Explanation of the first box – upstream process

1- Biological Drugs
Biological drugs are primarily recombinant therapeutic proteins produced through biological or biotechnological processes, using various expression systems such as mammalian cell lines, microorganisms, insects, and plants.

1-1- Cells and Microorganisms for Producing Biological Drugs

1-1-1- Bacteria

The use of biological protein drugs in human health dates back to the 19th century, when diphtheria antitoxin isolated from the serum of immunized animals, such as horses or sheep, was first used. Later, in the 20th century, molecules like insulin and growth hormone— all derived from animal sources— were utilized.

Given the risks associated with non-human animal proteins, the biological drug industry began producing biological drugs using recombinant DNA techniques in microorganisms. A classic example of this approach is the production of insulin (for treating type I and II diabetes) in Escherichia coli. Initially, insulin was purified from bovine and porcine pancreas, which was not only expensive but also led to immune responses in patients due to animal-derived insulin. However, in the recombinant DNA technique, the human target gene is isolated from the host and attached to a vector (plasmid), and this plasmid containing the human gene is used to transform bacterial cells, enabling them to produce high quantities of the recombinant target protein.

 

1-1-2- Filamentous Fungi

The wide variety of molecules produced by filamentous fungi justifies their use for the production of biological drugs. For example, paclitaxel (a natural anti-cancer substance) is isolated from certain endophytic fungi, or β-d-galactosidase (lactase – EC. 3.2.1 23), an extracellular enzyme produced by filamentous fungi, is used for production. This enzyme catalyzes the conversion of lactose to glucose and galactose, and is used in the treatment of lactose intolerance.

Another biological drug derived from fungi is the enzyme asparaginase. This enzyme is used to treat various diseases, such as acute lymphoblastic leukemia and non-Hodgkin lymphoma. Since tumor cells rely on an external source of asparagine for replication, a drug that depletes asparagine from the bloodstream leads to the death of cancer cells.

Despite the effectiveness of biological drugs produced by filamentous fungi, the application of biologically produced molecules from such organisms is still limited due to the high cost of purifying certain molecules and challenges in culturing filamentous fungi. The table below lists some biological drugs produced by fungi.

Compound	Organism
Taxol	Taxomyces andrenae
Beta-galactosidase	A. foetidus
Lovastatin	Monascus rubber
	A. terreus
l-asparaginase	A. terreus
Ergot alkaloids	Claviceps purpurea
Griseofulvin	P. griseofulvum
Proteases	Aspergillus sp
	Penicillium sp
Amphotericin B	Penicillium nalgiovense

1-1-3- Yeasts

Yeasts are used as host organisms for the production of recombinant compounds, synthesizing a wide variety of substances such as aromatics, terpenoids, sterols, alcohols, sugar derivatives, citric acid, lactic acid, organic and fatty acids, terpenes, peptides, and several important therapeutic proteins.

Over 40 different recombinant proteins have been produced by Saccharomyces cerevisiae, including several biological drugs, which are listed in the table below.

Type	Protein	Therapeutic application
Blood related	Human Serum Albumin	Surgery (plasma expander)
	Hirudin	Blood coagulation disorders
	Human transferrin	Anemia
Hormones	Insulin Precursor	Diabetes
	Glucagon	Diabetes
Antigen	Hepatitis surface antigen	Hepatitis vaccination

Yeasts, particularly Saccharomyces cerevisiae, are used as hosts for the production of recombinant compounds, synthesizing a variety of substances such as aromatics, terpenoids, sterols, alcohols, sugar derivatives, citric acid, lactic acid, organic and fatty acids, terpenes, peptides, and several important therapeutic proteins.

Biological drugs produced by Saccharomyces cerevisiae include insulin and its analogs, human serum albumin, hepatitis vaccines, and virus-like particles, for example, for vaccination against human papillomavirus.

The advantages of using Saccharomyces cerevisiae for the production of biological drugs include:

• Proper folding of many human proteins produced
• Extracellular secretion of produced proteins, facilitating the purification process
• Many post-translational modifications, such as disulfide bond formation, sialylation, and glycosylation.

However, despite these advantages, there are some limitations, such as the yeast’s inability to perform high-mannose N-glycosylation, which can reduce the half-life and efficacy of proteins.

