Bioreactor Design Concepts for Viral Vaccine Production

By Sachin Kumar

Bioreactor Design Concepts for Viral Vaccine Production covers a range of interdisciplinary chapters from the engineering perspective of bioreactor design to the biotechnological perspectives of vector design for vaccine development. The book covers bioreactor concepts such as static systems, single-use systems, stirred tanks, perfusion, wave and packed-beds. It reviews options for efficient and economical production of human vaccines and discusses basic factors relevant for viral antigen production in mammalian cells, avian cells, and insect cells. This book will be a great resource for those interested in implemented novel bioreactor design or experimental schemes towards intensified or/and enhanced vaccine production.

• Covers the fundamentals of bioreactor designs
• Provides strategies for designing a successful vector-based vaccine
• Discusses the applications of biological kinetics, thermodynamics and basic substrate requirements for viral vaccine production

• Language: English
• Publisher: Academic Press
• Release date: May 12, 2024
• ISBN: 9780443153792

Book preview

Chapter 1: Introduction of bioreactor

Surajbhan Sevda¹, and Sachin Kumar²     ¹Environmental Bioprocess Laboratory, Department of Biotechnology, National Institute of Technology Warangal, Warangal, Telangana, India     ²Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, Assam, India

Abstract

Fermented food products and beverages have been used since ancient times, and these methods have changed with the geographical area. The first scientific way was introduced by Louis Pasteur in the 18th century. The bioprocess industry now evolved into the food industry, pharmaceutical industry, biofertilizer, and biological wastewater treatment. In the 21st century, more people are moved to urban places so the need for packaged food also increases. The bioreactor evolved from the conventional fermenter to the single-use bioreactor for various applications such as producing curd from milk to vaccines and therapeutic protein production at a larger scale. The importance of bioreactors has emerged in the various industries. This chapter describes bioreactor evolution from a conventional process to modern vaccine production.

Keywords

Antibody; Bioprocess; Bioreactor; Fermenter; History of bioprocess; Vaccine

1. Introduction

In the past century, the world population has increased rapidly to more than 8 billion in 2023 compared to 2 billion people in the world. The bioreactor/fermenters are considered as the heart of any bioprocess, and these are used for the production of antibiotics, vaccines, single-cell protein, fermented food, chemicals, wastewater treatment, and bioenergy generation at a larger scale (Demirden et al., 2022; Dyment et al., 2020; Xie et al., 2015). In ancient times, alcoholic beverages such as wine and beer are produced using yeast with traditional wooden-type bioreactors. All over the world, fermented food such as Kefir, Kombucha, Sauerkraut, Tempeh, Miso, and many fermented vegetables are produced using controlled growth microorganisms and the use of enzymes for conversion from raw material to fermented products. In 1857, Louis Pasteur discovered that microorganisms can cause spoilage the food, and also in controlled conditions, they can produce lactic acid and ethanol (Berche, 2012; Cavaillon & Legout, 2022). The concept of pasteurization has also discovered that heating the media will destroy all the present microorganisms and the product can get a longer shelf-life. Louis Pasteur also gave the germ theory, and it proved the importance of microorganisms in the industrial bioprocess. In 1928, Alexander Fleming discovered accidently penicillin that has an antibacterial effect on gram-positive pathogens, and it was used widely in the Second World War (Cavaillon & Legout, 2022). Penicillin was considered the first antibiotic, and within 10 years, it was started production in industrial bioreactors (Stanbury et al., 2017a). In 1942 penicillin mass production started, and it was the start of modern industrial biotechnology for health care (Zhang et al., 2017).

The future of industrial biotechnology looks very promising, with many new technological advancements and applications on the horizon (Zhang et al., 2017). Here are some of the key trends that are shaping the future of industrial biotechnology:

1. Microbiome engineering: The human microbiome, which consists of trillions of microorganisms living in and on our bodies, plays a critical role in our health and well-being. Microbiome engineering involves the manipulation of these microbial communities to improve human health and treat diseases (Lear et al., 2021; Lovley & Holmes, 2021). This is a rapidly growing field with many potential applications in areas such as personalized medicine and disease prevention.

2. CRISPR-Cas genome editing: The CRISPR-Cas system is a powerful genome editing tool that allows for the precise and efficient modification of genetic material (Rai et al., 2021). It has many potential applications in industrial biotechnology, including the development of new microbial strains with improved properties and the engineering of crops for improved productivity and sustainability.

3. Advanced fermentation techniques: Advances in fermentation technology are enabling the development of new and improved fermentation processes for industrial biotechnology applications (Asimakopoulos et al., 2018; Rabaey & Rozendal, 2010; Rittmann & Herwig, 2012). These include the use of continuous fermentation, fed-batch fermentation, and other advanced techniques that improve productivity, reduce waste, and lower production costs.

4. Circular economy: The circular economy is a system in which waste is minimized and resources are used in a more sustainable and efficient manner (Catone et al., 2021; Okolie et al., 2022; Shemfe et al., 2018). Industrial biotechnology is well suited to contribute to the circular economy, with many applications in areas such as waste reduction, recycling, and the development of renewable feedstocks (Molina et al., 2020).

