Key Factors that Impact the Bioreactor Vessels

      KEY FACTORS THAT IMPACT THE DESIGN, CONSTRUCTION CONSIDERATIONS, AND STEPS INVOLVED IN QUALIFICATION AND VALIDATION OF BIOREACTOR VESSELS     by (Name)     The Name of the Class (Course) Professor (Tutor) The Name of the School (University) The City and State where it is located The Date   Key Factors That Impact the Design, Construction Considerations, and Steps Involved in Qualification and Validation of Bioreactor Vessels A bioreactor vessel is an enclosed and controlled space where biological reactions can occur without external disturbance (Zhong, 2011). It is a valuable tool in any laboratory that researches or relies on biological processes, including proteomics, cell culture, and cell biology. Bioreactors can either be made out of glass or by use of stainless steel. Those made using stainless steel are useful because they allow the maintenance of high internal pressure. On the other hand, the choice of glass is motivated by the need to observe the contents without necessarily opening the bioreactor. Further, assemblies can be added to the reactor vessel to allow processes like stirring, adding or removing samples, and monitoring conditions like temperature and pressure. When choosing a bioreactor vessel, some factors to consider include the size needed, the type of reactions to be accommodated, and whether they are single-use or reusable. While these considerations help in the selection process, they do not shape the design, construction, and qualification processes. Instead, they are the outcomes of these stages of application of bioreactors. As a result, the current paper explores the key factors that impact a bioreactor vessel design, the applicable construction considerations, and the steps involved in the qualification and validation of bioreactor vessel designs. The paper will first discuss the developments that have taken place to arrive at modern bioreactors, the key classes and types of bioreactors, and the steps in a typical bioprocess reaction. The purpose of these sections is to provide a visual image of what bioreactors are, what they entail, how they work, and the subsequent control systems. These sections will inform the subsequent sections on the factors that influence design, construction, qualification. Section 1: About Bioprocess and Bioreactors A bioprocess is a process that relies on complete living cells to obtain desired products. Bioprocesses often involve cell culture in which cells are removed from an organism and placed in conditions where such cells can grow and multiply to assist in research and experiments. Fermentation (of food particles – brewing) is also a fundamental aspect of the bioprocess. While bioprocesses may occur naturally in the environment, bioreactors allow for the development of an artificial environment with an optimum condition that allows the isolation and growth of particular cells or microbes. Background and Development on Bioreactors The term bioreactor can be used interchangeably with the term fermenter (Rasche, 2019). Researchers and scientists who cultivate yeast, bacteria, and fungi rely on fermenters, while those who cultivate mammalian cells often use bioreactors. Fermentation, particularly for food and drink, is the earliest concept dating from 4,200 BC in Mesopotamia (Singh, 2008). In essence, bioreaction and fermentation are not new concepts to human society. Instead, the process and tools involved have evolved with advancements in research and technology. For the first time in Europe, large-scale bioreactors were used in the 1930s to produce compressed yeast. The earlier crude version of the reactor consisted of a large cylindrical tank with a network of perforated pipes at the base through which air could be introduced. In later modifications, mechanical impellers were added to the designs to increase the mixing and enhance the breakup and dispersal of air bubbles. These new developments led to the compressed air requirement of today. Further, baffles on the walls were added to subsequent designs to prevent vortex formation on the liquid inside the bioreactor. Strauch and Schmidt patented the first such bioreactor were patented in 1934 by Strauch and Schmidt, whose system had aeration tubes and introduced steam and water for sterilization and cleaning. However, following the emergence of the culture technique for penicillin production, there was an increasing need for aseptic conditions that allowed good aeration and agitation. This was a critical factor in the British government had established that the surface culture was increasingly inadequate, especially with the rise in demand for bioreaction processes. Since none of the existing fermentation plants were immediately equipped for deep fermentation, the first pilot plant was established in India in 1950. Since then, technological advancement besides extensive research has enabled the design of different types of bioreactors. For instance, there was the emergence of reusable and disposable bioreactors. According to Pollard and Kistler (2017), the traditional stainless-steel bioprocess flow has been revolutionized by the emergence of single-use disposable bioreactors. Single us is a faster, cheaper, and lower-cost alternative to stainless steel. The combination of continually rising upstream titers with enhanced single-use technology is the key driver of the growth in popularity of single-use bioreactors. In essence, it allows for future strategies to influence modular facilities with scale-out instead of a scale-up approach to design and implementation (Pollard & Kistler, 2017). However, despite the rise of single-use bioreactors, they face several challenges currently being targeted through design processes. Some of the key challenges include expanding plug-and-play capabilities, standardization of testing protocols, and lowering bag failure. A disposable bag is used in single-use bioreactors instead of a cultural vessel made of glass or stainless steel. Pharmaceutical organizations, in particular, prefer the use of bags for the preparation of media and buffer, virus inactivation, purification, filtration, and cell harvesting. The bags consist of multilayered polymer films and are gamma-sterilized on arrival. Inside the bag, the lining that comes in contact with cell culture is often polythene. For protection, the bag is usually encased in a sturdy structure like a steel cylinder, rocker, or cuboid. The two types of single-use bioreactors are rockers and stirrers. The difference between the two is that while rockers rely on a rocking motion for mixing and agitation of a medium, stirrers mimic the conventional bioreactors that have an integrated mechanism for stirring (Pollard & Kistler, 2017). The key advantages of single-use bioreactors include eliminated need for cleaning, decreased turnaround and downtime, lower risks of cross-contamination, reduced costs of operation, and simple installation. Unlike disposable bioreactors, reusable glass or steel bioreactors have fixed vessel configurations that have predefined port assemblies. In single-use bioreactors, pre-sterilized plastic cultivation chambers that can get discarded are used. While single-use bioreactors are currently limited to less than 2000 liters in scale, reusable ones are ideal on an industrial scale. The advantages of disposable bioreactors combined with the robustness of reusable reactors have resulted in hybrid systems. As a result, there are three main types of bioreactors: stirred tank, airlift, and bubble column. The size of each of these types is determined by operation and process. For instance, fermentation of diagnostic enzymes requires about 1-20000 liters bioreactor, while production of amino acids on a large scale may require up to 5,000,000 liters of bioreactors. Critical Components of Stirred-Tank Bioreactors When it comes to cultivation systems for growing eukaryotic and prokaryotic cells in a laboratory, Rasche (2019) argues that shake flasks, T-flasks, and cell culture dishes are the first to come to mind. However, when large quantities of cell cultivation are required, bioreactors are the ideal alternative. While there are many types of bioreactors, as already mentioned, the stirred-tank bioreactor is the most commonly utilized on an industrial scale. There are several critical components of a stirred tank bioreactor (figure 1).   Figure 1: Stirred-tank Courtesy of -  (Rasche, 2019) The vessel houses the medium in which the cultivation of cells is done. The tank also has a head plate which is used to close the vessel. Modern systems typically have a computerized external component that acts as a control system in adjusting the culturing conditions and includes control software. Further, a component is attached to the head or the vessel whose purpose is to measure and adjust culturing conditions. The motor at the top of the vessel provides the culturing rotational mechanism. In contrast, the sensors provide feedback on the conditions inside the vessel by providing information on critical areas like pressure, temperature, and PH. The impeller provides the stirring mechanism which is required for the cultivation of cells and microbes.   Creation of Optimal Cultivation Conditions Bioreactors are similar to shakers and incubators because they allow alteration and creation of optimal environmental conditions for the growth of microbes and cells. However, the difference emerges based on how these conditions are achieved. The key areas to consider while creating optimal conditions or environments include culture mixing, tempering, establishing aerobic and anaerobic conditions, PH control, and parameters control. Culture Mixing Instead of shaking as a way of mixing, a stirring tank bioreactor has an impeller used to stir the culture. The impellor shaft provides a connection between the motor and the impellor (Rasche, 2019). There is a need to mix yeast, bacteria, and suspension cell cultures and cultures attached to the growth matrix in such bioreactors. Tempering Tempering involves the attainment of the ideal bioreaction temperature. The temperature sensor continuously provides feedback on the temperature level in the vessel, which allows for adjustments or alterations. Regulation of temperature is made by placing the vessel in a thermowell with either a water jacket or a heating blanket. The tempering process also allows cooling to take place. Aerobic and Anaerobic Conditions Industrial fermenters or bioreactors can be divided into two major classes: aerobic and anaerobic (Zhong, 2011). Little special equipment is required in anaerobic bioreactors except for removing heat generated by the fermentation process to retain a constant, consistent, and desired temperature. On the other hand, aerobic bioreactors require several special additional features that allow adequate aeration and mixing. Transfer of oxygen in shakers, for instance, relies on shaking, which acquires oxygen from the immediate surrounding environment. This is because shaking allows exposure of the liquid surface to oxygen (Rasche, 2019). In bioreactors, however, oxygen coming from a compressed cylinder air cylinder is