1-1-4-Mammalian Cells

Using mammalian cells for the production of biological drugs has historically been associated with the following challenges:

• More difficult production of recombinant mammalian cells compared to microorganisms
• More difficult scaling up of mammalian cell culture compared to microorganisms
• Lower yield of mammalian cells compared to microorganisms
• Complexity of the cell culture medium required for mammalian cell growth
• The need for serum for mammalian cell growth
• Sensitivity of mammalian cells to environmental stresses
• Slower growth rate of mammalian cells compared to other microorganisms

Despite these challenges, mammalian cells are capable of post-translational modifications, including glycosylation, making them suitable for the production of complex therapeutic antibodies. After decades of optimization in cell lines, culture media, and bioreactors, protein expression levels of more than 10 g/L and cell densities of over 20 X 10^6 cells/mL are now achievable for mammalian cells.

1-1-5-Insect Cells

A relatively small portion (only 2-3%) of all newly approved active pharmaceutical compounds are produced using insect cells as hosts. Insect cells are used to produce recombinant proteins and virus-like particles (VLPs). The selected system for insect cell cultivation is typically suspension culture. It should be noted that insect cells, such as Spodoptera frugiperda (Sf9) and Trichoplusia ni (Hi5), show oxygen consumption rates up to 13 times higher than that of CHO cells.

Insect cells are mainly used with the baculovirus expression vector system (BEVS) and are rapidly emerging as a platform for recombinant protein production. Insect cells offer several advantages over mammalian cells, such as easier cultivation, higher tolerance to osmolarity, fewer by-products, and higher expression levels. However, since proteins produced by insect cells have shorter N-glycans and lower sialylation, they may not be suitable for all applications.

2- Biological Drug Production Process

The production technology for biological drugs can be divided into upstream and downstream processes.

2-1-Upstream Production Process

The upstream production process refers to the cell or microorganism growth stage needed for the production of biological drugs or other biological molecules. This includes selecting the cell line, choosing the culture medium, adjusting growth parameters, and optimizing the production process to achieve optimal conditions for cell growth and biological drug production. The primary goal of the upstream process is to convert the culture medium into the desired metabolic products. This requires highly controlled conditions (such as temperature, pH, dissolved oxygen, agitation rates, etc.) and the use of appropriate culture equipment (bioreactors, fermenters, roller bottles, etc.) at large scale.

Upstream biological drug production is typically carried out in two general ways:

2-1-1-Adherent Cell Culture

Adherent cell culture is a type of cell culture where cells need to attach to a two-dimensional surface for growth, facilitating cell adhesion and spreading. Most vertebrate-derived cells (except blood cells) can be cultured this way. In this type of culture, the following equipment is used for cell culture and volume expansion:

2-1-1-1-Petri dishes and T-Flasks

Typically, adherent cells grow as a monolayer in culture vessels such as Petri dishes and T-Flasks. T-Flasks vary in size and can provide surface areas ranging from 25 cm² to 225 cm² for cell culture. These culture vessels are also used for small-scale seed cell production.

2-1-1-2- Multi-tray systems

Multi-Tray In cell culture systems that require surface areas up to 25,400 cm², multi-tray systems are used. These systems utilize stacked trays, providing a large, multi-layer surface for cell attachment and growth. The principle of multi-tray systems is based on increasing the surface area available for cells to adhere to by stacking trays, similar to a large version of a T-Flask.

While these systems have advantages over traditional T-Flasks in terms of surface area, they still retain many of the limitations of T-Flask systems. Essentially, the multi-tray system is just a larger, multi-layered version of a T-Flask. These systems remain static, without agitation, aeration, or movement of the culture medium, which can lead to challenges in ensuring uniform conditions for cell growth.

One concern with multi-tray systems is the potential difference in gas exchange between the upper and lower layers of the trays. This can result in variations in oxygen and carbon dioxide levels, which may affect the performance and quality of the cells. Such differences in gas exchange can lead to suboptimal growth conditions, and, ultimately, reduced cell viability and production yield.

2-1-1-3-Roller Roller Bottles

Roller bottles are widely used in biotechnology applications, particularly in vaccine development. Unlike static systems, roller bottles provide agitation of the culture medium through rollers placed on the bottle, which allow for continuous movement. This motion promotes better gas exchange and nutrient distribution within the culture.