5. Artificial intelligence and machine learning: The use of artificial intelligence and machine learning is becoming increasingly important in industrial biotechnology (Onukwuli et al., 2021). These tools can be used to optimize process parameters, predict product quality and yield, and identify new and improved microbial strains for industrial applications.

Overall, the future of industrial biotechnology looks very promising, with many new and exciting developments on the horizon. It is an interdisciplinary field that combines biology, chemistry, engineering, and other disciplines to create sustainable and eco-friendly products and processes that benefit society and the environment (Foley et al., 2010; Karthikeyan et al., 2019; Machado & Atsumi, 2012; Tzirita et al., 2019; Vanhercke et al., 2014).

2. History of industrial biotechnology

Industrial biotechnology is a field that involves the use of biological systems and processes to create products and processes that are useful to humans. The history of industrial biotechnology dates back to ancient times, with humans using microbes for the production of foods and beverages such as bread, beer, and cheese (Adlakha & Yazdani, 2015; Fang et al., 2022; Lv et al., 2019; Van Groenestijn et al., 2009).

In the 19th century, Louis Pasteur developed the germ theory of disease, which led to the development of methods for controlling microbial growth and preventing food spoilage (Berche, 2012; Smith, 2012). This laid the foundation for the modern field of microbiology, which has played a key role in the development of industrial biotechnology (Fig. 1.1).

In the early 20th century, industrial biotechnology began to emerge as a distinct field with the development of processes for the production of chemicals such as acetic acid, citric acid, and ethanol using microbes (Kracke et al., 2019; Lovley & Nevin, 2011; Wong, 2014). These processes involved the use of fermentation, a process in which microorganisms convert sugars into other products such as alcohol and organic acids.

In the mid-20th century, the discovery of antibiotics and other microbial products led to the development of new applications for industrial biotechnology, including the production of pharmaceuticals and biologics (Bu et al., 2013; Chen et al., 2022; Chisti, 1992; Lopez et al., 2021). This led to the establishment of the biotech industry, which has grown rapidly in recent decades. In the 21st century, industrial biotechnology has continued to evolve, with new applications emerging in areas such as biofuels, bioplastics, and industrial enzymes (Algade Amadu et al., 2022; Delattre et al., 2016; Lacina et al., 2003). Advances in genetic engineering and synthetic biology have also enabled the development of new and improved microbial strains for industrial biotechnology applications.

Overall, the history of industrial biotechnology reflects a long-standing human interest in using biological systems to create useful products and processes, and the field continues to evolve and grow in new directions. Becze and Liebmann (1944) used the first large-scale (above 20-L capacity) fermentor for the production of yeast (Becze & Liebmann, 1944). However, during the First World War, a British scientist named Chain Weizmann (1914–1918) developed a fermenter for the production of acetone (Dixon, 1996).

Figure 1.1  Louis Pasteur (a) at the time of his research on fermentation and wine and (b) at the time of his research of the vaccine against rabies. From Berche (2012).

As the importance of aseptic conditions was recognized, steps were taken to design and construct piping, joints, and valves in which sterile conditions could be achieved and manufactured when required (Karthikeyan et al., 2022). For the first time, large-scale aerobic fermentors were used in central Europe in the year 1930s for the production of compressed yeast (Becze & Liebmann, 1944). The fermentor consisted of a large cylindrical tank with air introduced at the base via a network of perforated pipes (Karthikeyan et al., 2022). In later modifications, mechanical impellers were used to increase the rate of mixing and to break up and disperse the air bubbles. This process led to the compressed air requirements. Baffles on the walls of the vessels prevented forming a vortex in the liquid. In the year 1934, Strauch and Schmidt patented a system in which the aeration tubes were introduced with water and steam for cleaning and sterilization (Stanbury et al., 2017a). The decision to use the submerged culture technique for penicillin production, where aseptic conditions, good aeration, and agitation were essential, was probably a very important factor in forcing the development of carefully designed and purpose-built fermentation vessels (Stanbury et al., 2017a).

In 1943, when the British Govt. decided that surface culture was inadequate, none of the fermentation plants were immediately suitable for deep fermentation (Stanbury et al., 2017b). The first pilot fermentor was erected in India at Hindustan Antibiotic Ltd., Pimpri, Pune in the year 1950 (Zhang et al., 2017). Industrial biotechnology is a field that involves the use of biological systems and processes to create products and processes that are useful to humans. It is an interdisciplinary field that combines principles from biology, chemistry, engineering, and other fields to design and optimize biological systems for industrial applications (Stanbury et al., 2017b). Industrial biotechnology has a wide range of applications in many industries, including pharmaceuticals, chemicals, food and beverage, agriculture, and energy (Fang et al., 2022; Meuwly et al., 2007; Torres et al., 2007). Here are some examples of industrial biotechnology applications.

1. Production of biofuels: Industrial biotechnology is used to produce biofuels such as ethanol, biodiesel, and biojet fuel from renewable biomass sources such as corn, sugarcane, and algae.

2. Manufacturing of chemicals: Industrial biotechnology is used to produce a wide range of chemicals such as organic acids, enzymes, and biopolymers using microbial fermentation (Karthikeyan et al., 2022).

