Introduction to Bioprocessing 12.1 Notes PDF

11/11/2024, 18:59 12.1. Notes | Eduvos Eduvos (Pty) Ltd (formerly Pearson Institute of Higher Education) is registered with...

11/11/2024, 18:59 12.1. Notes | Eduvos Eduvos (Pty) Ltd (formerly Pearson Institute of Higher Education) is registered with the Department of Higher Education and Training as a private higher education institution under the Higher Education Act, 101, of 1997. Registration Certificate number: 2001/HE07/008. Date: Monday, 11 November 2024, 6:59 PM Introduction to Bioprocessing 12.1. Notes 1. Learning Outcomes By the end of this lesson, you should be able to: Explain what mammalian cell manufacturing systems are and why they are used in bioprocessing. Identify and differentiate between various mammalian cell lines used in manufacturing systems such as Chinese Hamster Ovary (CHO) cells, Human Embryonic Kidney (HEK) cells, and others. Discuss the advantages and disadvantages of mammalian cell manufacturing systems. Describe the process of protein production in mammalian cells, from cell line development to protein purification. https://mylms.vossie.net/mod/book/tool/print/index.php?id=776254 1/25 11/11/2024, 18:59 12.1. Notes | Eduvos Prescribed Reading Websites: Schofield, D. 2021. Mammalian cell culture – Types and application. [Online] Available at: https://www.evitria.com/journal/cho-cells/mammalian-cell-culture/. [Accessed 22 August 2024] Eberle, C. 2022. CHO cells – 7 facts about the cell line derived from the ovary of the Chinese hamster. [Online] Available at: https://www.evitria.com/journal/cho-cells/cho- cells/. [Accessed 22 August 2024]. Synthego. 2024. HEK293 Cells: Background, Applications, Protocols, and More. [Online] Available at: https://www.synthego.com/hek293#hek293Origin. [Accessed 24 August 2024]. Mohta, A. 2023. Understanding Hybridoma Technology for Monoclonal Antibody Production. [Online] Available at: https://www.the-scientist.com/understanding- hybridoma-technology-for-monoclonal-antibody-production-71108. [Accessed 25 August 2024]. Online Journal Article: Olmos, E., Bensoussan, D. 2021. Human and animal cells as factories for therapeutic molecules. Bioproduction. pp19. hal-03633916. Available at: https://hal.univ-lorraine.fr/hal-03633916. [Accessed 22 August 2024]. 2. Relevance Connection The development of a mammalian cell line is a crucial step in creating mammalian cell manufacturing systems. Biopharmaceutical products, such as monoclonal antibodies and viral vectors, are often produced using mammalian cells and researchers need to determine the cell line best suited for producing it. Watch the video below, which provides a short introduction to mammalian cell line development. https://mylms.vossie.net/mod/book/tool/print/index.php?id=776254 2/25 11/11/2024, 18:59 12.1. Notes | Eduvos 3. Introduction The production of recombinant proteins in the Biopharmaceutical industry was estimated to hold a global market share of around USD 20.89 billion in 2023, with most recombinant therapeutic proteins produced via mammalian cells. Mammalian cell factories are often preferred over bacterial or yeast expression systems in the production of biologics, because of several reasons. Arguably the most important reason being the ability of mammalian cells to make human-compatible modifications postproduction, which greatly impacts the product quality and yield. Mammalian cells are generally used in virus-based vaccine manufacturing. Chinese Hamster Ovary (CHO) is the most widely used cell line for high-yield stable recombinant protein production, while Human Embryonic Kidney (HEK) is a popular choice for transient transfection low-yield protein manufacturing, viral-based vaccine and gene and cell therapy-related vector production. Other mammalian cell lines such as NSO, Sp2.0, Vero, MRC-5 and PerC.6 are also used in both recombinant protein and virus productions. 4. What are Mammalian Cell Cultures? Mammalian cell culture → Process of growing cells obtained from mammals in vitro, in flasks or bioreactors. https://mylms.vossie.net/mod/book/tool/print/index.php?id=776254 3/25 11/11/2024, 18:59 12.1. Notes | Eduvos In vitro → The technique of growing cells outside their original tissue, and instead, a growth medium is used to replicate the natural environment and support the cell growth process. Cells typically grown in a cell culture vessel such as a shake flask placed in an incubator that provides a controlled environment for optimal growth. To maintain cell integrity, the medium contains nutrients, a carbon source such as glucose, vitamins, amino acids, salts, oxygen and CO2, and growth factors. Mammalian cells are eukaryotic cells. THEREFORE, each cell itself is much more complex than a bacterial cell. Since mammals consist of trillions of individual cells in many organs and tissues, there is a lot of specialization among mammalian cells and the process of culturing these cells, is a lot more complicated. According to their specialization, mammalian cells are categorized into cell types, depending on their: Development as an embryo Physiological function Morphology Origin These categorizations are explained in more detail in the sub-sections that follow. 