Cell Factories and Mammalian Cell Culturing - Course Notes

Overview of Mammalian and Insect Cells as Cell Factories

• Context: These notes cover the curriculum for the 2025 semester taught by Edel McNeela at SETU regarding the use of mammalian and insect cells in biotechnology.

• Semester Curriculum Summary:

  • Module 1: Overview of Mammalian Cells (Introduction, Milestones, Primary and Continuous Cultures, Biopharma Examples).

  • Module 2: Growth, Transformation, and Immortalization.

  • Module 3: Cell Death (Apoptosis, Necrosis, Autophagy).

  • Module 4: Transfection (Methods, Plasmids, and Vectors).

  • Module 5: Selection and Screening for Biopharmaceutical Production.

  • Module 6: Cell Line Engineering and Targeted Gene Integration.

  • Module 7: Large-Scale Mammalian Cell Culture and Bioreactors.

  • Module 8: Bioassays.

  • Module 9: Insect Cells and Baculovirus Expression Systems.

The Synthetic Biology Toolbox and Industrial Applications

• The Synthetic Biology Toolbox Components:

  • Genetic Parts: Promoters and terminators.

  • Genetic Editing: Tools for modifying the genome.

  • DNA Sequencing: Reading genetic code.

  • Strain & Clone Development: Optimization of the cell lineage.

  • Adaptation: Adjusting cells to specific culture conditions.

  • High-Throughput Screening: Rapidly evaluating large numbers of clones.

  • Automation: Utilizing robotic systems for efficiency.

  • Genetic Architecture: Use of the standard $Promoter - GOI (Gene of Interest) - Terminator$ construct.

• Cells as Biofactories:

  • Industrial Biotechnology: Production of biofuels, citric acid, and succinic acid.

  • Biopharmaceutical Manufacturing: Production of insulin, antibodies, and vaccines.

Definitions and Classifications of Biopharmaceuticals

• Biologics: Products manufactured using, or extracted directly from, living systems (bacteria, plant, animal cells). These include proteins, vaccines, blood products, cellular therapies, and gene therapies.

• Biopharmaceuticals: Medical drugs produced using biotechnology and molecular biology methods. These include:

  • Peptides and Proteins: Recombinant proteins and monoclonal antibodies (mAbs).

  • Nucleic Acids: DNA, RNA, or antisense oligonucleotides.

• Broad Redefinition: Any product derived from biological sources or designed to mimic biological molecules for therapeutic or diagnostic purposes.

• Main Categories (August 2024 data):

  • Antibodies.

  • Vaccines.

  • Nucleic-acid Therapeutics.

  • Enzymes.

  • Recombinant Hormones.

  • Engineered Cell Therapeutics.

  • Miscellaneous.

• Environmental Sustainability: Biopharmaceuticals are noted as being less harmful to the planet compared to traditional chemical synthesis.

Market Trends and Regulatory Status

• FDA Approval Trends (1994–2024):

  • There is a general upward trend in the approval of Biologics License Applications (BLAs) compared to New Molecular Entities (NMEs).

  • Between 2016 and 2021, approvals show a diverse structural classification.

• FDA Approvals by Modality (2024):

  • Small Molecules: 30 approvals.

  • Monoclonal Antibodies (mAb): 16 approvals.

  • Proteins: 10 approvals (including 2 fusion proteins).

  • Peptides: 3 approvals.

  • Oligonucleotides: 2 approvals (antisense/inhibitor).

  • Other: Includes imaging pharmaceuticals, combination drugs, toxins, and bispecific mAbs.

• Protein Expression Market:

  • Value in 2024: $2.7 billion.

  • Projected value in 2035: $6.3 \text{ Billion}$.

  • Compound Annual Growth Rate (CAGR): $8.0\%$.

  • Global market includes mammalian, yeast, cell-free, prokaryotic, insect, and algal expression systems.

Expression Systems for Recombinant Protein Production

• Active Ingredients: Most commercially available biopharmaceuticals contain recombinant proteins.

• Host Selection Factors:

  • Expression levels.

  • Protein folding quality.

  • Post-Translational Modifications (PTMs): Crucial examples include Glycosylation and Phosphorylation.

  • Scalability.

  • Time to expression.

  • Costs (Maintenance and Production).

  • Secretory mechanisms.

  • Ease of genetic manipulation.

Comparative Analysis of Host Organisms

• Bacterial Cells (e.g., E. coli, B. subtilis):

  • Advantages: Low cost, rapid growth, high expression levels.

  • Disadvantages: Protein misfolding, lack of PTMs, secretion issues.

  • Example Product: Trurapi (insulin aspart) by Sanofi.

• Yeast Cells (e.g., S. cerevisiae, P. pastoris):

  • Advantages: Low cost, rapid growth, stable strains, can perform some PTMs.

  • Disadvantages: Some incompatible PTMs, cell aggregation, inadequate secretion.

  • Example Product: Vyepti (eptinezumab-jjmr) by Lundbeck Seattle.

• Insect Cell Lines (e.g., S2 from D. melanogaster, Sf9 from S. frugiperda):

  • Advantages: High expression, correct folding, performs PTMs.

  • Disadvantages: Some incompatible PTMs, time-consuming process.

  • Example Product: Supemtek (quadrivalent influenza vaccine) by Sanofi.