3. Development of biopharmaceuticals: Industrial biotechnology is used to develop and produce biopharmaceuticals such as vaccines, antibodies, and enzymes using genetically engineered microorganisms or mammalian cells.

4. Production of industrial enzymes: Industrial biotechnology is used to produce enzymes for a wide range of industrial applications such as laundry detergents, food processing, and biofuels (Karthikeyan et al., 2022).

5. Agricultural applications: Industrial biotechnology is used to develop and produce genetically modified crops that have improved resistance to pests, diseases, and environmental stresses.

Overall, industrial biotechnology offers a sustainable and eco-friendly approach to product and process development, and it continues to evolve and expand with new technological advancements and applications (Karthikeyan et al., 2022).

Modern industrial biotechnology involves the use of advanced technologies and approaches to develop and optimize biological systems for industrial applications. Fig. 1.2 shows the evolution of system biology for new vaccine/protein development.

Figure 1.2  Various relationships in a cell; various stages help in whole-cell understanding using the system biology approach. From Heux et al. (2015).

Here are some of the key features of modern industrial biotechnology:

1. Synthetic biology: Synthetic biology involves the design and construction of new biological systems and the reprogramming of existing biological systems to perform specific functions (Nikolaivits et al., 2021). It has revolutionized industrial biotechnology by enabling the creation of new microbial strains with improved properties such as higher productivity, greater resistance to environmental stresses, and better product quality (Fig. 1.2).

2. Genomics and proteomics: Advances in genomics and proteomics have enabled the rapid and accurate sequencing and analysis of microbial genomes and proteins (Talbot et al., 2008). This has facilitated the identification of new metabolic pathways, the optimization of existing metabolic pathways, and the development of new microbial strains for industrial biotechnology applications (Talbot et al., 2008).

3. High-throughput screening: High-throughput screening techniques allow for the rapid screening of large numbers of microbial strains for desirable properties such as high productivity, low cost, and improved product quality (Banerjee et al., 2019; Hou et al., 2009). This has accelerated the development and optimization of microbial strains for industrial biotechnology applications (Syed et al., 2021).

4. Process optimization: Modern industrial biotechnology involves the optimization of entire production processes, not just individual microbial strains (Bi et al., 2022; Kiesslich & Kamen, 2020). This includes the development of advanced process monitoring and control systems, the integration of different unit operations, and the use of modeling and simulation tools to optimize process parameters (Berrie et al., 2020; Cervera et al., 2013; Fang et al., 2022; Ripoll & Betancor, 2021).

5. Sustainability and circular economy: Modern industrial biotechnology is focused on developing sustainable and eco-friendly processes that minimize waste and reduce greenhouse gas emissions (Tuan et al., 2022). It also aims to develop processes that use renewable feedstocks and generate renewable products, thereby contributing to the circular economy.

Overall, modern industrial biotechnology is a rapidly evolving field that is changing the way we develop and manufacture products across many industries. It offers new opportunities for sustainable and eco-friendly product and process development, and it continues to expand with new technological advancements and applications.

3. History of the first vaccine production

The concept of vaccination, or inoculating individuals with a mild form of a disease to build up immunity against a more severe form, has been around for centuries. However, the first successful production of a vaccine against disease was achieved in 1796 by British physician Edward Jenner (Cavaillon & Legout, 2022). The first vaccine production was achieved by British physician Edward Jenner in 1796. Jenner developed the idea of using cowpox, a relatively mild disease, as a vaccine against smallpox, a much more severe and deadly disease. He tested his theory on an 8-year-old boy named James Phipps, inoculating him with cowpox and then exposing him to smallpox. Phipps did not develop smallpox, demonstrating that cowpox had indeed provided immunity to the disease (Cavaillon & Legout, 2022).

Jenner's discovery was groundbreaking, and vaccination quickly became an accepted method for preventing diseases. Jenner observed that milkmaids who contracted cowpox, a relatively mild disease, seemed to be immune to the much more severe disease of smallpox. He developed the idea of using cowpox as a vaccine against smallpox and tested his theory on an 8-year-old boy named James Phipps (Cavaillon & Legout, 2022). Jenner inoculated Phipps with cowpox, and when the boy recovered, he exposed him to smallpox. Remarkably, Phipps did not develop the disease, demonstrating that cowpox had indeed provided immunity to smallpox. This marked the first successful vaccination against a disease (Berche, 2012; Ng, 2012). Jenner's discovery was groundbreaking, and vaccination quickly became an accepted method for preventing diseases. Jenner's work also led to the development of vaccines against other diseases such as typhus, cholera, and rabies, and laid the foundation for modern immunology (Berche, 2012; Luter et al., 2017; Sobolewski, 2019). His work laid the foundation for modern immunology, and vaccines have since been developed against a wide range of diseases, including measles, polio, influenza, and COVID-19. In the 19th century, the production of vaccines became more widespread and standardized. One of the most important developments during this time was the discovery of germ theory, which explained how infectious diseases are caused by microorganisms such as bacteria and viruses (Homma et al., 2013; Mobley & Hausinger, 1989; Römling et al., 2013; Sciences et al., 2021).