injected into the culture. Oxygen is vital for the growth of cultures, and the amount of oxygen in the medium is measured with a DO sensor through the bioprocess control software. PH Control PH control relies on specimen exposure to carbon dioxide in incubators when culturing is done in dishes or flasks. However, bioreactors are connected to compressed gas cylinders that introduce the gas into the vessel. Thus, PG can be continuously measured and altered depending on the needs of the current bioreaction. In other instances, basic and acid solutions are used as an alternative to controlling PH levels. Parameters Control Different components and control software is responsible for controlling PH, temperature, dissolved oxygen, and pressure. These parameters are measured using sensors such as the DO sensor. The information is transmitted to the bioprocess computer system (software), which regulates the addition of PH agents or CO₂. A Typical Bioprocess Maintenance of cultures requires researchers and scientists to keep the cultures within incubators. Bioreactors are essential in specific products or experiments, which may last for an extended period depending on the application and the organism. The following steps summarize a typical bioprocess. Preculture In this stage, the inoculation of the medium by a preculture is done. Precultures are often grown in incubators or shakers before they are introduced in a bioreactor vessel. In essence, small precultures are used to inoculation of larger bioreactors. Bioreactor Preparation The preparation of a bioreactor for a bioprocess or experiment is done in parallel to the inoculation preparation (in the first stage). Typically, preparation involves sterilization of the feed lines, sensors, and the bioreactor, addition of essential mediums, the definition of process parameters setpoints, and the connection of the bioreactor with the bioprocess control system. Sterilization is an essential preparation process because it reduces the chances of contamination between mediums or cultures used previously in the bioreactor. Inoculation Inoculation is the process through which cells or microbes are introduced into the bioreactor. Similar to the bioprocess preparation, inoculation tools are also sterilized effectively to eliminate possibilities of contamination during the transfer process. Cultivation Period Cultivation time ranges from a few hours to several weeks (Zhong, 2011). Agitation, DO, PH, and temperature during this period are monitored and controlled in real-time through the bioprocess control software. Further, a researcher can analyze bits of the current culture; to determine the biomass and metabolites concentration (Rasche, 2019). This allows for the researcher to feed the culture by adding nutrient solutions depending on the analysis outcomes. Additionally, the culturing period consists of four phases. The lag phase is where the organisms or cells multiply slowly since they need to adapt to the new culture conditions. The exponential growth phase is the rapid growth and generation of cells, while the stationary phase is when growth and multiplication stop. Culturing stops because conditions like oxygen concentration and accumulation of by-products evolve into growth-limiting factors. The death phase is the last phase in artificial cell culture, where the density of viable cells begins to decrease. Harvest Stage Once the cell culture enters the stationary stage, the bioprocess is stopped, and culture is harvested. The stage is followed by the downstream process in which further processing of broth is done. The last stage involves bioreactor cleaning, where sterilization is used to kill and remove culture residues to avoid contamination when the bioreactor is utilized again. Figure 2 below is a summary of a typical cell culture process in a bioreactor.     Figure 2: Series of Events in a Typical Bioprocess (Rasche, 2019) Section 2: Design, Construction, and Quantification A design is a plan or drawing that represents the outlook and functions of an object or process. It provides the basis on which the theoretical aspects of an item or process are integrated with the tools, machinery, or equipment used to support such process or form part of the object. On the other hand, construction is how a design is transformed into a finished product or process to serve a specific purpose. It involves translating what is theoretically captured in the design process into a practical application or product (AcevedoJuan & Gentina, 2007). Quantification and validation involve the processes that ensure that the constructed product, besides its designs, meets a certain level of requirements such as efficacy or environmental concerns. Like any other complex system, a bioreactor requires the steps of design, construction, and quantification to be established as part of best-practice strategies. The several factors that shape these three aspects are discussed in detail in this section. Factors that Influence Design of Bioreactors According to Yang (2011), the design of bioreactors has made an essential process in the short period in which lung engineering has existed. As a result, modern bioreactors can recreate various aspects of physiology and provide necessary cues to the cultured tissue. For instance, pulsatile perfusion has been redesigned and improved, enabling control of pressure profiles and flow rates. Further, negative pressure ventilation systems have been developed to minimize potential cell damage and increase physiological breathing. Additionally, Vilas Boas and Simoes (Vilas Boas & Simõe...