4.1. Development as an Embryo Histology → the science of microscopic anatomy This discipline lists hundreds of distinct types of human cells alone. Histologic cell types are classified how they originated during early embryonic development: Endoderm derived: e.g. barrier cells, hormone-secreting cells, cells of the liver, digestive tract, lungs Ectoderm derived: e.g. epithelial cells, cells of the nervous system Mesoderm derived: e.g. metabolism and storage cells, reproductive system, muscle cells, blood cells, skeletal cells, kidney cells https://mylms.vossie.net/mod/book/tool/print/index.php?id=776254 4/25 11/11/2024, 18:59 12.1. Notes | Eduvos Figure 1: The three germ layers of embryos which will give rise to different organ systems. Source: https://www.invitra.com/en/two-months-pregnant/embryonic-skin-development/. 4.2. Physiological Function Cells can also be categorized depending on their main physiological function: Hormone-releasing cells: e.g. thyroid gland cells, pituitary gland cells, adrenal gland cells Exocrine secretory cells: e.g. salivary gland cells, pancreatic cell Nervous system cells: e.g. sensory transducer cells, neurons, glial cells Storage cells: e.g. liver lipocytes, adipocytes Extracellular matrix cells: e.g. connective tissue fibroblasts, tendon fibroblasts, osteoblasts/osteocytes (bone cells) Contractile cells: e.g. skeletal muscle cells, cardiac muscle cells, smooth muscles 4.3. Morphology Cells can also be categorized based on morphology: Fibroblastic / fibroblast-like cells: Bipolar or multipolar and elongated in shape (see image below). Grow attached to a substrate and often align into parallel assemblies. https://mylms.vossie.net/mod/book/tool/print/index.php?id=776254 5/25 11/11/2024, 18:59 12.1. Notes | Eduvos In living organisms, fibroblasts secrete the extracellular matrix and are part of the connective tissue. In addition, neuronal cells can rank among fibroblast-like cells due to their multipolar shape. Figure 2: Fibroblasts with bipolar or multipolar shape. Source: https://www.leica-microsystems.com/science- lab/introduction-to-mammalian-cell-culture/#gallery-1. Epithelial-like cells: Have a polygonal shape. In general show more regular dimensions. (See image below) Grow attached to a substrate in discrete patches. Exhibit a plasma membrane with two sub-areas differing in structure and function: Apical membrane → faces the culture medium in vitro, faces inside of hollow organ (lumen) in vivo. Basolateral membrane → lies between the individual cells and the culture vessel in vitro, lies between the individual cells and adjacent tissue inside of the hollow organ in vivo. Figure 3: The two sub-areas of a plasma membrane. Source: https://courses.washington.edu/pbio375/epithelial-histology/epithelial-histology.html. Both membrane domains are separated by tight-junctions. https://mylms.vossie.net/mod/book/tool/print/index.php?id=776254 6/25 11/11/2024, 18:59 12.1. Notes | Eduvos In vivo, epithelial cells line the body structures. Figure 4: Epithelial cells size and shape. Source: https://www.leica-microsystems.com/science- lab/introduction-to-mammalian-cell-culture/#gallery-1. Lymphoblast-like cells: Show spherical outline and are typically grown in suspension (see image below). In contrast to fibroblastic or epithelial-like cells, do not attach to the surface. Good example → blood cells. Figure 5: Lymphoblasts size and shape. Source: https://www.leica-microsystems.com/science- lab/introduction-to-mammalian-cell-culture/#gallery-1. https://mylms.vossie.net/mod/book/tool/print/index.php?id=776254 7/25 11/11/2024, 18:59 12.1. Notes | Eduvos 4.4. Origin Cells can also be categorized depending on their origin: Immortalized cells: Derived from cancerous tissues or transfected with oncogenes. Cells that divide indefinitely. This attractive attribute makes them very easy to culture as a cell line. They are also robust and affordable. On the other hand, one should consider that their mutated genetic background is not very close to nature anymore. A