Biomanufacturing Transcript PDF

PROPRIETARY. DO NOT SHARE. Transcript: Biomanufacturing Section 1: Cell Lines Welcome Welcome to Biotech Primer’s Biomanufacturing class. This class is divided into 5 sections: Cell Lines, Cell Banks, Upstream Bioprocessing, Downstream Bioprocessing, and Advancements in Biomanufacturing. Let’s begi...

PROPRIETARY. DO NOT SHARE. Transcript: Biomanufacturing Section 1: Cell Lines Welcome Welcome to Biotech Primer’s Biomanufacturing class. This class is divided into 5 sections: Cell Lines, Cell Banks, Upstream Bioprocessing, Downstream Bioprocessing, and Advancements in Biomanufacturing. Let’s begin with Cell Lines — What are they? What do they do? How are they made? Section 1: Cell Lines Objectives At the end of this section, you should be able to: • Define biomanufacturing. • Compare and contrast the two types of cells used to manufacture biologics. • Explain the steps of cell line development. Biomanufacturing Defined Biomanufacturing is the process of making biological products from living cells. Biological products include enzymes, vaccines, hormones, and monoclonal antibodies. Collectively these are called by different names including biologic product, protein product, therapeutic protein, or biologic. Regardless of the name used, these are medicines used by hundreds of millions of people worldwide to cure a multitude of diseases. Research and Development Before a therapeutic protein is ready to be manufactured, several steps must take place. Let’s start at the very beginning, with Research and Development. The job of Research and Development is three-fold: First, R&D must identify the drug target. Second, they must identify the protein that will interact with that drug target. Third, R and D must identify the gene of interest, the gene that codes for the protein. Once the protein has been identified R&D will create a cell line, a cell that will produce that protein. Before we discuss cell lines, let’s take a closer look at cells in general. 1 Copyright 2024 Biotech Primer Inc. PROPRIETARY. DO NOT SHARE. Therapeutic Protein Examples This screen shows a few examples of therapeutic proteins created by biomanufacturing. Insulin, a small, simple therapeutic protein indicated for diabetics, is manufactured within the prokaryotic cell, a bacterium named E. Coli. Humira, a large, complex monoclonal antibody indicated for rheumatoid arthritis, is manufactured within a eukaryotic cell, specifically a mammalian cell called a CHO cell. Types of Cells Cells can be divided into two main branches — prokaryotes and eukaryotes. Prokaryotes are unicellular. They do not have a nucleus, so their DNA floats freely within the cell. Prokaryotes are small, measuring only a few microns. Bacteria are an example of a prokaryote. We use bacteria — a prokaryote — to manufacture small, simple therapeutic proteins. Eukaryotes are both unicellular and multicellular. Eukaryotes have a nucleus, so their DNA is protected by an additional membrane. Eukaryotes are large, measuring 10 microns and up. Examples include unicellular yeast and multicellular mammalian cells. Both yeast and mammalian cells are used to create larger, more complex therapeutic proteins. Cell Lines Biologic drugs are produced in cells. Cell lines are cells engineered to produce a particular product called "cell lines". For established cell lines, recombinant DNA is introduced into bacteria in the form of plasmids. The plasmid contains the gene coding for the therapeutic protein. Once the bacteria receive the plasmid, it will make the recombinant protein from the non-bacterial gene as if it was one of its genes. Bacteria divide quickly — some strains every 20 minutes or so. So, it is essential to quickly obtain a culture with only bacteria harboring the introduced plasmid. Otherwise, they may be lost by growth competition by non-plasmid-containing bacteria. So, scientists include an antibiotic-resistant gene in the plasmid. This is a gene from antibioticresistant bacteria that produces a protein that makes the bacterium resistant to the killing properties of an antibiotic, like ampicillin or neomycin, or tetracycline. This is usually a protein that 2 Copyright 2024 Biotech Primer Inc. PROPRIETARY. DO NOT SHARE. destroys the antibiotic. So, when the antibiotics are added to the bacterial culture, those bacteria that do not have the plasmid die, and those that have the plasmid survive and divide to produce a culture of bacteria that all possess the recombinant plasmid. Biologics are produced in cells. Cell lines are cells engineered to produce a particular product such as therapeutic proteins. Bacterial Cell Line Development Let’s take a closer look at cell line development in bacteria. If more background information is needed, consider taking our Introduction to Genetic Engineering. Let’s begin with the recombinant plasmid that has been genetically engineered to contain the gene of interest and antibiotic-resistant gene. Recall the gene of interest codes for the therapeutic protein. First, we must put the plasmid into the bacterial cell. Two common methods used are heat shock and electroporation. Both methods create pores in the membrane allowing the plasmid to enter the bacteria. Not all the bacteria will survive the process or take up the plasmid, so the next step kills off the bacteria that do not contain the plasmid. Not all bacteria take up the plasmid. The bacteria that do not contain the plasmid cannot produce therapeutic