal specification of < 0.25 EU/mL
Fluid Path Cleanliness: Periodically, rinse effluents from representative samples of Allegro systems are tested for particulates. Fluid path rinses meet the current limits under USP <788> Particulate Matter in Injections
The materials of construction of the Allegro STR biocontainer meet:
USP<88> Biological Reactivity Test In Vivo for Class VI - 50 °C Plastics
USP<87> Biological Reactivity Tests In Vitro, Cytotoxicity
USP<85> Bacterial Endotoxin Tests
USP<661> Physico-chemical Testing for Plastics
USP<788> Particulate Matter in Injections
Included components that are certified TSE/BSE free
Are supplied double bagged and irradiated at a minimum dose of 25 KGy
Preventive Maintenance and Service Packages
The Allegro STR single-use bioreactors are supported with extensive maintenance and service packages Pall has a global network of technicians and engineers available to assist with installation, qualification, training, technical support, preventive maintenance and on-site repairs. Contact Pall to arrange to discuss any of these services.
Pall Accelerator Process Development Services provide customers with expert advice and support for the implementation of fully integrated single-use processing solutions into new or existing processes, including process optimization, equipment selection, transfer of existing processes, scale-up and troubleshooting advice. Details can be found in the Accelerator Process Development Services brochure (USD 3079).
Preventive maintenance packages are available to ensure the continuous reliable operation of the Allegro STR bioreactors, minimizing downtime. These include a full functional test, exchange of wear parts and calibration of all sensors.
The Allegro STR bioreactors are available as standard models and both the bioreactor hardware and consumables can be customized. Please contact a Pall representative to find the ideal solution for your application. Our teams are specialized in upstream and downstream processing and will gladly help find the right technology for any part of your manufacturing process. It is possible to combine the Allegro products to meet any requirement in the upstream part of the process:
Media sterilization and aseptic transfer
Liquid transfer into the bioreactor (e.g. glucose, anti-foam, base, etc.)
Seed train solutions
Cell harvest and separation
Please contact Pall for a Total Solution discussion on your process.