very prominent example is the HeLa cell line which was derived from a cervix carcinoma. Compared to the 46 chromosomes of normal human cells, their nuclei have around 80 chromosomes. Other commonly used immortalized cell lines are HEK, A549, Jurkat, MDCK, COS, or Vero cells. Figure 6: Scanning electron micrograph of an apoptotic HeLa cell. Source: https://en.wikipedia.org/wiki/HeLa. Primary cells: Taken directly from living tissue. For cultivation → original tissue fragment dissociated enzymatically, chemically, or mechanically into single cells which can be seeded on culture flasks. Much harder to cultivate than immortalized cell lines: Survival rate low. Many don’t divide. Genetic manipulation can be challenging. https://mylms.vossie.net/mod/book/tool/print/index.php?id=776254 8/25 11/11/2024, 18:59 12.1. Notes | Eduvos Are preferred in some cases because their characteristics are much closer to natural cells. THEREFORE → scientific results obtained with primary cells can be translated into the in vivo world with more confidence. Figure 7: Primary neuronal cell culture. Source: https://www.leica-microsystems.com/science- lab/introduction-to-mammalian-cell-culture/#gallery-1. Stem cells: Body’s founder cells. Can divide and differentiate from non-specialized cells into specialized tissue cells with dedicated characteristics and functions. Several classes: Pluripotent stem cell → most versatile and able to differentiate into any kind of body cell. Multipotent stem cells → limited differentiation range. Bipotent stem cells → can only develop into two different cell types. Source of stem cells varies. Classically they are obtained from embryos – embryonic stem cells. Others can be extracted from certain tissues à e.g. the bone marrow contains blood stem cells. Due to ethical concerns, gaining stem cells from living animals or humans is hard to justify. That is why the opportunity to induce already differentiated body cells to transform into stem cells gained a lot of interest. Somatic cells can be reprogrammed by transfection with only four genes (c-Myc, klf-4, oct-4, sox-2) → resulting in induced Pluripotent Stem Cells (iPSC). Although challenging, give researchers highest flexibility. Depending on the ingredients included in the cell medium, like certain nutrients and growth factors, stem cells can be triggered to develop into a distinct cell type, e.g. neurons. Especially in the clinical environment, iPSCs are of great importance, as researchers are able to obtain disease specific or even patient specific cells to find a cure. https://mylms.vossie.net/mod/book/tool/print/index.php?id=776254 9/25 11/11/2024, 18:59 12.1. Notes | Eduvos Figure 8: Different types of cells that can arise from stem cells. Source: https://www.mayoclinic.org/tests- procedures/bone-marrow-transplant/in-depth/stem-cells/art-20048117. Figure 9: General applications of mammalian cell cultures. Source: https://www.evitria.com/journal/cho- cells/mammalian-cell-culture/. 5. What is a Mammalian Cell Line? A population of mammalian cells that can be grown due to their ongoing cell division. Generally, a normal cell has a limited lifespan and does not divide indefinitely. To generate a cell line, cells must undergo immortalization. https://mylms.vossie.net/mod/book/tool/print/index.php?id=776254 10/25 11/11/2024, 18:59 12.1. Notes | Eduvos Immortalization processes to achieve this can be: Mutations of genes that deregulate the cell cycle Enhancing proliferation processes Natural sources of immortal cell lines exist à cancerous tissues/cells In contrast, stem cells have the natural ability to divide indefinitely, e. g. embryonic stem cells from blastocysts. 6. Industrial Applications of Mammalian Cell Factories Most common cell line is the CHO (Chinese Hamster Ovary) cell → accounts for approximately 75 % of recombinant protein production. Insect cells (infected with a baculovirus carrying the gene coding for the protein of interest) also attractive tools for the transient production of proteins on a smaller scale. A list of the main mammalian cell lines and their recognized applications is given in Table 1. Table source: Olmos & Bensoussan (2021). More recently, new lines of human origin are emerging to produce adenoviral vectors or recombinant proteins (HEK293, PER.C6). Present a higher advantage: Allow production of a non-immunogenic glycan. Main drawback: Are susceptible to human virus contaminations requiring viral inactivation. https://mylms.vossie.net/mod/book/tool/print/index.php?id=776254 11/25 11/11/2024, 18:59 12.1. Notes | Eduvos HEK293 (Human Embryo Kidney 293) and its