protein so those bacteria must be removed from the cell culture to make room for the bacteria that do house the plasmid. This is done with the help of the antibiotic resistance gene. The bacteria that have not taken up the plasmid will die in the presence of the antibiotic. Therefore, by simply adding the antibiotic to the cell culture the bacteria that have not taken up the plasmid are killed, while at the same time, the bacteria that have taken up the plasmid remain and multiply. All the remaining bacteria can create therapeutic proteins coded by the gene of interest. Mammalian Cell Line Development Mammalian cells are more sensitive to environmental changes, and therefore will not survive the process of heat shock or electroporation that was used to transfer a plasmid into bacterial cells. Therefore, a structure known as a liposome is used. A liposome is a sac into which a plasmid is 3 Copyright 2024 Biotech Primer Inc. PROPRIETARY. DO NOT SHARE. injected. The liposome’s membrane is composed of lipids just like the cell membrane. Because of this, the liposome can enter the cell through the membrane. Once inside the cell, the plasmid is released from the liposome and enters the cell nucleus. We cannot use antibiotic selection because mammalian cells are resistant to antibiotics. Therefore, an essential nutrient gene is added to the plasmid. An essential nutrient is needed by the eukaryotic cell to live. Because this essential nutrient is removed from the growth media only cells that produce the nutrient themselves can survive. Only cells that have taken up the plasmid can create therapeutic protein. Bacterial vs Mammalian Cell Lines Now that we have explained how prokaryotic and eukaryotic cell lines are made, let’s explore the main differences between the two. Bacterial cells grow and divide quicker than mammalian cells. Bacteria divide every 20 to 30 minutes. Mammalian cells divide once a day on average. Because of this reproduction time difference, a full production campaign in bacterial cells is completed in days, while a full production campaign in mammalian cells takes weeks. Additionally, the growth media for bacterial cells is relatively inexpensive when compared to the media needed to culture mammalian cells. Bacteria are more stable, while mammalian cells are much more sensitive to changes in the cell culture environment. Changes in temperature, pH, cell density, and other factors affect mammalian cells more than bacterial cells. So, after looking at this list you might be wondering why anyone would want to use mammalian cells: they are slower, more costly, and more difficult to work with. And you would be right, if it were possible, it would always be easier and less expensive to use bacterial cells. However, bacterial cells have limitations due to their size and simple structure— bacterial cells can only produce small, simple proteins. Therefore, if the desired therapeutic protein is large and requires complex folding or post-translational modifications, a mammalian cell line must be used to create that biologic. 4 Copyright 2024 Biotech Primer Inc. PROPRIETARY. DO NOT SHARE. Monoclonal Antibodies Produced in Mammalian Cell Line The creation of monoclonal antibodies is an excellent example of bacterial vs mammalian cells for protein creation. Because mammalian cells can perform post-therapeutic modifications, such as glycosylation, bacterial cells are unable to produce therapeutic monoclonal antibodies. To create therapeutic monoclonal antibodies eukaryotic cells must be used. Glycosylation patterns can also be different between cell lines used in biomanufacturing so determining the best cell line to use can be challenging. Eukaryotic Cell Lines As mentioned, eukaryotic cells are needed to manufacture more complex proteins that require post-translational modifications. Several cell types are currently being used in biomanufacturing to produce proteins. Some examples include: • Pichia cells are yeast cells used to manufacture human-type plasminogen activators, or t-PA indicated for stroke. • Sf9 cells are insect cells used to manufacture virus-like particles, or VLPs, used in vaccines. Mammalian cells include: • NS0 cells are used to create monoclonal antibodies. • PerC6 cells produce a variety of biopharmaceutical products, including vaccines, gene therapy products, monoclonal antibodies, and other therapeutic proteins. Currently, the most common mammalian cell in biomanufacturing is the Chines Hamster Ovary or CHO cell. CHO cells currently produce many monoclonal antibodies on the market. Section 1: Cell Lines Summary In this section, we learned: • Biomanufacturing is the process of making biological products from living cells. • The two types of cells used to manufacture biologics are prokaryotic and eukaryotic cells. An example of a prokaryotic cell is a bacterial cell. Bacteria can 5 Copyright 2024 Biotech Primer Inc. PROPRIETARY. DO NOT SHARE. manufacture small, simple biologics. An example of a eukaryotic cell is a mammalian cell. Mammalian cells can manufacture large, complex biologics. • The steps of cell line development include creating a plasmid that houses the gene of interest and a selection gene. Placing that plasmid into the production cell. Selecting for and growing the production cell. 6 Copyright 2024 Biotech Primer Inc. PROPRIETARY. DO NOT SHARE. Section 2: Cell Banks Welcome In the last section, we demonstrated how cell lines are produced in both prokaryotic