Single-use bioreactor
Encyclopedia
A single-use bioreactor or disposable
bioreactor is a bioreactor
with a disposable bag instead of a culture vessel. Commercial single-use bioreactors are made by several well known producers and are available since the end of the 1990s.
and are now rapidly replacing conventional bioreactors more and more.
Instead of a culture vessel made from stainless steel or glass a Single-use bioreactors is equipped with a disposable bag. These disposable bags are usually made of a three layers plastic foil. One layer is made from Polyethylene terephthalate
or LDPE) to provide mechanical stability. A second layer made as a gas-proof barrier using e.g. PVA
, PVC
) and finally a contact layer made from PVA
or PP
). For medical applications these single-use materials that get in touch with the product have to be certified by the European Medicines Agency
or similar authorities responsible for other regions.
On the other hand are bioreactors that are agitated by rocking motion. This type of bioreactor does not need any mechanical agitators inside the single-use bag.,. Both variants the stirred and the rocking motion single-use bioreactor are used up to a scale of 1000 L volume. Beside this both construction principles one more variant exists, the Kuhner Shaker, originally designed for media preparation, but also useful for cell cultivation. PBS Biotech employs a novel Air Wheel technology in their single use bioreactors that uses air buoyancy for rotational power and provides low shear and rapid homogeneous mixing of cell cultures without the use of any motors, magnetic drives or mechanically driven devices.
Single-Use Bioreactors consists of a less number of parts compared with conventional bioreactors, so the initial and maintenance costs are reduced.
Limiting factor for the use of some Single-Use Bioreactors is the achievable oxygen transfer, represented by the specific mass transfer coefficient (kL) for the specific phase area (a), resulting in the volumetric oxygen mass transfer coefficient (kLa). Theoretically this can be influenced by a higher energy input (increasing the stirrer speed, respectively the rocking frequency). Considering the fact, that Single-Use Bioreactors are mainly used for cell culturing, the energy input is limited by the delicate nature of cells. Higher energy input leads to higher sheer forces causing the risk of cell damages.
Single-Use Bioreactors are currently available with at a volume of about 1000 L that’s why scale up is limited compared to conventional bioreactors, however, a handful of suppliers are now delivering units at the 2,000 liter scale and some suppliers (Sartorius, Xcellerex, Thermo Scientific HyClone and PBS Biotech) are providing a family of single-use bioreactors from bench-top to full-scale production. Three challenges exist for faster and greater single use bioreactor adoption 1) higher quality and lower cost disposable bags and containers, 2) more reusable and disposable sensors and probes that can provide high quality analytics including real-time cell culture level data points, and 3) a family of bioreactors from lab to production that has full scale-up of the bioprocess. Suppliers are working to improve plastic bag materials and performance and also develop a broader range of sensors and probes that provide scientists greater insight to cell density, quality and other metrics needed to improve yields and product efficacy. New perfusion devices are also becoming popular for certain cell culture applications.
comparing Single-Use Bioreactors and conventional bioreactors does not exist, but many ecological reasons are supporting the concept of Single-Use Bioreactors. For a complete life cycle assessment not only the manufacturing, but also the repeated use need to be considered. Even the main part of a Single-Use Bioreactor is not a disposable, but will be continuously reused. The plastic bag that is used instead of a culture vessel is a disposable, as well as all parts of the bag, Sensors, tubings and stirrers. The bag and all its parts are mainly made from plastics that are derived from mineral oil. Current recycling concepts are mainly focussed on incarnation, to recover the energy originated form the mineral oil as heat and electricity. Most of the mineral oil will be burned anyway in power plants or engines of cars. Burning of single use components of bioreactors will do a detour through biochemical engineering during their life cycle that does not have a big influence. The making of conventional culture vessels form stainless steel or glass costs more energy than making plastic bags. Using conventional bioreactors the culture vessel need to be cleaned and sterilised after each fermentation
. Cleaning will need large amounts of water, but also acids, alkali and detergents. Sterilisation with steam at 121 degrees and 1 bar pressure will need again large quantities of energy and large amounts of distilled water. Distilled water in terms of the pharmaceutical grad is called water for injection that must be prepared by spending lots or energy as well.
