Disposable bioreactor
A single-use bioreactor is a bioreactor in which the parts that come into contact with the culture medium are made of single-use material. In English it is referred to as a disposable bioreactor or single-use bioreactor . Commercial single-use bioreactors have been offered by bioreactor manufacturers since the late 1990s.
Disposable technology in bioreactors
In animal cell culture technology , single-use bioreactors are increasingly replacing traditional bioreactors. In contrast to conventional systems, pre-sterilized disposable bags are used in single-use bioreactors. The disposable bag replaces the traditional culture vessel made of glass or stainless steel. For bioprocess applications, three-layer composite films are mostly used as single-use materials. These consist of a mechanical support layer (e.g. PET , LDPE ), a gas-impermeable barrier layer (e.g. EVA , PVDC ) and a contact layer (e.g. EVA , PP ). For many applications, the single-use materials must be certified by a drug regulatory agency .
Designs
There are basically two different designs of single-use bioreactors, which differ in the technology of mixing. On the one hand, stirred single-use bioreactors, which are modeled on conventional bioreactors. However, the agitators are also designed as disposable products and are already integrated and pre-sterilized in the disposable bag. When using the bag, these are mechanically or magnetically coupled to the stirrer motor of the actual bioreactor. On the other hand, there are rocked, single-use bioreactors without any further mechanical fittings in the bags, so-called “wave” or “rocking motion” bioreactors. Today, both variants are sensibly used up to a volume of 1000 L. Another device, the Kuhner shaker, was originally intended for media production, but can also be used for cell cultivation.
Type of litigation
In addition to the classic methods such as batch , continuous or feed-based cultivation , perfusion culture is also used. In bioreactors with a perfusion module, the culture space is divided into two compartments: a growth space in which the cells are located and a cell-free metabolite / nutrient space. The two rooms are separated by a semipermeable membrane. Due to their throw-away principle, single-use bioreactors are particularly suitable for perfusion culture. There are both small-volume laboratory reactors and systems on a 100 l scale.
Acoustic Wave Separation (AWS), a revolutionary technology for cell culture clarification filtration for fed-batch and perfusion applications, enables highly efficient, continuous removal of cells in a closed system without centrifugation. AWS technology applies acoustic forces to a flow channel to create a 3D standing wave. When a cell culture passes through the flow channel, the cells are stopped at the nodes of the sound waves and then aggregated. Eventually, due to the decrease in buoyancy, they precipitate. Important advantages are no temperature rise, no damage to cells or proteins and a robust process with high yield.
A distinction is made between flat membrane bioreactors and hollow fiber cartridges . The necessary nutrients enter the growth area through the semi-permeable membrane and are available to the cells. At the same time, growth-limiting metabolic products diffuse into the metabolite / nutrient space. This enables very high cell densities to be achieved. The structure of the bioreactor, however, is more complex compared to the other culture systems. For example, a hollow fiber reactor consists of thousands of hollow fibers that simulate a capillary system. As a result, scale-up is only possible to a limited extent.
Measure and regulate
Measuring and regulating a cell culture process in a single-use bioreactor is an undreamt-of challenge, since the plastic bag in which the cultivation takes place is a closed, pre-sterilized system. Thermostat sensors, pH and conductivity measuring electrodes, glucose and oxygen electrodes, pressure sensors, etc. a. therefore cannot simply be inserted into the bag when required, but must be built into the bag beforehand. This gives rise to complex problems because, on the one hand, the bags are manufactured, stored and delivered dry and, on the other hand, further calibrations are not possible before the bag is used. In addition, a decision has to be made during the manufacture of the bags as to which configuration of the possible sensors makes sense and should be installed in advance. These challenges led to the development of completely new measurement methods. The pH value, for example, is measured using a small plate only a few millimeters in size, which is located in the bag behind a protective membrane and carries a special dye. This dye changes under the influence of a changing pH value. This change can in turn be measured from the outside of the bag using a laser-optical method, and the pH value can be determined from this. These and other non-invasive processes and measurement techniques have been developed to series production readiness in the past few years, even if the individual sensors still have specific problems that need to be improved in the long term.
Disposable process solutions
In addition to the single-use bioreactors, complete single-use process solutions are offered for entire process lines. These include media and buffer production, cell harvest, filtration, purification, and virus inactivation.
Advantages and disadvantages
Disposable materials have several advantages over stainless steel systems. The single-use technology avoids time-consuming cleaning and sterilization processes, which in pharmaceutical production in particular leads to less complex qualification and validation processes and thus to considerable cost savings. As with other single-use technologies, the use of single-use bioreactors reduces the risk of cross-contamination and thus increases bio and process safety. They are therefore of particular interest for the production of biological preparations.