and eukaryotic cell lines. In this section, we will learn how cell lines are used to establish cell banks. Section 2: Cell Banks Objectives At the end of this section, you should be able to: • Define cell banking. • Compare and contrast the master cell bank with the working cell bank. • Explain why cell banking is necessary to ensure drug safety and efficacy. Identifying Best Clone Once a cell line is created, that line is screened to identify the best clone to move forward with into production. This involves measuring each prospective cell line’s production capacity — which means calculating how many grams of protein can be produced per liter of cells and calculating the number of impurities produced. Once the best clone is identified, it is used to make the master cell bank. Cell Banks The term Cell Banking refers to the process of storing cells of a specific genome, for future use in the production of biologic drugs. Cell banks are critical to ensuring the continuity of product identity and quality. When cells are stored at extremely low temperatures — 210 degrees Celsius — all cell growth is stopped. Essentially capturing a moment in time. Decades after a production cell line is created, a company can access the original cell line by going back to the stored cells. Why is the ability to go back to the original cell line important? All cells grow and change with time so companies must have access to the original cell line. Two Types of Cells Banks There are two types of cell banks — master cell banks (MCBs) and working cell banks (WCBs). 7 Copyright 2024 Biotech Primer Inc. PROPRIETARY. DO NOT SHARE. Master Cell Bank Production A master cell bank is created by expanding the original production cell line for a few generations and creating a few hundred vials of cells, each vial containing about one million cells. Because of its critical importance, the master cell bank will usually be divided into two or three and stored in distinct geographic locations. That way, if storage conditions are not able to be maintained due to natural disasters or other unforeseen circumstances in one location, the company will not lose its entire master cell bank. Working Cell Bank Production A working cell bank is created by taking one vial from the master cell bank, expanding it for a few generations, and again freezing down hundreds of vials that each contain a few million cells. Working cell banks are the cell banks from which cells are taken to start each new manufacturing campaign. Why Is Accessing the Original Cell Line Important? Differences in protein structure may exist even between two proteins made from the same gene but produced by different cell lines. To ensure the same therapeutic protein is produced year in and year out the same cell line must be used. Section 2: Cell Banks Summary In section two, we learned: • Cell banking refers to the process of storing cells of a specific genome, for future use in the production of biologic drugs. • A master cell bank was created by expanding the original production cell line for a few generations. A working cell bank is created from the master cell bank and is used to start each new manufacturing campaign. • Cell banking is necessary to ensure drug safety and efficacy. Cell banks ensure the same therapeutic protein is made each time a production campaign is run. 8 Copyright 2024 Biotech Primer Inc. PROPRIETARY. DO NOT SHARE. Section 3: Upstream Processing Welcome Now that you understand how cell lines and cell banks are made and the reasons behind these two processes, let’s discuss upstream bioprocessing. Upstream bioprocessing is the first step in the biomanufacturing process. Let’s begin. Section 3: Upstream Processing Objectives By the end of this section, you should be able to: • Define upstream bioprocessing. • List the three main goals of upstream bioprocessing. • Explain how to optimize these goals. • Describe the steps taken in upstream bioprocessing. The Campaign The scale-up of a cell culture process can be very difficult and time-consuming, taking as long as 3 to 6 weeks before the product is obtained. The entire process of producing a biotech product from start to finish is often called a “campaign” and is usually divided into two main parts — upstream and downstream. Upstream Bioprocessing Defined Upstream bioprocessing is when biological materials are grown, under controlled conditions, to manufacture certain types of products. Upstream bioprocessing refers to the initial stage in which cells are grown, for example, either bacterial or mammalian cell lines in bioreactors. Upstream Bioprocessing Development Goals The goals for upstream bioprocess development include optimizing cell density, optimizing cell viability, and optimizing protein production. Routes to Optimization of Goals To achieve these upstream bioprocessing development goals - media composition, engineered production cells, and bioreactor conditions must be maximized. 