A comparison of the life cycle assessment of conventional and Single-Use Bioreactors looks much more favourable for the Single-Use Bioreactors as expected before. According a report of A. Sinclair et al. Single-Use Bioreactors will help to safe 30% of electrical energy for operation, 62% of the energy input for the production of the system, 87% of water and finally 95% of detergents, all compared to conventional bioreactors.
Disposable
A disposable is a product designed for cheapness and short-term convenience rather than medium to long-term durability, with most products only intended for single use. The term is also sometimes used for products that may last several months to distinguish from similar products that last...
bioreactor is a bioreactor
Bioreactor
A bioreactor may refer to any manufactured or engineered device or system that supports a biologically active environment. In one case, a bioreactor is a vessel in which a chemical process is carried out which involves organisms or biochemically active substances derived from such organisms. This...
with a disposable bag instead of a culture vessel. Commercial single-use bioreactors are made by several well known producers and are available since the end of the 1990s.
Single-use technology at bioreactors
Single-use bioreactors are widely used in the field of mammalian cell cultureCell culture
Cell culture is the complex process by which cells are grown under controlled conditions. In practice, the term "cell culture" has come to refer to the culturing of cells derived from singlecellular eukaryotes, especially animal cells. However, there are also cultures of plants, fungi and microbes,...
and are now rapidly replacing conventional bioreactors more and more.
Instead of a culture vessel made from stainless steel or glass a Single-use bioreactors is equipped with a disposable bag. These disposable bags are usually made of a three layers plastic foil. One layer is made from Polyethylene terephthalate
Polyethylene terephthalate
Polyethylene terephthalate , commonly abbreviated PET, PETE, or the obsolete PETP or PET-P, is a thermoplastic polymer resin of the polyester family and is used in synthetic fibers; beverage, food and other liquid containers; thermoforming applications; and engineering resins often in combination...
or LDPE) to provide mechanical stability. A second layer made as a gas-proof barrier using e.g. PVA
Polyvinyl acetate
Polyvinyl acetate, PVA, PVAc, poly, is a rubbery synthetic polymer with the formula n. It belongs to the polyvinyl esters family with the general formula -[RCOOCHCH2]-...
, PVC
Polyvinyl chloride
Polyvinyl chloride, commonly abbreviated PVC, is a thermoplastic polymer. It is a vinyl polymer constructed of repeating vinyl groups having one hydrogen replaced by chloride. Polyvinyl chloride is the third most widely produced plastic, after polyethylene and polypropylene. PVC is widely used in...
) and finally a contact layer made from PVA
Polyvinyl acetate
Polyvinyl acetate, PVA, PVAc, poly, is a rubbery synthetic polymer with the formula n. It belongs to the polyvinyl esters family with the general formula -[RCOOCHCH2]-...
or PP
Polypropylene
Polypropylene , also known as polypropene, is a thermoplastic polymer used in a wide variety of applications including packaging, textiles , stationery, plastic parts and reusable containers of various types, laboratory equipment, loudspeakers, automotive components, and polymer banknotes...
). For medical applications these single-use materials that get in touch with the product have to be certified by the European Medicines Agency
European Medicines Agency
The European Medicines Agency is a European agency for the evaluation of medicinal products. From 1995 to 2004, the European Medicines Agency was known as European Agency for the Evaluation of Medicinal Products.Roughly parallel to the U.S...
or similar authorities responsible for other regions.