• Mammalian Cell Lines (e.g., CHO, HEK293):

  • Advantages: Correct folding, human-compatible PTMs (Glycosylation), established regulatory approvals.

  • Disadvantages: High cost, lengthy production timeframe, potential for unstable cell lines.

  • Example Product: Rituxan/MabThera (rituximab) by Genentech/Roche.

The Importance of Mammalian Expression Systems

• Complexity: Mammalian systems produce large, complex proteins with PTMs (specifically glycosylation) that are most similar to native human proteins.

• Market Dominance:

  • As of 2020–2022, approximately $75\%$ of biopharmaceutical approvals utilize mammalian-based production cells, a significant rise from approximately $33\%$ in 1989.

• Glycosylation: A key PTM for monoclonal antibodies (mAbs) that ensures efficacy and reduces immunogenicity in humans.

Classification and Characteristics of Cell Cultures

• Primary Culture:

  • Maintenance of cells dissociated directly from parental tissue (via chemical, mechanical, or biological methods).

  • Maintained in medium using glass or plastic containers.

  • Finite lifespan in vitro.

  • Retain differentiated phenotypes.

  • Usually anchorage-dependent and exhibit contact inhibition.

• Sub-classification of Primary Cultures by Morphology:

  • Epithelial Cell Type.

  • Endothelial Cell Type.

  • Lymphoblast Cell Type.

  • Neuronal Cell Type.

  • Fibroblast Cell Type.

• Secondary Cell Cultures (Cell Lines):

  • Result from the sub-culture (passage) of primary cells.

  • Requires transfer to a new vessel to provide fresh nutrients and space.

Finite vs. Continuous Cell Lines

• Finite Cell Lines:

  • Limited lifespan and number of generations.

  • Slow growth rate.

  • Doubling time: $24-96 \text{ hours}$.

• Continuous Cell Lines:

  • Transformed or immortalized.

  • Property of ploidy.

  • Absence of contact inhibition (can divide indefinitely).

  • Rapid growth rate.

  • Doubling time: $12-24 \text{ hours}$.

  • Transformation definition: Spontaneous or induced permanent phenotypic changes resulting from heritable changes in DNA.

  • Immortalization: Can be achieved via telomerase or viral/chemical methods.

• Cell Strains: A subpopulation of a cell line positively selected from a culture through cloning or other specific methods.

Attachment Properties of Mammalian Cells

• Adherent (Anchorage-dependent):

  • Require a surface to attach to for growth.

  • Growth is limited by surface area.

  • Dissociated enzymatically or mechanically.

• Suspension (Anchorage-independent):

  • Do not require attachment.

  • Growth is limited by cell concentration.

  • Often derived from blood system cells or adapted cell lines.

Historical Milestones in Animal Cell Technology

• Early Foundations:

  • 1885: Roux maintained chicken embryos in saline solution.

  • 1907-1910: Harrison observed nerve fiber outgrowth in culture (frog nerve fibers).

• Medium and Technique Advancement:

  • 1910s-20s: Carrel introduced aseptic techniques and the "Carrel" flask; Rous and Jones used trypsin for cell suspension.

  • 1940s-50s: Addition of antibiotics to medium.

  • 1952: HeLa (first human cell line) established from cervical cancer cells.

  • 1950s: Development of synthetic media like MEM (Minimum Essential Medium).

  • 1958: Chinese Hamster Ovary (CHO) cell line established.

• Expansion of Capability:

  • 1960s: Hayflick and Moorhead demonstrated the finite lifespan of human cells; Ham grew cells in serum-free medium.

  • 1975: Kohler and Milstein produced antibody-secreting hybridomas.

  • 1980s: Discovery of telomerase (Blackburn and Greider); first mAb approved for human therapy; first CHO-derived protein (tPA) licensed.

• Modern Era:

  • 1997: Birth of Dolly the sheep.

  • 2000s-Present: Discovery of human induced pluripotent stem cells (iPSCs); advancements in 3D cell culture, microfluidics, 3D bioprinting, and organoid research.

Applications of Cell Culture

• Studying normal cell physiology and biochemistry.

• Testing effects of drugs and toxic compounds.

• Studying mutagenesis and carcinogenesis.

• Drug screening and development.

• Stem cell, cellular, and gene therapy.

• Large-scale manufacture of biological compounds (vaccines and therapeutic proteins).

Specific Mammalian Cell Lines in Biopharma

• CHO (Chinese Hamster Ovary): Established 1958. Accounts for 81% of mammalian cell lines used in manufacturing.

  • Advantages: Allows gene amplification for higher yields; low susceptibility to human viruses; grows in suspension; adaptable to serum-free/animal-free culture; well-characterized and regulated; capable of human-compatible PTMs.

  • Disadvantages: Less effective than human cells for certain PTMs; long development time.

• HeLa: Established 1952. Crucial for the polio vaccine.

• NS0 and Sp2/0: Murine myeloma cells; established 1970s-80s. Important for specific mAb production (NS0 accounts for 8%, Sp2/0 for 2%).

• HEK293 (Human Embryonic Kidney): Established 1973. Human cell line used in approximately 3% of production.

• BHK21 (Baby Hamster Kidney): Established 1961. Used for veterinary vaccines and recombinant clotting factors (4% share).

• Per.C6: Immortalized human embryonic retinal cells; established in the 1990s.