9 Copyright 2024 Biotech Primer Inc. PROPRIETARY. DO NOT SHARE. Media generally comprise an appropriate concentration of energy and compounds which regulate cell growth and reproduction. The production cells are engineered for maximum protein production. The bioreactor’s type, volume, and growth parameters are chosen to maximize cell density and cell viability. A Closer Look: Growth Media Considerations Growth media, also known as cell culture, is a liquid or gel designed to support the growth of cells. The composition and concentration of nutrients are considered carefully. These compounds may include glutamine, amino acids, carbohydrates, lipids, vitamins, salts, and buffers. Growth media must meet the following requirements: • It must support cell growth. • It must promote the production of the desired therapeutic product. • Be compliant with all regulatory guidelines. • Ensure it is safe through validation of the absence of harmful components such as chemicals, viruses, and bacteria. • It must address environmental concerns for the cells by considering pH, pressure, shear force when the cells are stirred, oxygen, and carbon dioxide levels. • Be cost-effective. • Come from a reliable supplier to ensure the media is always available. A Closer Look: Bioreactor Considerations Let’s take a quick look at bioreactors. A bioreactor is an apparatus for growing organisms such as bacteria, yeast, and mammalian cells, under controlled conditions. If cells show decreased growth the culprits may include cellular debris, waste, or contamination of viruses, microbes, and endotoxins. Bioreactors are used in industrial processes to produce pharmaceuticals, vaccines, therapeutic antibodies, and other biologics. 10 Copyright 2024 Biotech Primer Inc. PROPRIETARY. DO NOT SHARE. Scale-Up Upstream refers to the production of the protein product, most often by using cells – usually bacterial or, mammalian - growing in culture. Large-scale protein manufacture for clinical use requires the production of large volumes of protein-producing cells. Because cell growth is optimal at a specific cell density, production is performed in a stepwise manner. Collectively, the steps of growing the cells are called upstream processing. Protein manufacturing is a highly controlled, sterile process that safeguards the product from contamination. Not only is contamination carefully avoided, but multiple steps are also included to inactivate and remove adventitious agents throughout the process. Each new batch is grown from the same stock of cells to assure consistent product by using identical starting material. The cell stock is stored frozen in small vials. Production is initiated by removing a vial of stock culture from storage and thawing the cells. First, the cells are grown in a small volume of a few hundred milliliters. The next steps involve a scale-up process, which utilizes bioreactors of increasing size. A bioreactor is a sterile vessel that grows cell cultures in large volumes and that maintains all the proper growth conditions, which are closely monitored. This stepwise scale-up process is required to obtain optimum cell growth and protein production. Scale-up leads to final high-volume growth conditions at industrial scale operations, utilizing large production bioreactors. For CHO cells, a common type of mammalian cell used in biologics production, bioreactors can be as large as 20,000 L in volume. Section 3: Upstream Processing Summary We learned in section three: • Upstream bioprocessing is when biological materials are grown, under controlled conditions, to manufacture certain types of products. 11 Copyright 2024 Biotech Primer Inc. PROPRIETARY. DO NOT SHARE. • The three main goals of upstream bioprocessing include cell density, cell viability, and maximum protein production. • Upstream bioprocessing goals are optimized by ensuring the correct growth media is used. The production cells are engineered to produce most products, and the bioreactor operates at maximum capacity. • The steps taken in upstream bioprocessing include growing production cells in increasingly larger bioreactors until the maximum number of cells are producing the maximum amount of product. 12 Copyright 2024 Biotech Primer Inc. PROPRIETARY. DO NOT SHARE. Section 4: Downstream Bioprocessing Welcome Now that you understand what occurs during the first half of a campaign, let’s look at the second step called downstream bioprocessing. Section 4: Downstream Bioprocessing Objectives At the end of this section, you should be able to: • Define downstream bioprocessing. • Explain the purpose of the harvesting process. • Explain the purpose of the purification process. Downstream Bioprocessing Defined Having completed cell growth, or upstream bioprocessing, the product will now be purified from the cells, formulated, and packaged. Collectively, this is known as downstream processing. Harvesting Process Upstream and downstream bioprocessing operations are linked via the cell harvesting step. Cell harvesting refers to collecting cells from media surfaces and isolating them from the cell culture medium. In the downstream phase of manufacturing, the protein product is isolated from the cells that produced it. The final protein product — which will be the active ingredient in the biologic drug — must be purified away from all other cellular components to be therapeutically useful. Some proteins, such as monoclonal antibodies and growth factors, are secreted by the cell and will be present in the cell culture medium. Other proteins, such as enzymes, will be retained inside the cell. Proteins found inside the cell require special protocols to extract them for purification. Usually, this involves bursting the cells open to release the protein product, which then must be purified away from the other components that were inside the cell. In this scenario, cell harvesting is a crucial unit operation involving the removal of cells, cell debris, and other soluble and insoluble impurities that are detrimental to subsequent chromatographic separation processes. 