different variants, transfected with viral DNA, is the most prominent human cell line used for protein expression. Other cell-lines and their applications: HT-1080 (human fribrosarcoma) → factor VII CAP (from human amniocytes) → adenoviral vectors PER.C6 (from human retinoblast) → immunoglobulins HuH-7 (from human hepatoma) → factor IX 7. Chinese Hamster Ovary (CHO) Cells Figure 10: Chinese hamster ovary cells. Source: https://www.evitria.com/journal/cho-cells/cho-cells/. Chinese hamster ovary (CHO) cells have been cultured and manipulated for the use in molecular biology and pharmaceutical biotechnology for decades. Many different cell lines are used in molecular biology research labs, CHO cells are the most commonly used host for the expression of recombinant monoclonal antibodies (mAbs). As the name suggests, CHO cells derived from the ovary of a small rodent called the Chinese hamster (Cricetulus griseus). Figure 11: A Chinese hamster. Source: https://www.enzo.com/note/what-are-the-advantages-of-using- chinese-hamster-ovary-cho-cells/. https://mylms.vossie.net/mod/book/tool/print/index.php?id=776254 12/25 11/11/2024, 18:59 12.1. Notes | Eduvos Morphology: can be epithelial-like or elongated. Cell Culturing of CHO: Doubling time: typically, 24 hours. Can be adherent monolayer or grown in suspension. Growth Medium: Ham's F12K or Dulbecco’s Modified Eagle’s Medium (DMEM), supplemented with 10% Fetal Bovine Serum. Growth media should be replaced every 2-3 days. Growth Conditions: grown incubator at 37°C, pH 7.0, dissolved oxygen 30%. Storage Conditions: long-term storage à kept in the liquid nitrogen vapor phase. At -80°C not recommended, will result in loss of viability over time. Freezing: must be frozen slowly & freezing media should contain complete growth media supplemented with 5% cryoprotectant such as DMSO. Use an isopropanol storage box to ensure cells freeze slowly (-1°C per minute) before moving cells into the liquid nitrogen vapour phase. Thawing: thawed rapidly by gentle agitation in a 37°C water bath for 2 minutes. Then added to pre- warmed media, centrifuged at low speed to remove freeze media and subsequently resuspended in growth media. Biosafety Level: biosafety level classified as BSL-2. Figure 12: CHO cell size. Source: https://www.evitria.com/journal/cho-cells/cho-cells/. Figure 13: Phase-contrast micrograph of CHO cells. Source: https://en.wikipedia.org/wiki/Chinese_hamster_ovary_cell. Due to their similarity to the human cell system, CHO cells are used in biological and medical research involving: Genomic and chromosome studies https://mylms.vossie.net/mod/book/tool/print/index.php?id=776254 13/25 11/11/2024, 18:59 12.1. Notes | Eduvos Toxicity assays Gene expression CHO cell lines are the best choice as a mammalian host for industrial production of recombinant protein therapeutics. Figure 14: Utilization of CHOs in Research and Manufacturing. Source: https://www.evitria.com/journal/cho- cells/cho-cells/. Why? → They are highly efficient in terms of yield. Can produce recombinant protein on a scale of 3-10 grams per liter of CHO cells culture. The following sub-sections provide more information on CHOs, including their advantages and disadvantages, as well as the procedure for antibody production in CHO cell lines. 7.1. Advantages and Disadvantages Main Advantages: Easy to Culture: Grows well in suspension and as adherent culture, rendering the cells ideal for GMP procedures. Their tolerance to variations in pH, oxygen levels, temperature or pressure make them the ideal cell for large-scale culture. High Productivity and yield: High recombinant protein yields and specific productivity. Thanks to genetic optimization, protein yields of 3-10 grams per liter of cell culture. Defined Culture Conditions: Can be adapted for defined, serum-free culture conditions, as well as allowing for animal-free and protein-free production and better safety and stability profiles. Various Selection Systems: Antibiotic and metabolic selection by DHFR (Dihydrofolate Reductase) - or GS (glutamine synthase) - deficiency to obtain stable clones of high productivity with ease. Post-Translational Modifications: https://mylms.vossie.net/mod/book/tool/print/index.php?id=776254 14/25 11/11/2024, 18:59 12.1. Notes | Eduvos Protein synthesis machinery within a CHO cell produces proteins that are characterized by similar post-translation modifications and glycosylations as human cells. Therefore, these proteins suitable for human applications, which is why they are used in the production of therapeutic proteins. These modifications within recombinant protein production