Louis Pasteur, a French microbiologist, made several important contributions to the field of vaccination during this period (Fig. 1.1). In 1885, he developed a vaccine against rabies, a deadly viral disease that affects the nervous system. Pasteur's rabies vaccine was made by attenuating (weakening) the virus and injecting it into patients to stimulate their immune systems (Smith, 2012). Pasteur also developed vaccines against other diseases, including anthrax and cholera. His work paved the way for the development of other vaccines in the 20th century (Berche, 2012).

Another significant development during the 19th century was the establishment of vaccine production facilities. In the United States, the first vaccine production facility was established in 1891 by the New York City Department of Health. The facility produced vaccines for smallpox, diphtheria, and tetanus (Brig-Ortiz, 2022; Demirden et al., 2022). Overall, the 19th century was a period of significant progress in the production of vaccines, with many new vaccines being developed and standardized production facilities being established. In the 19th century, vaccines were primarily generated through the process of attenuation, or weakening of the disease-causing microorganism. This process involved growing the microorganism in a culture and then exposing it to conditions that would weaken its virulence. The weakened microorganism could then be used to create a vaccine that would provide immunity to the disease without causing serious illness (Demirden et al., 2022; Eckels, 2014; Frisio & Ventura, 2021; Shafyi & Mohammadi, 2018). One of the most famous examples of attenuation in vaccine generation was Louis Pasteur's development of a rabies vaccine. Pasteur weakened the rabies virus by exposing it to air for several days and then injected the weakened virus into animals to create immunity. This approach was successful, and Pasteur's rabies vaccine became widely used in the late 19th century (Smith, 2012). Another approach to vaccine generation in the 19th century was the use of live attenuated vaccines. These vaccines were made by growing the microorganism in a culture under conditions that would make it less virulent, but still capable of causing an immune response. Live attenuated vaccines were developed for diseases such as smallpox, measles, and mumps (Demirden et al., 2022; Josefsberg & Buckland, 2012). In addition to these approaches, other vaccine-generation methods were also used in the 19th century, including the use of killed microorganisms and the use of bacterial toxins. The development of these vaccines paved the way for future advances in vaccine generation in the 20th and 21st centuries (Alvarez et al., 2020). In the 21st century, vaccine production has become more advanced and sophisticated, with the development of new technologies and methods for generating vaccines (Demirden et al., 2022; Nikolaivits et al., 2021; Wunderlich et al., 2014). One of the most significant developments in vaccine production has been the use of genetic engineering techniques. This approach involves modifying the genetic material of a microorganism, such as a virus or a bacterium, to create a vaccine that is safe and effective. Genetic engineering has been used to develop vaccines against diseases such as hepatitis B, human papillomavirus (HPV), and COVID-19 (Fig. 1.3). Another important development in vaccine production has been the use of recombinant DNA technology.

This approach involves inserting genes from a disease-causing microorganism into a harmless bacterium or virus, which can then be used to generate a vaccine (Fig. 1.3). Recombinant DNA technology has been used to develop vaccines against diseases such as hepatitis A and B, as well as human papillomavirus (HPV) (Cervera et al., 2013; Zhang et al., 2017). In addition to these methods, other approaches to vaccine production have been developed in the 21st century, including the use of virus-like particles (VLPs) and conjugate vaccines. VLPs are particles that mimic the structure of a virus but do not contain any viral genetic material and can be used to stimulate an immune response without causing illness (Fig. 1.3). Conjugate vaccines are made by attaching a bacterial antigen to a carrier protein, which enhances the immune response and provides long-lasting immunity (Frisio & Ventura, 2021; Josefsberg & Buckland, 2012; Tapia et al., 2019). Overall, the 21st century has seen significant advancements in vaccine production, with the development of new technologies and methods that have led to the creation of safe and effective vaccines for a wide range of diseases (Fig. 1.3). Industrial-scale vaccine production involves the manufacturing of large quantities of vaccines using standardized and quality-controlled processes (Fang et al., 2022; Tapia et al., 2017). The production of vaccines on an industrial scale is a complex process that involves several stages, including:

1. Antigen production: The first step in vaccine production is the production of the antigen, which is the component of the vaccine that stimulates an immune response (Alvarez et al., 2020). The antigen can be produced using a variety of methods, including growing the microorganism in a culture, using recombinant DNA technology, or purifying the antigen from the microorganism (Fig. 1.3).

2. Formulation: Once the antigen has been produced, it is combined with other components, such as adjuvants, stabilizers, and preservatives, to create a vaccine formulation that is stable and effective (Trabelsi et al., 2012).

3. Filling: The vaccine formulation is then filled into vials, syringes, or other containers, and sealed to prevent contamination (Bartczak et al., 2022).

Figure 1.3  Various steps of vaccine production in the upstream process. From Fang et al. (2022).

4. Quality control: Throughout the vaccine production process, rigorous quality control measures are in place to ensure that the vaccine is safe and effective (Shafyi & Mohammadi, 2018). This includes testing the vaccine for purity, potency, and safety.

5. Distribution: Once the vaccine has been produced and tested, it is distributed to healthcare providers and other organizations for use (Alvarez et al., 2020; Injac, 2022).