13 Copyright 2024 Biotech Primer Inc. PROPRIETARY. DO NOT SHARE. Purification Involves Column Chromatography Chromatography refers to a variety of methods used to separate individual molecules from a complex mixture based on their size, structure, or their electric charge. Shown on the screen are methods of column chromatography. Click each item to know more. Protein Chromatography Protein chromatography is used to separate the therapeutic protein or production cells have produced away from the other proteins the cells were also producing. The mixture is passed through a solid matrix, the stationary phase which separates different molecules. The protein mixture is passed through the column under gravity. Applying high pressure or HPLC increases speed and resolution. Proteins that are not retained by the column flow straight through, while proteins that are retained follow later or must be washed off – eluded – under different conditions. Protein elution from the column can be monitored from the ability of proteins to absorb UV light using a spectrophotometer – giving an elution “profile”. Fractions corresponding to the different elusion times are collected automatically in a fraction collector. Ion Exchange Chromatography Ion exchange chromatography separates proteins based on their charge. Some amino acids like glutamate and aspartate, have a negative charge at neutral pH, while others like arginine and lysine have a positive charge. Therefore, proteins can have a net negative, positive, or neutral charge, depending on their amino acid composition. The ion exchange matrix consists of beads coated with a negatively, or in this case, positively charged chemical. Anion exchange is when negatively charged proteins bind to positively charged beads. Net positive and neutral proteins flow straight through the matrix while net negative proteins are retained because the negatively charged proteins are attracted to the positively charged coated beads. 14 Copyright 2024 Biotech Primer Inc. PROPRIETARY. DO NOT SHARE. These can then be eluted by applying a gradient salt solution. As we do this, ions in the buffer begin to exchange with and displace proteins that had been absorbed into the beads. The displaced proteins then flow out of the bottom of the column. By increasing the salt concentration, the molecules with the weakest ionic interactions are disrupted first and eluted earlier in the salt gradient. Those molecules that have a very strong ionic interaction require a higher salt concentration and elute later in the gradient. All the material that elutes from the column is collected in a numbered series of test tubes. These individuals’ aliquots are called “fractions”. Affinity Chromatography Affinity chromatography works on a similar principle to ion exchange, but instead of charged molecules, molecules with a unique shape attach to the beads. Only proteins with a “pocket” complementary to the unique shape attached to the bead will bind to the matrix in the column. Proteins are separated by their structure. After the unbound proteins have been washed away, the bound proteins can be eluted with a soluble form of the shape chemical which replaces the bound form. Size Exclusion Chromatography Size exclusion chromatography, commonly called gel filtration, works by a different principle, and separates proteins based on their size – larger proteins flow through the matrix faster than the smaller ones. The matrix contains porous beads with pores of a defined size. Large proteins cannot enter the beads and simply flow around them. Smaller proteins can enter the beads and pass through them which takes longer. Proteins of intermediate size either do or do not flow through the beads. The result is that different-sized proteins gradually get separated from one another and can be collected in different column fractions once they have passed through the column. For gel filtration columns to work effectively, they must be much longer and thinner than any type of columns to enable the resolution of different size proteins. 15 Copyright 2024 Biotech Primer Inc. PROPRIETARY. DO NOT SHARE. Purification Process Purification is a series of processes intended to isolate the protein product from a complex mixture. In either case — whether your protein product remains inside the cell or is secreted into the cell culture medium, all is extracted and loaded onto a column. A column is a tool that can separate proteins based on their charge, shape, and size. This process is called chromatography. Chromatography refers to a variety of methods used to separate individual molecules, including therapeutic protein, from a complex mixture. Shown on the screen are methods of column chromatography. Click each of them to learn more. Protein Chromatography Protein chromatography is used to separate the therapeutic protein our production cells have produced away from the other proteins the cells were also producing. The mixture is passed through a solid matrix - the stationary phase - which separates different molecules. The protein mixture is passed through the column under gravity. Applying high pressure or HPLC increases speed and resolution. Proteins that are not retained by the column flow straight through, while proteins that are retained follow later or must be washed off - eluted - under different conditions. Protein elution from the column can be monitored from the ability of proteins to absorb UV light using a spectrophotometer - giving an elution "profile". Fractions corresponding to the different elution times are collected automatically in a fraction collector. Ion Exchange Chromatography Ion exchange chromatography separates proteins based on their charge. Some amino acids like glutamate and aspartate have a negative charge at neutral pH, while others like arginine and lysine have a positive charge. Therefore, proteins can have a net negative, positive or neutral charge, depending on their amino acid composition. The ion exchange matrix consists of beads coated with a negatively or, in this case, positively charged chemical. Anion exchange is when negatively charged proteins bind to positively charged beads. 