One of the main advantages of roller bottles is their ability to provide a larger surface area for cell attachment and growth compared to standard T-Flasks. This is particularly important in applications requiring larger-scale cell culture or when high cell densities are needed. The rolling motion also reduces the risk of cell clumping and helps maintain a uniform environment for optimal cell growth.

However, while roller bottles improve agitation and increase surface area, they still have limitations in terms of scalability and the requirement for precise control over the growth environment. Additionally, they typically require more space and careful monitoring to ensure that the cells are not exposed to shear stress or uneven conditions that could affect their growth and productivity
.

2-1-2- Suspension Cell Culture (Bioreactor or Fermentor)

The suspension cell culture process at large scale involves several stages, including the seed train (carried out in shake flasks or spinner flasks), inoculum train (conducted in bioreactors), and the production phase.

1. Seed Train: This initial stage is where small volumes of culture are prepared and expanded to increase the number of cells for inoculation into larger culture systems. Shake flasks or spinner flasks are commonly used in this phase to grow the cells in a controlled environment.
2. Inoculum Train: At this stage, the cells grown in the seed train are transferred into larger bioreactors, where they are further expanded and prepared for the production phase. Bioreactors offer a controlled environment to optimize cell growth, as they allow for precise control over parameters like pH, temperature, oxygen levels, and agitation.
3. Production Phase: Once the inoculum has been successfully expanded, the culture enters the production phase, where cells produce the desired biologic product (e.g., therapeutic proteins). This phase is critical as it involves maximizing protein expression while ensuring high cell viability and quality of the final product.

Success in scaling up the process is typically evaluated based on key process indicators, such as:

• Cell density: A higher cell density typically leads to higher product yields.
• Viability: Monitoring the percentage of viable cells is essential for ensuring that the process remains productive.
• Protein expression levels: The amount of therapeutic protein expressed by the cells is a key quality metric.
• Product quality: Characteristics such as purity, glycosylation patterns, and bioactivity are essential to meet regulatory standards for biologic products.

Bioreactors and fermentors offer advanced capabilities to manage large-scale cell cultures and are integral to the production of biologic drugs.

Spinner Flask

Shake Flask

2-1-2-1- Upstream Cell Culture Challenges in Large-Scale Bioreactors

When scaling up cell culture to large-scale bioreactors, several challenges may arise, especially regarding the efficient supply of oxygen and the removal of CO2 produced by cells. These challenges largely depend on the operational parameters set for the final bioreactor. Some of the critical parameters that need to be optimized include:

Critical Operational Parameters for Bioreactors:

• Cell Culture Volume: The total volume of the cell suspension in the bioreactor plays a major role in oxygen transfer and nutrient exchange.
• Feed Volume and Feed Rate: The quantity and frequency at which nutrients are added to the bioreactor affect the growth and metabolism of the cells.
• Agitation Rate: Proper mixing and agitation ensure the uniform distribution of nutrients and gases, as well as the prevention of cell clumping.
• Aeration and Dissolved Oxygen Levels: Sufficient aeration and control of dissolved oxygen are crucial for maintaining cell viability, as most mammalian cells require oxygen for optimal growth.
• pH: Maintaining the correct pH is vital to ensure cellular processes proceed efficiently and to avoid conditions that could inhibit cell growth or protein production.
• Temperature: Optimal temperature control is necessary to maintain cellular metabolism and prevent stress that could affect growth and productivity.

Additional Operational Challenges in Large-Scale Bioreactors:

Apart from the technical parameters, several other challenges are associated with large-scale cell culture in bioreactors:

• Batch-to-Batch Variation in Raw Materials: The composition and quality of raw materials (e.g., media components, growth factors) may vary from batch to batch, potentially affecting consistency in cell growth and product yield.
• Uniformity of Culture Medium in Different Volumes: Ensuring that the medium is mixed evenly in larger volumes is challenging. This can impact nutrient availability and waste product removal, leading to variations in cell growth.
• Cell Stability: Cells may undergo genetic changes or mutations over time that can affect their ability to produce the desired product consistently. Ensuring cell line stability is a key consideration.
• Contamination Risk: Large-scale bioreactors are vulnerable to contamination by other microorganisms, which can severely impact production yields and product quality.
• Human Error: Given the complexity of the processes involved, human errors during monitoring, adjustments, or sampling can lead to inconsistencies and operational failures.

Overall, achieving optimal conditions in large-scale bioreactor systems requires careful control and monitoring of these factors to ensure high-quality, consistent production of biological products.