Industrial-scale vaccine production requires specialized facilities and equipment, as well as highly trained personnel to carry out the production and quality control processes (Fig. 1.3). The production of vaccines on an industrial scale has been instrumental in controlling and eradicating infectious diseases and continues to be an important tool in public health efforts around the world. Vaccine production in bioreactors is a common approach in industrial-scale vaccine production (Besnard et al., 2016; Syed et al., 2021). Bioreactors are specialized vessels that are designed to support the growth of microorganisms, such as bacteria or yeast, and can be used to produce large quantities of antigens or other vaccine components (Cervera et al., 2013; Shen et al., 2016).

The production of vaccines in bioreactors involves several stages, including:

1. Inoculation: The first step is to inoculate the bioreactor with the microorganism that will be used to produce the vaccine antigen (Marcelino et al., 2006). This can be done by adding a small amount of the microorganism to the bioreactor, which will then grow and multiply.

2. Growth: Once the microorganism has been inoculated into the bioreactor, it will begin to grow and multiply, using the nutrients in the culture medium. The bioreactor is designed to provide the ideal conditions for growth, including temperature, pH, and oxygen levels.

3. Harvesting: Once the microorganism has grown to the desired level, the vaccine antigen can be harvested from the culture (Coronel et al., 2020; Dormido et al., 2014). This is typically done by filtering the culture to remove the microorganisms and then purifying the antigen from the culture fluid.

4. Formulation and filling: Once the vaccine antigen has been purified, it can be combined with other vaccine components to create a vaccine formulation (Trabelsi et al., 2012). The vaccine formulation is then filled into vials, syringes, or other containers and sealed to prevent contamination.

5. Quality control: Throughout the vaccine production process, rigorous quality control measures are in place to ensure that the vaccine is safe and effective (Shafyi & Mohammadi, 2018). This includes testing the vaccine for purity, potency, and safety.

Bioreactor technology has revolutionized vaccine production, allowing for the production of large quantities of vaccines in a highly controlled and efficient manner (Fig. 1.3). The use of bioreactors has contributed to the development and distribution of vaccines for a wide range of diseases, including influenza, hepatitis B, and human papillomavirus (HPV). The COVID-19 pandemic has created an urgent need for the large-scale production of effective vaccines to control the spread of the disease (Frisio & Ventura, 2021; Injac, 2022). The production of COVID-19 vaccines on a large scale has been a significant challenge due to the unprecedented speed and scale of vaccine development, as well as the need to ensure vaccine safety and efficacy.

Several different approaches have been used to produce COVID-19 vaccines on a large scale. These include:

1. mRNA vaccines: The Pfizer-BioNTech and Moderna COVID-19 vaccines are both mRNA vaccines, which are produced using a process called in vitro transcription (Pardi et al., 2018; Schlake et al., 2012). This involves using enzymes to synthesize RNA molecules that encode the spike protein of the SARS-CoV-2 virus, which is the antigen used in the vaccine (Pardi et al., 2018). The mRNA is then packaged into lipid nanoparticles, which protect the mRNA and facilitate its delivery into cells.

2. Viral vector vaccines: The Oxford-AstraZeneca and Johnson & Johnson COVID-19 vaccines are both viral vector vaccines, which use a harmless virus (in this case, an adenovirus) to deliver the genetic material that encodes the spike protein of the SARS-CoV-2 virus into cells (Fulber & Kamen, 2022; Tapia et al., 2017). The cells then produce the spike protein, which triggers an immune response.

3. Protein subunit vaccines: The Novavax COVID-19 vaccine is a protein subunit vaccine, which uses recombinant DNA technology to produce large quantities of the spike protein of the SARS-CoV-2 virus (Heidary et al., 2022). The spike protein is then combined with an adjuvant to enhance the immune response.

To produce COVID-19 vaccines on a large scale, specialized facilities and equipment are needed, as well as highly trained personnel to carry out the production and quality control processes (Fig. 1.3). The production of COVID-19 vaccines on a large scale has required significant investment from governments and pharmaceutical companies, as well as collaborations between public and private sector organizations (Heidary et al., 2022).

Despite the challenges, the large-scale production of COVID-19 vaccines has been a remarkable achievement and has helped to bring the pandemic under control in many parts of the world (Plotkin et al., 2017).

There are several leading companies that specialize in vaccine production, with many of them playing a key role in the development and manufacturing of COVID-19 vaccines (Plotkin et al., 2017). Here are some of the major companies involved in vaccine production (Douglas & Samant, 2017).

1. Pfizer/BioNTech: Pfizer and BioNTech collaborated to develop the first authorized COVID-19 vaccine in the United States and the European Union, which is an mRNA vaccine called Comirnaty.

2. Moderna: Moderna also developed an mRNA-based COVID-19 vaccine, which is authorized for emergency use in several countries, including the United States and the European Union.

3. AstraZeneca: AstraZeneca developed a viral vector-based COVID-19 vaccine called Vaxzevria, which has been authorized for emergency use in many countries.

4. Johnson & Johnson: Johnson & Johnson developed a single-dose viral vector-based COVID-19 vaccine called Janssen COVID-19 Vaccine, which has also been authorized for emergency use in many countries.