are not possible in other popular in vitro cell types such as E. coli. The variety of post-translational modifications of the produced biologic often allows for a biosimilar, if not human-identical products with excellent pharmaceutical activity and biocompatibility. Genetic Cell Engineering: Well-proven genetic tools are available to optimize CHO cells, from gene introduction to knock-out, knock-down and post-translational silencing. FDA-Approval: Used for nearly 50 biotherapeutics already approved in the USA and EU. Low Virus Susceptibility: Due to the hamster origin, the risk of propagation of human viruses is decreased, reducing production loss and increasing biosafety. Thanks to these characteristics as well as constant progress, optimization and improvement, CHO cell cultures today can be used for large-scale transient expression after plasmid transfection in a relatively short time span. CHO cell antibody production can be tailored to the respective customer’s requirements. Disadvantages: Initial difficulties associated with CHO cells have been streamlined over the years due to constant development and optimisation of methods. Today, main concern → the use of living animals in the process. Although original CHO cells were extracted from Chinese hamsters, it is now possible to recreate them in vitro without the involvement of a living creature in the process. 7.2. Procedure for Antibody Production in CHO Cell Lines Transfection of the cell To achieve post-translational modifications → vectors/plasmids integrated to cell genome. A highly transcribed region of the genome is targeted in the process to overcome the sensitivity of gene silencing. Gene expression https://mylms.vossie.net/mod/book/tool/print/index.php?id=776254 15/25 11/11/2024, 18:59 12.1. Notes | Eduvos The vector genome usually contains one insert for antibody genes (your gene of interest) and one insert for the genes to enable plasmid replication in bacteria. To achieve high levels of gene expression, strong promoters like dihydrofolate reductase or glutamine synthetase used. Protein synthesis and Glycosylation CHO cell is led to produce corresponding proteins and folded correctly through glycosylation. Glycosylation: Process by which a carbohydrate is covalently attached to a target macromolecule, typically protein à serves various functions (some proteins do not fold correctly unless they are glycosylated). Post-translational modifications Established into the amino acid formulation. Improvement of CHO cell culture medium formulation Done through substitution of glucose and glutamine – the transfected cells are grown into fed-batch culture to achieve high production yields through glucose, glycan and glycoprotein. Screening of cell culture Through processes such as proteomics and chromatography to prevent cell death and achieve high purification levels. Quality control and cell banking Cloned cells are tested for their identity, purity, stability, safety, and production ability and only the most suited cells are selected. Cell banking: Cryopreservation of the identified cell lines to preserve and store these valuable cell lines for various applications in future. Maintenance Once a cell line is selected → needs to be maintained in optimal conditions. Involves regular subculturing → small number of cells transferred to fresh culture vessels to ensure continuous cell growth. Maintenance medium usually contains nutrients, growth factors and supplements necessary for cell viability and growth. Cells incubated at controlled temperature, humidity and carbon dioxide levels to provide an ideal environment for their proliferation. Cell line stability Crucial → ensure consistent monoclonal antibody production over time. https://mylms.vossie.net/mod/book/tool/print/index.php?id=776254 16/25 11/11/2024, 18:59 12.1. Notes | Eduvos Regular monitoring and characterization of the cell line, such as genetic stability assessments and phenotypic analysis, are performed to confirm its stability and uniformity. 8. Human Embryonic Kidney Cells HEK293 cell line → one of the most widely used cell lines in research. Establishment in 1970s. Robust and fast-growing cell line and its derivatives → used extensively in receptor signalling, cancer research, and large-scale protein production. Also commonly used in CRISPR-based genome engineering studies. HEK293: Immortalized human embryonic kidney cells. Cell line was transformed with sheared adenovirus 5 (Ad5). 