16 Copyright 2024 Biotech Primer Inc. PROPRIETARY. DO NOT SHARE. Net positive and neutral proteins flow straight through the matrix, while net negative proteins are retained because the negatively charged proteins are attracted to the positively charged coated beads. These can then be eluted by applying a gradient salt solution. As we do this, ions in the buffer begin to exchange with and displace proteins that had been absorbed into the beads. The displaced proteins then flow out of the bottom of the column. By increasing the salt concentration, the molecules with the weakest ionic interactions are disrupted first and eluted earlier in the salt gradient. Those molecules that have a very strong ionic interaction require a higher salt concentration and elute later in the gradient. All the material that elutes from the column is collected in a numbered series of test tubes. These individual aliquots are called "fractions". Affinity Chromatography Affinity chromatography works on a similar principle to ion exchange, but instead of charged molecules, molecules with a unique shape attach to the beads. Only proteins with a "pocket" complementary to the unique shape attached to the bead will bind to the matrix in the column. Proteins are separated by their structure. After the unbound proteins have been washed away, the bound proteins can be eluted with a soluble form of the shaped chemical which replaces the bound form. Protein A Affinity Chromatography Protein A affinity chromatography is a specific type of affinity chromatography that uses a resin in the chromatography column that contains Protein A. Protein A is specifically used to purify monoclonal antibodies. This column is often the first step used when purifying monoclonal antibodies and is a very efficient purification step. Protein A binds to the Fc or constant region of the antibody. This Fc region is virtually identical for every antibody in a species. This allows protein A to be used to purify monoclonal antibodies. Size Exclusion Chromatography Size exclusion chromatography, commonly called gel filtration, works by a different principle and separates proteins based on their size, larger proteins flow through the matrix faster than smaller ones. 17 Copyright 2024 Biotech Primer Inc. PROPRIETARY. DO NOT SHARE. The matrix contains porous beads with pores of a defined size. Large proteins cannot enter the beads and simply flow around them. Smaller proteins can enter the beads and pass through them, which takes longer. Proteins of intermediate size either do or do not flow through the beads. The result is that different-sized proteins gradually get separated from one another and can be collected in different column fractions once they have passed through the column. For gel filtration columns to work effectively, they must be much longer and thinner than other types of columns to enable the resolution of different size proteins. Formulation Process Once purified, the protein product must be formulated, or mixed with other ingredients to provide an adequate volume and consistency for patient administration. The formulation is defined as the process in which different chemical substances, including the active drug, are combined to produce a final medicinal product. Biologic Formulation: Critical to Stability Key elements of formulation keep the protein stable and protect it from degradation. The buffer enables the final formulation to maintain the correct pH. pH is a key parameter. pH has a major impact on protein stability, which is critical in maintaining the correct protein structure, which in turn enables the protein to function correctly. Surfactants protect the protein from unfolding at the air/water or the glass/water interface. A range of different stabilizers are added to help maintain the protein’s shelf life. Because formulation influences product attributes such as bioavailability and stability, it is critical to the final biologic’s clinical safety and efficacy. Packaging Process The packaging process consists of filling vials with drug products and finishing the process with a physical inspection of the primary product container and the liquid or lyophilized drug product within the container before distribution. Primary packaging is in direct contact with the drug such as the vial, syringe, or stopper. Secondary, packaging is the carton which is designed to protect 18 Copyright 2024 Biotech Primer Inc. PROPRIETARY. DO NOT SHARE. the drug product and the primary container. A label is applied to the primary container, the secondary container, and package inserts. Testing Protocols The isolation and purification of the protein product must be verified through confirmed testing