5. Novavax: Novavax developed a protein subunit-based COVID-19 vaccine called NVX-CoV2373, which has shown promising results in clinical trials.

6. Sanofi/GSK: Sanofi and GlaxoSmithKline (GSK) have collaborated to develop a protein subunit-based COVID-19 vaccine called Vidprevtyn, which is currently in Phase III clinical trials.

These are just a few of the major companies involved in vaccine production. There are many other companies, including smaller biotech firms and contract manufacturing organizations, that play important roles in vaccine development and production (Campos-Ferreira et al., 2021; Douglas & Samant, 2017).

4. Bioreactor: Definition

For lab-scale and high-scale industrial-scale production, bioreactors play a very important role in producing desirable products using a defined substrate in the presence of a specific microbial strain (Bellani et al., 2020; Gallo-Ramírez et al., 2015; Robinson & Nigam, 2003; Strik et al., 2008). The produced product from the microbial strain may be intracellular or extracellular in nature.

The industrial process is divided into two parts: (a) upstream process and (b) downstream process. The produced product from a biotech process is defined in two categories such as (a) high volume low-cost product (example: ethanol, Baker's yeast, fermented food) and (b) low volume high-cost product (vaccines, antibiotics, and proteins). In the development of a new bioprocess for a new product formation, the below steps are used to design a particular specific process.

1. Identification of a substrate, which provides carbon and energy sources for growing a particular microbial strain. The substrate should also be available thought out the process and it available at a reasonable cost.

2. Identification of a high-yield product-producing microbial strain. Now due to advances in genetic engineering, a wild strain is also modified genetically to achieve high product yield and substrate utilization.

3. The selection of a specific bioreactor depends on the type of bioprocess, mode of operation, product quality, and cost.

4. Optimization of product purification using different downstream processing.

So, the bioreactor plays a key role in all the industrial bioprocess. The bioreactor is defined as a reactor that provides a suitable condition for culture to grow in well-defined conditions. A bioreactor also provides a suitable mixing inside so the substrate is available in the same concentration to all cells. Bioreactors also provide a proper environment with respect to heat control inside the reactor. Basically, various size bioreactors are used in different steps of a bioprocess such as (a) small-scale culture, (b) bench-top bioreactor, (c) pilot-scale bioreactor, and (4) industrial-scale bioreactor.

In general, bioreactors have the following features:

1. Sterilization capabilities: Bioreactors must be capable of being sterilized before use to ensure that the culture environment is free of any unwanted contaminants.

2. Control systems: Bioreactors are equipped with sophisticated control systems that allow for precise regulation of temperature, pH, dissolved oxygen levels, and other key variables that can affect cell growth and viability.

3. Mixing systems: Bioreactors typically have mixing systems that are designed to provide gentle and even mixing of the cell suspension. This is important for ensuring that all cells have access to the nutrients and oxygen they need for growth.

4. Sampling ports: Bioreactors may have sampling ports that allow for easy and noninvasive sampling of the cell suspension for analysis and monitoring purposes.

5. Harvesting capabilities: Bioreactors may be equipped with systems for harvesting the cells once they have reached the desired level of growth.

For cultivating microorganisms in the bioreactor, practical knowledge of a particular microbe's microbiology and kinetic analysis is needed (Guo et al., 2022; Oke & Ijardar, 2021). In the optimization steps from small-scale to the industrial-scale bioreactor, parameters such as medium composition, pH, temperature and other environmental parameters need to be optimized for high product yield (Huang & McDonald, 2009). Modern bioreactors are equipped with very advanced instruments to monitor the real-time values for operational parameters such as dissolved oxygen, carbon dioxide, pH, substrate and product concentration, and other process variables (Jin & Hong, 2018).

5. Bioreactor design

Bioreactors are widely used in industrial biotechnology for the production of a wide range of products, including proteins, antibodies, vaccines, and cell therapies (Palomares & Ramírez, 2009). A bioreactor is a vessel or system that provides a controlled environment for the growth and proliferation of cells or microorganisms. Various types of bioreactors are used for different growing conditions of microorganisms.

5.1. Bioreactor for cell suspension

A bioreactor for cell suspension is a type of bioreactor that is used to cultivate cells in suspension. In a cell suspension reactor, cells are grown in a liquid medium that is agitated to ensure proper mixing and aeration. In the case of cell suspension, bioreactors are designed to support the growth and expansion of cells in suspension culture (Huang & McDonald, 2009). The cells are suspended in a nutrient-rich media and are agitated to ensure efficient oxygen and nutrient transfer. There are different types of bioreactors for cell suspension culture, including stirred-tank reactors, wave bioreactors, and airlift bioreactors (Dryden et al., 2021). Stirred-tank bioreactors are the most commonly used bioreactors for cell suspension culture. They are designed with an impeller that rotates at high speeds to provide agitation and oxygen transfer. The cells are suspended in the media and are constantly mixed to ensure an even distribution of nutrients and oxygen (Marcelino et al., 2006; Tapia et al., 2017).