293 → 293rd experiment Addition of Ad5 E1A and E1B genes to the HEK genome → immortalized the cell line by interfering with cell cycle control and preventing apoptosis. Addition of Ad5 also allows high levels of recombinant protein production. (making it very popular) How? → Plasmid vectors contain the CMV promoter. Morphology → epithelial Cell size → Typically between 11 and 15µm Dependent upon culturing conditions. Cells grown in an adherent monolayer culture will appear flatter and have a larger diameter than cells in suspension culture, which will be spheroid. https://mylms.vossie.net/mod/book/tool/print/index.php?id=776254 17/25 11/11/2024, 18:59 12.1. Notes | Eduvos Figure 15: Left and middle → Low - and High–density images of HEK cells; Right → Immunofluorescent HEK293 cells. Sources: https://www.beckman.com/resources/product-applications/lead-optimization/cell-line- development/human-embryonic-kidney-293. What is HEK293T? Derivitive of original cell line Genome contains SV40 large T antigen → enables production of recombinant proteins within plasmid vectors containing the SV40 promoter. For this reason, commonly used for retroviral production. Cell Culturing of HEK293 Doubling time: double rapidly → every 34 to 36 hours Typically grown as adherent monolayer but can be adapted for growth in suspension. Growth Medium: high-glucose growth media such as Eagle’s Minimum Essential Medium (EMEM) or Dulbecco’s Modified Eagle’s Medium (DMEM), supplemented with Fetal Bovine Serum (FBS) to a final concentration of 10%. Growth media should be replaced every 2-3 days. Growth Conditions: grown in a humidified incubator at 37°C, pH 7.0 – 7.2, dissolved oxygen 50%, supplemented with 5% CO2. Storage Conditions: long-term storage → kept in the liquid nitrogen vapor phase. At -80°C not recommended, will result in loss of viability over time. Freezing: must be frozen slowly & freezing media should contain complete growth media supplemented with 5% cryoprotectant such as DMSO. Use isopropanol storage box to ensure cells freeze slowly (-1°C per minute) before moving cells into the liquid nitrogen vapor phase. Thawing: thawed rapidly by gentle agitation in a 37°C water bath for 2 minutes. Then added to pre- warmed media, centrifuged at low speed to remove freeze media and subsequently resuspended in growth media. Biosafety Level: Due to the presence of adenovirus in the cells, the biosafety level is classified as BSL-2. The following sub-sections provide more information on the advantages and disadvantages pf HEK293, as well as the applications of HEK. 8.1. Advantages and Disadvantages of HEK293 Advantages Rapid doubling time. https://mylms.vossie.net/mod/book/tool/print/index.php?id=776254 18/25 11/11/2024, 18:59 12.1. Notes | Eduvos Easy to culture. Reproducibility of results: Results are generally consistent and highly reproducible. Protein Production: Highly efficient at producing large amounts of recombinant proteins, therefore ideal for producing therapeutic proteins in commercial quantities. Gene Expression: Can be used for transient and stable expression of desired genes. Can also be transfected with a tetracycline repressor to make HEK293 T-REx cells, so that gene expression can be switched on when needed → useful when testing cell membrane receptors and similar ion channels, which will cause cells to become ‘leaky’ if they are continually expressed. Transfection Amenability: Highly amenable to transfection and can be transfected using variety of chemical and physical methods. Disadvantages Bacterial Contamination: Bacteria one of the most common sources of contamination and will usually be obvious as it will change the pH, colour and turbidity of the culture. Special case: Mycoplasma infections → not immediately apparent. Why is this dangerous? → If left unchecked, mycoplasma will quickly spread to all other cultures, resulting in loss of cell viability and problems with gene expression which will affect your experiments. How to avoid? → Make sure have flawless aseptic technique, test for mycoplasma infections regularly and keep new cultures received from other labs in a separate quarantine incubator until they can be tested. What if I encounter contamination? → Discard any contaminated flasks and give incubator a deep- clean. Viral Contamination: As with all human cell lines, HEK cells come with associated risk of human-specific viruses, and this cell line is particularly susceptible to viral infections. Unlike common bacterial infections, viral contamination is not always obvious and is usually detected through virus-specific PCR-based assays. Culture Period: Even for immortalized cell lines, culturing for extended periods will cause the health of cells to degrade → affects growth rate and translation efficiency of cells, which can have negative consequences for downstream experiments, including reproducibility of results. While the exact maximum passage number (amount of a times a cell line can be sub-cultured) is debated, it’s generally considered best to stay below 20 passages. 