Compared to traditional bioreactors, single-use bioreactors often only consist of very few parts. This means that the acquisition costs are lower and the maintenance effort is greatly reduced. Decisive for the usefulness of such reactor systems is the achievable introduction of oxygen, which is determined by the so-called specific mass transfer coefficient (kL) based on the specific phase interface (a), called k L a values . Theoretically, this can be influenced by a higher energy input (increase in the stirrer speed or the shaker frequency). Since single-use bioreactors are mainly used for cell cultivation, this is limited by the nature of the quite sensitive cell systems. Higher energy input leads to higher shear forces (friction between cells), which can destroy the cells. Another technical limit is the stability of the bags, since with increasing volume and the resulting filling level, the internal pressure on the relatively thin materials increases, and the load can lead to leakages . In the case of single-use bioreactors, there is currently the disadvantage that they are limited in terms of scale-up, their working volume is currently limited to 2000 l.
Disposable bioreactors are not necessarily more environmentally harmful than conventional bioreactors because of the disposable aspect of the bags used for cultivation. A complete life cycle assessment comparing single-use bioreactors with conventional bioreactors made of stainless steel or glass is not yet available, but there are many arguments in favor of using single-use bioreactors from an ecological point of view. Similar to returnable bottles and PET bottles, you don't just have to look at the manufacture of the products, but also at the entire life cycle, especially when it comes to use and reuse. The core of the single-use bioreactor is not a single-use product anyway, but is used again and again. Only the culture vessel is a plastic bag that is only used once. The plastic bag, including probes and connections, as well as any built-in stirrer elements, consists almost exclusively of petroleum-based plastics. Current recycling concepts assume incineration after use. The energy stored in the product, which comes from crude oil, is recovered in the form of electricity or heat . Since the majority of crude oil is burned in power plants or car engines anyway, the additional environmental impact of the combustion of disposable systems from bioprocess engineering, which only make a detour on their life cycle to combustion, is insignificant. Thus, finally, the energy for production and transport still remains to be considered. The manufacture of conventional bioreactor culture vessels made of steel or glass is undoubtedly more energy-intensive than the manufacture of a plastic bag. When using the conventional bioreactor, the culture vessel has to be laboriously cleaned after each fermentation ; this requires large amounts of water, but also acids , bases or cleaning agents and disinfectants. Finally, the bioreactors are sterilized with superheated steam at 121 ° C under a pressure of 1 bar, which in turn consumes large amounts of water and energy. The water used, especially in pharmaceutical applications where the purity of the products is important, is distilled water , which in turn has to be specially produced beforehand using a lot of energy. On closer inspection, single-use bioreactors are therefore obviously in a much better position than would originally be assumed. According to an investigation, the use of single-use bioreactors can save 30% of the electrical energy for operation, 62% of the energy used for production, 87% of water and 95% of cleaning agents compared to conventional bioreactors.
Individual evidence
- ^ Barbaroux M., Sette A .: Properties of Materials Used in Single-Use Flexible Containers: Requirements and Analysis. . In: BioPharm International . 11, 2006.
- ↑ WAVE-Biotech Homepage ( Memento of the original from February 28, 2011 in the Internet Archive ) Info: The archive link was inserted automatically and has not yet been checked. Please check the original and archive link according to the instructions and then remove this notice.
- ↑ BIOSTAT® CultiBag RM Homepage ( Memento of the original from March 1, 2013 in the Internet Archive ) Info: The archive link was inserted automatically and has not yet been checked. Please check the original and archive link according to the instructions and then remove this notice.
- ↑ Kuhner Shaker
- ↑ http://www.bionity.com/de/produkte/128287/kontinuliche-prozessfuehrung-in-der-zellklaerung.html
- ↑ Morrow KJ: Disposable Bioreactors Gaining Favor: New Components and Systems Improve Process Reliability and Reduce Cost. . In: Genetic Engineering News . 26, No. 12, 2006, pp. 42-45.
- ↑ Winfried Storhas: Bioreactors and peripheral devices: A guide for university education, for manufacturers and users . Springer-Verlag , 1994, ISBN 978-3-540-67054-4 , pp. 60-61.
- ↑ Sinclaire A., Leveen L. et al .: The Environmental Impact of Disposable Technologies Archived from the original on July 11, 2011. Information: The archive link was automatically inserted and not yet checked. Please check the original and archive link according to the instructions and then remove this notice. In: BioPharm International Supplements . 2008.