Wave bioreactors are a newer technology using wave-like motion to provide agitation and oxygen transfer. The cells are suspended in a bag that is attached to a platform that moves up and down to create waves (Bartczak et al., 2022). This system is ideal for the large-scale production of cell therapies and vaccines. Airlift bioreactors use a column with a draft tube to create a circulating flow of media and cells. The cells are suspended in the media and are lifted to the top of the column where they are exposed to oxygen (Sánchez Mirón et al., 2002). This system is ideal for low-shear stress cultures and the production of high-density cell suspensions. Overall, bioreactors for cell suspension culture are essential for the production of many biopharmaceuticals and cell therapies (Minteer et al., 2014; Tavassoli et al., 2018). The selection of the appropriate bioreactor system will depend on the specific application and the desired cell culture conditions. Fig. 1.4 shows various kinds of bioreactors used for industrial-scale and lab-scale production.

There are several types of cell suspension reactors, including.

1. Stirred-tank bioreactor: This is the most commonly used type of reactor for cell suspension (Fig. 1.4). It consists of a tank with an impeller that agitates the cell suspension and provides oxygen and nutrients to the cells (Tapia et al., 2017, 2019).

2. Bubble column bioreactor: This type of reactor uses bubbles to provide agitation and oxygen transfer to the cells (Hu et al., 2017; Seo et al., 2012). The cells are suspended in a liquid medium, and the column is filled with gas that bubbles through the medium, providing oxygen to the cells.

3. Airlift bioreactor: This reactor uses a draft tube to create a circulating flow of liquid and cells (Sánchez Mirón et al., 2002). The cells are lifted to the top of the reactor by air bubbles, where they are exposed to oxygen (Fig. 1.4).

4. Wave bioreactor: This type of reactor uses a wave-like motion to provide agitation and oxygen transfer to the cells (Bartczak et al., 2022; Zhan et al., 2019). The cells are grown in a plastic bag that is attached to a platform that moves up and down to create waves.

5. Spin filter bioreactor: This is a newer type of reactor that uses a rotating filter to separate the cells from the medium (Siegel et al., 1992). The cells are grown on the filter and are continuously exposed to fresh medium and oxygen (Niño-López & Gelves-Zambrano, 2015).

Figure 1.4  Various bioreactors used for vaccine production and culturing stem cells. From Zhang et al. (2019).

The choice of bioreactor for cell suspension will depend on a variety of factors, including the type of cells being grown, the required oxygen transfer rate, the required mixing intensity, and the scale of production (Fulber & Kamen, 2022; Huang & McDonald, 2009; Shen et al., 2019). Each type of reactor has its advantages and disadvantages, and the appropriate choice will depend on the specific needs of the application (Fig. 1.4). A bioreactor provides a controlled environment for the growth and proliferation of cells in a liquid medium. Bioreactors for cell suspension typically have several features that are specifically designed to support the growth and maintenance of microorganisms or cells in suspension (Huang & McDonald, 2009).

Overall, bioreactors for cell suspension are an essential tool for the production of a wide range of biopharmaceuticals and industrial products that require the cultivation of microorganisms or cells in suspension (Fig. 1.4). The choice of bioreactor will depend on a variety of factors, including the specific needs of the application, the type of microorganism or cell being grown, and the desired scale of production.

5.2. Hollow-fiber and membrane bioreactor

A hollow-fiber bioreactor (HFB) is a type of bioreactor used for cell culture, which uses semipermeable hollow fibers to create a separation between the cell culture medium and the surrounding environment (De Bartolo, 2016; Miyoshi et al., 2018; Pankhania et al., 1994). The hollow fibers are typically made from materials such as polyethylene or polycarbonate and are arranged in a bundle within the bioreactor (Coronel et al., 2020; Miyoshi et al., 2018). The bundle is then enclosed in a container that can be pressurized with gas to provide aeration to the cell culture medium (Pankhania et al., 1994). The cell culture medium is then circulated through the hollow fibers, providing a large surface area for the cells to grow and exchange nutrients and waste products with the surrounding medium (Miyoshi et al., 2018; Nikolay et al., 2020). One of the key advantages of the HFB is the high surface area to volume ratio provided by the fibers, which allows for efficient oxygen and nutrient transfer and waste removal (Fig. 1.4). The semipermeable nature of the fibers also allows for the separation of the cell culture medium and the surrounding environment, which can reduce the risk of contamination and increase the yield of the cell culture (De Bartolo, 2016; Nikolay et al., 2020; Pankhania et al., 1994). HFBs are used in a variety of applications, including the production of vaccines, therapeutic proteins, and other biopharmaceuticals (De Bartolo, 2016). They are particularly useful for the cultivation of anchorage-dependent cells, which require a surface to attach to grow. The fibers provide a suitable surface area for the cells to attach to, while still allowing for efficient nutrient and waste exchange (Miyoshi et al., 2018).

Overall, HFBs are an important tool for cell culture in biotechnology, offering advantages in terms of efficient nutrient transfer, reduced contamination risk, and high yield of cell culture (De Bartolo, 2016). A membrane bioreactor (MBR) is a type of bioreactor that combines biological treatment with membrane filtration. It is commonly used in wastewater treatment, but can also be used in other applications such as bioremediation and bioprocessing (Miyoshi et al., 2018; Mutamim et al., 2013; Salerno et al., 2022). The MBR typically consists of a tank or basin that contains an activated sludge system, which is a mixture of microorganisms that degrade the organic matter in the wastewater (Fig. 1.4). The membrane filtration component of the MBR is a semipermeable membrane that acts as a barrier to separate the activated sludge from the treated water (Salerno et al., 2022).