8.2. HEK Applications https://mylms.vossie.net/mod/book/tool/print/index.php?id=776254 19/25 11/11/2024, 18:59 12.1. Notes | Eduvos Virus/Vaccine Production Go-to ‘packaging cells’ for viral production because they express the SV40 large T-antigen → allows them to replicate viral plasmids with the SV40 promotor. How to generate a vaccine? → Transfect ‘helper’ plasmids (viral packaging machinery) along with the viral plasmid containing the gene of interest, HEKs then produce and package viral particles. Viral particles then filtered to remove unwanted material (HEK cells, etc.) and are ready to use on another cell or organism. This pipeline is utilized to transduce cell lines for basic science research all the way up to the production of clinical viral therapies. See Sars-Covid-19 vaccine example below from Kaewborisuth et al. (2022) Figure 16: Development of a cell line for the production of chimeric SARS-CoV-2 VLP. Source: Kaewborisuth et al. (2022). What is happening in Figure 16? → Lentiviruses carrying either a SARS-CoV-2 S gene with a deletion of the RRAR motif (ΔFurin) and fused to an influenza virus HA transmembrane and cytoplasmic tail (HAcyt), or an influenza A M1 gene were used to transduce HEK293T cells. The resulting HEK293T cells express S on the cell surface, and associate with M1 proteins. The M1 proteins induce the cell membrane budding, resulting in the release of S-M1 VLP into the cell culture supernatant while the cell line is maintained in culture. (Kaewborisuth et al. 2022). Protein Production Very easy to transfect in bulk and produce recombinant proteins at high levels. Where clinical applications of recombinant proteins are necessary, HEKs are human in origin, therefore, eliminate non-human Post-Translational Modifications (which is a concern with other protein production systems, such as yeast). https://mylms.vossie.net/mod/book/tool/print/index.php?id=776254 20/25 11/11/2024, 18:59 12.1. Notes | Eduvos HEKs can also grow in suspension, which allows for large scalability, and can also be adapted for serum- free growth, which significantly reduces the cost of production. Research Applications Two areas of research were useful due to intrinsic properties. HEK-293 cells are tumorigenic → good candidate cell line model for cancer-specific research. Membrane receptors and ion channels often exogenously expressed and studied in HEKs due to the cell line’s ability to faithfully produce these proteins and its relatively low expression of native channels. 9. Hybridomas A more specialized approach: Hybridoma Technology → Fusion of B-cells with myeloma cells to generate immortalized antibody producing hybridoma cells. B-cells / B-Lymphocyte in the spleen: a key cell of the adaptive immune response that is responsible for humoral immunity in mammals and that produces antibodies. Myeloma cells: cancer of the plasma cells. By creating a hybrid of two cell types, a cell line can be created that is immortalized and that produces antibodies for the large-scale production of monoclonal antibodies (mAbs). The following sub-sections further explore hybridoma technology, as well as the advantages, disadvantages, and application of hybridomas. 9.1. Hybridoma Technology: Step-by-Step Step 1: Immunization A mammal, typically a mouse, is injected with a target antigen, stimulating an immune response. Antigen injection can occur in a series over the course of several weeks. Then, mouse's spleen is harvested to obtain B cells that produce desired antibody. Step 2: Cell Fusion Antibody-producing B cells fused with myeloma cells in cell culture. https://mylms.vossie.net/mod/book/tool/print/index.php?id=776254 21/25 11/11/2024, 18:59 12.1. Notes | Eduvos Polyethylene glycol (PEG) facilitates fusion of both cells’ plasma membranes → forming single hybridoma cell with two or more nuclei. Alternatively, electrofusion can merge the cells using a pulsed electrical field. Step 3: Hybridoma Cell Growth Less than 1% of initial cells fuse to form hybridoma cells. Unused B cells in the culture stop dividing naturally, while chemotherapy destroys the unfused myeloma cells. Researchers use HAT (hypoxanthine-aminopterin-thymidine) medium to allow the selective proliferation of immortal monoclonal antibody-producing cell lines. Aminopterin in the HAT medium → stops nucleotide synthesis, while