protocols. The manufacturer rigorously tests representative samples because testing destroys the product. Testing occurs throughout the process starting from the released material to the final product. Test for Safety, Quality, Identity, Purity, and Potency – SQIPP. Maintain a batch record, which is a detailed record of each step in the manufacturing process, and document the results. Quality Control is responsible for environmental monitoring – making sure that any area in which the product is handled is contamination-free - and all product testing. Quality Assurance is responsible for reviewing all test results and for ensuring that all policies, procedures, and processes are followed. We have come to the end of the third section where we learned that in some cases, production cells must burst open to release the protein product. The purification process follows next. Chromatography refers to a variety of methods used to separate individual molecules from a complex mixture based on their size, structure, and electrical charge. We learned the detailed processes for protein chromatography, Ion exchange chromatography, affinity chromatography, and size exclusion chromatography, and gave an overview of the testing protocols that need to be strictly followed. Section 4: Downstream Bioprocessing Summary In summary: • Downstream bioprocessing refers to the harvesting, purification, formulation, and packaging of the protein product. • The purpose of the harvesting process is to collect cells from media surfaces and isolate them from the cell culture medium. 19 Copyright 2024 Biotech Primer Inc. PROPRIETARY. DO NOT SHARE. • The purpose of purification is to isolate the protein product from a complex mixture. • The purpose of the formulation is to combine different chemical substances, including the active drug, to produce a final medicinal product. 20 Copyright 2024 Biotech Primer Inc. PROPRIETARY. DO NOT SHARE. Section 5: Advancements in Biomanufacturing Welcome Over the next few screens, we will first define what continuous bioprocessing is, describe upstream and downstream continuous bioprocessing, and finally, discuss some of the advantages and disadvantages of continuous bioprocessing. Section 5: Advancements in Biomanufacturing Objectives At the end of this section, you should be able to: • Define Critical Quality Attributes (CQAs). • Describe continuous bioprocessing. • Discuss Single-Use Systems in bioprocessing. Critical to Quality Attributes (CQAs) for Protein Therapeutics When manufacturing a therapeutic protein, certain specifications must be met to ensure that the protein will be safe and efficacious. These specifics are known to be critical to the quality attributes (CQAs) of the protein. The International Council for Harmonization (ICH) Q8 (R2) defines CQAs as “a physical, chemical, biological, or microbiological property or characteristic that should be within an appropriate limit, range, or distribution to ensure the desired product quality. CQAs are generally associated with the drug substance, excipients, intermediates (in-process materials), and drug product.” These attributes must be measured for the protein. CQAs include Protein Concentration or the amount of protein found in the solution. Aggregation can be a big issue in biomanufacturing because if proteins clump together into aggregates they will not perform their function in the body and could also be a safety concern because clumps are more likely to elicit an immune response. Potency is a measure of how well the protein works to elicit the desired response at that concentration. Clarity or particulates measures any foreign particles that might still be present in the protein solution that should have been removed in the purification process. 21 Copyright 2024 Biotech Primer Inc. PROPRIETARY. DO NOT SHARE. Chemical changes are a measure of how much change such as oxidation or fragmentation the protein has undergone. These could affect protein efficacy. Residual Protein A in the solution is something that needs to be measured if a protein A affinity column was used to purify the protein. This is important because protein A comes from staphylococcus aureus so it could elicit an immune response in the patient. Similarly, the presence of Host Cell Proteins (HCPs) which are proteins that come from the cell that was used to manufacture the protein must be examined as these could also trigger an immune response. Lastly, it is always necessary to check the sample for any bioburden such as viruses, mycoplasma, or bacteria that may not have been removed in the purification process. It is very important to keep these CQAs in mind as emerging processes such as continuous bioprocessing or single-use technologies are explored because changes to the process can certainly affect these attributes or the manufacturer’s ability to measure them. Continuous Bioprocessing In this section, we will first discuss continuous bioprocessing by defining what continuous bioprocessing is, describing upstream and downstream continuous bioprocessing, and finally discussing some of the advantages and disadvantages of continuous bioprocessing. What is Continuous Bioprocessing? A continuous bioprocess is one in which there is a constant input of raw materials leading to a constant output of product which can be maintained until demand is satisfied. To create a continuous bioprocess all the units of normal biomanufacturing are simply physically connected in