Types of single-use bioreactors
In general there are two different principles for the construction of Single-Use Bioreactors, differing in the agitation of the culture medium. On one hand are stirred Single-Use Bioreactors, similar to conventional bioreactors, but with stirrers from single-use materials that are already integrated in the plastic bag. The closed bag and the stirrer are pre-sterilized. for the use the bag will be mounted in the bioreactor and the stirrer will be connected mechanically or magnetically.On the other hand are bioreactors that are agitated by rocking motion. This type of bioreactor does not need any mechanical agitators inside the single-use bag.,. Both variants the stirred and the rocking motion single-use bioreactor are used up to a scale of 1000 L volume. Beside this both construction principles one more variant exists, the Kuhner Shaker, originally designed for media preparation, but also useful for cell cultivation. PBS Biotech employs a novel Air Wheel technology in their single use bioreactors that uses air buoyancy for rotational power and provides low shear and rapid homogeneous mixing of cell cultures without the use of any motors, magnetic drives or mechanically driven devices.
Measurement and control
Measurement and control of a cell culture process using a Single-Use Bioreactor is challenging, as the bag in which the cultivation will be performed is a closed and pre-sterilized system. Sensors for measuring the temperature, conductivity, glucose, oxygen, or pressure must be built into the bag during the manufacturing prior to sterilisation. They can’t be assembled prior to use of the bioreactor as in the conventional case. Consequently some challenges must be taken into consideration. The bag will be assembled, delivered and stored dry, with the consequence that e.g. normal pH-electrodes do not work. Calibration or additional assembly is not possible. This led to the development of preconfigured bags with new analytical probes. The pH-value will be measured using a patch that is just a few millimetres in size. This patch consists of a protecting membrane with a dye behind. Under influence of the changing pH value the dye changing its colour too. The change of the colour can be detected with an optical laser from outside of the bag. This and some other methods of non-invasive measurement have been developed during the past years. Even if these methods are already in use with satisfactory performance many of them are lacking in technical development. Further technical developments should lead to better solutions.Single-use process
Beside Single-Use Bioreactors, complete Single-Use Processes are offered by the world leading manufacturer, Sartorius Stedim Biotech GmbH. Single-use process steps available are: media and buffer preparation, cell harvesting, filtration, purification and virus inactivation. Other manufacturers include PBS Biotech, Inc. with its novel Air-Wheel mixing mechanism and low shear and scalability from lab to cGMP production and is fully integrated, easy to use www.pbsbiotech.com as well as Millipore, which is primarily upstream equipment www.millipore.com GE Healthcare with its pillow like disposable bag on a rocking table controller www.gehealthcare.com or Thermo Scientific www.thermo.com with its traditional stirred or impeller shaft based mixing approach does also have some of this technologies available. Only Sartorius Stedim, PBS Biotech, Thermo Fisher Hyclone and Xcellerex have ability to provide a fully scalable solution for a complete lab to production bioprocess. BioProcess Systems Alliance (BPSA) is the single use association advocating broader application of single-use to include CGMP use which will require FDA support. The agency has stated in public forums it is a major backer of single use technology in general as the technology provides significant benefits and value to include mitigation of cross contamination and reduced industry costs. FDA has stated it will not issue guidance to the industry on single use but may issue Q and As on its web site. When an FDA approved single use production site is approved, likely in next 6 to 18 months the gates will open for broader and faster single use adoption worldwide. Already single use is shown to be huge cost saver and increase process flexibility and operational efficiency for biosimilar applications and for biotech startups and CROs. Emerging markets are expected to embrace single use vs stainless given their lack of legacy infrastructure and desire to drive capex and opex costs out of the biotech operation. The market today is approx. $200M and could grow to over $1B or more in next 4-5 years.Advantages and disadvantages
Compared with conventional systems single-use solution does have some advantages. Application of single-use technologies reduces cleaning and sterilisation demands. Some estimates show cost savings of more than 60% with single use system compared to fixed asset stainless steel bioreactors. In pharmaceutical productions complex qualification and validation procedures can be made easier, and will finally lead to significant cost reductions. Like other single-use technologies reduces the application of Single-Use Bioreactors the risk of cross contaminations, and enhances the biological and process safety. This makes single-use applications suitable especially for any kind of biopharmaceutical products.Single-Use Bioreactors consists of a less number of parts compared with conventional bioreactors, so the initial and maintenance costs are reduced.