The membrane can be made from a variety of materials, such as polymeric or ceramic materials, and can be configured in different ways, including flat sheets or hollow fibers. The membrane pore size can also vary depending on the application, ranging from microfiltration to nanofiltration (Salerno et al., 2022). The HFBs and MBRs are both types of bioreactors that use membranes for separation, but there are some key differences between the two. One of the main differences between HFBs and MBRs is the type of membrane used. HFBs use semipermeable hollow fibers made from materials such as polyethylene or polycarbonate, while MBRs use flat or tubular membranes made from various materials including ceramics, polymers, or metals (Salerno et al., 2022). The pore size of the membranes can also differ between the two, with HFBs typically having larger pore sizes compared to MBRs (Miyoshi et al., 2018; Nikolay et al., 2020). Another difference between the two bioreactors is their primary application. HFBs are primarily used for cell culture applications, such as the production of therapeutic proteins or vaccines (Fig. 1.4). The large surface area of the hollow fibers provides a suitable surface for anchorage-dependent cells to attach to and grow, while still allowing for efficient nutrient exchange. In contrast, MBRs are primarily used for wastewater treatment applications, where they provide high-quality effluent with low levels of suspended solids and pathogens (Nikolay et al., 2020). The operation and maintenance of the two bioreactors also differ. HFBs require more precise control of parameters such as temperature, pH, and dissolved oxygen levels to ensure the health and viability of the cells being cultured. MBRs, on the other hand, require more frequent cleaning and maintenance to prevent fouling and clogging of the membrane. Overall, while both HFBs and MBRs use membrane separation, they are designed for different applications and have different membrane types and operation and maintenance requirements (Nikolay et al., 2020). One of the main advantages of MBRs is their ability to produce high-quality effluent with low levels of suspended solids and pathogens. The use of a membrane barrier also eliminates the need for secondary clarifiers, reducing the overall footprint of the treatment process (Miyoshi et al., 2018). MBRs are also highly efficient in removing organic matter, nitrogen, and phosphorus from wastewater, making them a popular choice for wastewater treatment in industries such as food and beverage, pharmaceuticals, and chemical manufacturing (Mutamim et al., 2013; Teplyakov et al., 2002).

However, MBRs can be more expensive to operate and maintain compared to traditional wastewater treatment systems, due to the cost of membrane replacement and the need for more frequent cleaning and maintenance (Fig. 1.4). Additionally, fouling and clogging of the membrane can occur over time, which can impact the overall efficiency of the system (Nikolay et al., 2020; Salerno et al., 2022). Overall, membrane bioreactors are an important technology for wastewater treatment and other applications where high-quality effluent is required (Nikolay et al., 2020). While they may be more expensive to operate, the benefits of high-efficiency treatment and reduced footprint can make them a valuable investment for many industries.

5.3. Fluidized-bed and fixed-bed bioreactor

A fluidized-bed bioreactor (FBB) is a type of bioreactor that uses a suspension of solid particles in a liquid to provide a high surface area for biological activity (Berrie et al., 2020). In an FBB, the particles are fluidized, or kept in constant motion, by a flow of liquid through the reactor. This creates a fluidized bed of particles that provides an ideal environment for microbial growth and metabolism (Berrie et al., 2020). The FBB is commonly used in wastewater treatment applications, where it provides efficient removal of organic matter, nitrogen, and phosphorus from wastewater. The high surface area of the fluidized particles allows for the attachment and growth of microorganisms, which can degrade organic matter in the wastewater. The fluidized bed also promotes mixing and mass transfer, which enhances the efficiency of the bioreactor (Berrie et al., 2020). FBBs can also be used in other applications, such as the production of enzymes, biopolymers, and other bioproducts. The high surface area of the fluidized bed allows for efficient contact between the microorganisms and the substrate, which can enhance the production of desired products (Fig. 1.4).

One advantage of FBBs is their high efficiency in treating wastewater and producing bioproducts, due to the large surface area provided by the fluidized bed of particles. FBBs are also more compact and have a smaller footprint compared to other types of bioreactors, making them suitable for applications where space is limited (Berrie et al., 2020). However, FBBs can be more difficult to operate and maintain compared to other types of bioreactors, due to the need to maintain the fluidized bed of particles and prevent particle aggregation or loss. Additionally, the particles in the FBBs can become clogged over time, which can impact the overall efficiency of the system. Overall, fluidized-bed bioreactors are an important technology for wastewater treatment and other bioprocessing applications, where high efficiency and a small footprint are desired (Fig. 1.4). While they may require more maintenance compared to other bioreactors, their ability to provide high surface area for microbial activity makes them a valuable technology for many applications. A fixed-bed bioreactor (FBBR) is a type of bioreactor that uses a solid support material, such as sand or activated carbon, to provide a surface for microbial attachment and growth (Rajendran et al., 2014). The solid material is packed into the reactor and provides a fixed bed for the microbial population to