series, which allows the process to become what is called a closed system where there are no openings to the outside environment. The advantage of this closed system is that it reduces the risk of contamination from the outside environment. In continuous bioprocessing upstream, harvest, clarification, and downstream steps occur in an uninterrupted flow, meaning that there are no discrete or hold steps that occur between steps of a traditional biomanufacturing process. Partially or semi-continuous processes can also be 22 Copyright 2024 Biotech Primer Inc. PROPRIETARY. DO NOT SHARE. performed in which a large portion of the processing is continuous, but there are a minimal number of separate steps performed. The reason that continuous bioprocessing is being examined as a possible future state of the art is that the reduction in discrete steps could save time and money in the manufacturing process. Upstream Continuous Bioprocessing In upstream continuous bioprocessing, cells are scaled in either a fed-batch bioreactor or a perfusion bioreactor. These profusion bioreactors maintain a constant volume and a constant cell density by adding and removing cells as needed, resulting in the constant production of products. This differs from traditional biomanufacturing because cells are not allowed to build up in the bioreactor, which caused cells to go above the optimal density for creating a therapeutic protein. Using this continuous method bioreactors can run for up to 90+ days and up to 2,000 liters of sample volume. The product is removed but cells remain in the bioreactor and are allowed to multiply until optimal density is reached. After the optimal cell density is reached, cell media and “extra” cells are removed (“cell bleed”) as needed. This leads to a steady state being achieved in which the rate of flow into the system is equal to the rate of the flow out of the system and a continuous synthesis of protein product is created. Continuous Chromatography After the product is harvested from the bioreactor, the next step is to go through the chromatography step for purification of the protein as discussed in the previous section. In continuous chromatography, the columns are placed in series with the eluate of the first column being loaded directly onto the next column allowing the next purification step to occur immediately. The process usually involves 2- 6 different columns and can produce high yields and high resolution of the protein. There are many formats for continuous chromatography which could also include running the sample through the same type of column several times to improve yield. The advantage here is that multiple chromatography steps can occur in series without the need to collect the sample from the column. 23 Copyright 2024 Biotech Primer Inc. PROPRIETARY. DO NOT SHARE. Continuous Bioprocessing Summary This screen shows a summary of continuous bioprocessing. As mentioned, the advantages of continuous bioprocessing could be increases in yield, purity, and speed of production which ultimately leads to saving time and money for the manufacturer. A possible disadvantage to moving towards continuous bioprocessing is that as the speed of the process increases, and hold steps are removed, there may not be as much time to test the sample for purity and safety. Therefore, as continuous bioprocess is explored, faster and more efficient ways to test the sample to ensure it meets all CQAs in real-time will be critical. Single-Use Technologies In addition to looking into making bioprocessing more continuous, there is also a push currently for the use of single-use systems in biomanufacturing. Single-use systems are also referred to as disposable systems, and these products are intended for one-time or single use and then are disposed of after that use. These products are usually made from a particular type of plastic suitable for this purpose. Single-use systems include single-use bioreactors, filtration systems, and chromatography systems. While the process may seem wasteful due to the production of more plastic waste, the use of these systems does reduce the energy and water used to clean the traditional systems between runs up to 50%. Because these cleaning steps are no longer needed there is much less chance of cross-contamination between manufacturing runs as well. Single-Use Systems: Benefits and Risks This screen gives a summary of the advantages and disadvantages of single-use systems with the advantages shown in green and the disadvantages shown in red. Section 5: Advancements in Biomanufacturing Summary To summarize this section, we first learned that: • Critical to Quality Attributes or CQAs are specifications that must be met when manufacturing a therapeutic protein and therefore must be considered when changes are made to the manufacturing process. 24 Copyright 2024 Biotech Primer Inc. PROPRIETARY. DO NOT SHARE. • Continuous bioprocessing is a process in which constant inputs of raw materials lead to a constant output of product. The advantage of continuous bioprocessing could be reduced cost and time needed for the manufacturing process. • Single-use technologies are being adopted by biomanufacturing companies to reduce the need for cleaning between manufacturing runs. The reduced time, energy, and water consumption needed for cleaning must be balanced with the larger creation of plastic waste that is created when using these single-use systems. 25 Copyright 2024 Biotech Primer Inc.