Limiting factor for the use of some Single-Use Bioreactors is the achievable oxygen transfer, represented by the specific mass transfer coefficient (kL) for the specific phase area (a), resulting in the volumetric oxygen mass transfer coefficient (kLa). Theoretically this can be influenced by a higher energy input (increasing the stirrer speed, respectively the rocking frequency). Considering the fact, that Single-Use Bioreactors are mainly used for cell culturing, the energy input is limited by the delicate nature of cells. Higher energy input leads to higher sheer forces causing the risk of cell damages.
Single-Use Bioreactors are currently available with at a volume of about 1000 L that’s why scale up is limited compared to conventional bioreactors, however, a handful of suppliers are now delivering units at the 2,000 liter scale and some suppliers (Sartorius, Xcellerex, Thermo Scientific HyClone and PBS Biotech) are providing a family of single-use bioreactors from bench-top to full-scale production. Three challenges exist for faster and greater single use bioreactor adoption 1) higher quality and lower cost disposable bags and containers, 2) more reusable and disposable sensors and probes that can provide high quality analytics including real-time cell culture level data points, and 3) a family of bioreactors from lab to production that has full scale-up of the bioprocess. Suppliers are working to improve plastic bag materials and performance and also develop a broader range of sensors and probes that provide scientists greater insight to cell density, quality and other metrics needed to improve yields and product efficacy. New perfusion devices are also becoming popular for certain cell culture applications.
Environmental aspects
Single-Use Bioreactors are because of the amount of disposable material compared with conventional bioreactors. A complete life cycle assessmentLife cycle assessment
A life-cycle assessment is a technique to assess environmental impacts associated with all the stages of a product's life from-cradle-to-grave A life-cycle assessment (LCA, also known as life-cycle analysis, ecobalance, and cradle-to-grave analysis) is a technique to assess environmental impacts...
comparing Single-Use Bioreactors and conventional bioreactors does not exist, but many ecological reasons are supporting the concept of Single-Use Bioreactors. For a complete life cycle assessment not only the manufacturing, but also the repeated use need to be considered. Even the main part of a Single-Use Bioreactor is not a disposable, but will be continuously reused. The plastic bag that is used instead of a culture vessel is a disposable, as well as all parts of the bag, Sensors, tubings and stirrers. The bag and all its parts are mainly made from plastics that are derived from mineral oil. Current recycling concepts are mainly focussed on incarnation, to recover the energy originated form the mineral oil as heat and electricity. Most of the mineral oil will be burned anyway in power plants or engines of cars. Burning of single use components of bioreactors will do a detour through biochemical engineering during their life cycle that does not have a big influence. The making of conventional culture vessels form stainless steel or glass costs more energy than making plastic bags. Using conventional bioreactors the culture vessel need to be cleaned and sterilised after each fermentation
Fermentation (biochemistry)
Fermentation is the process of extracting energy from the oxidation of organic compounds, such as carbohydrates, using an endogenous electron acceptor, which is usually an organic compound. In contrast, respiration is where electrons are donated to an exogenous electron acceptor, such as oxygen,...
. Cleaning will need large amounts of water, but also acids, alkali and detergents. Sterilisation with steam at 121 degrees and 1 bar pressure will need again large quantities of energy and large amounts of distilled water. Distilled water in terms of the pharmaceutical grad is called water for injection that must be prepared by spending lots or energy as well.
A comparison of the life cycle assessment of conventional and Single-Use Bioreactors looks much more favourable for the Single-Use Bioreactors as expected before. According a report of A. Sinclair et al. Single-Use Bioreactors will help to safe 30% of electrical energy for operation, 62% of the energy input for the production of the system, 87% of water and finally 95% of detergents, all compared to conventional bioreactors.