hypoxanthine and thymidine can be used by cells, such as B cells, carrying the HGPRT (hypoxanthine-guanine phosphoribosyl transferase) enzyme. Hybridoma cells with functional HGPRT enzyme can survive and grow, while myeloma cells lacking it eventually die. Step 4: Screening Hybridoma cells screened for the monoclonal antibody of interest using an enzyme-linked immunosorbent assay (ELISA). Indirect ELISAs identify antibodies with the appropriate specificity by immobilizing the antigen on a surface and incubating it with a hybridoma cell supernatant. Techniques such as western blot, flow cytometry, and immunoprecipitation-mass spectrometry also used to screen their hybridoma cultures. Step 5: Hybridoma Expansion Finally, desired hybridoma cells cloned to obtain stable cell population and growing the culture to collect large amounts of monoclonal antibodies. This can be achieved through one of two methods: In vitro growth of hybrid cells in tissue culture. In vivo growth following inoculation of hybridoma cells into a mouse’s abdomen. https://mylms.vossie.net/mod/book/tool/print/index.php?id=776254 22/25 11/11/2024, 18:59 12.1. Notes | Eduvos Figure 17: Step-by-step procedure for creating hybridoma cells. Source: https://www.the- scientist.com/understanding-hybridoma-technology-for-monoclonal-antibody-production-71108. Figure 18: Artificial production of monoclonal antibodies. Source: https://www.britannica.com/science/monoclonal-antibody. The technique above involves fusing certain myeloma cells (cancerous B cells), which can multiply indefinitely but cannot produce antibodies, with plasma cells (noncancerous B cells), which are short-lived but produce a desired antibody. The resulting hybrid cells, called hybridomas, grow at the rate of myeloma cells but also produce large amounts of the desired antibody. In this way, researchers obtain large quantities of antibody molecules that all react against the same antigen. The essential production steps are shown. In step 2, HGPRT is hypoxanthineguanine phosphoribosyltransferase, an enzyme that allows cells to grow on a medium containing HAT, or hydroxanthine, aminopterin, and thymidine. As shown in step 4, only hybridomas can live in the HAT medium; unfused myeloma cells, lacking HGPRT, die in the medium, as do unfused plasma cells, which are naturally short-lived. Hybdridomas can grow and can, therefore, be selected. 9.2. Advantages and Disadvantages Advantages https://mylms.vossie.net/mod/book/tool/print/index.php?id=776254 23/25 11/11/2024, 18:59 12.1. Notes | Eduvos Precise antigen targeting. A never-ending supply of consistent antibodies. High sensitivity and specificity for use in biological assays. Elimination of the need for animal models (in vitro method). Disadvantages Long production time. Resource-intensive and expensive workflow. Not suitable for generating short peptides and fragment antigens. Susceptibility to contamination and poor cell viability. Risk of virus contamination and disease transmission. Absence of stable myeloma cells for human antibody production. 9.3. Hybridoma Applications Diagnostic Applications Owing to their high specificity, antibodies produced by hybridoma technology have a wide range of diagnostic applications, including the following: Enzyme-linked immunosorbent (ELISA): detecting HIV antibodies, hepatitis B surface antigen, and pregnancy hormone. Immunofluorescence assay (IFA): detecting autoimmune disorders, influenza virus, and Chlamydia trachomatis. Western blot: analyzing cancer biomarkers. Flow cytometry: assessing immune cells in HIV, leukemia, and lymphoma. Immunohistochemistry (IHC): analyzing cancer biomarkers. Rapid antigen tests: detecting malaria, dengue, Zika virus, and COVID-19. Immunotherapy Various FDA-approved indications of monoclonal antibodies exist (see table below). Common indications include the following: https://mylms.vossie.net/mod/book/tool/print/index.php?id=776254 24/25 11/11/2024, 18:59 12.1. Notes | Eduvos Cancer treatment: anticancer immunotherapy against prostate, breast, lung, bladder, liver, gastric, colorectal, and endometrial cancer. Autoimmune disorders: management of rheumatoid arthritis, Crohn's disease, lupus, psoriasis. Infectious diseases: prevention and treatment of respiratory syncytial virus and COVID-19. Organ transplant: rejection prevention of kidney, liver, lung, and heart transplants. Noteworthy FDA-approved monoclonal antibodies Table obtained from: https://www.the-scientist.com/understanding-hybridoma-technology-for-monoclonal- antibody-production-71108. https://mylms.vossie.net/mod/book/tool/print/index.php?id=776254 25/25