Host Cells

• Understanding host cells is crucial when synthesizing recombinant proteins for biopharmaceutical products.

Types of Host Cells Used in Biopharmaceutical Production

• Bacteria are simple to use but may result in insufficient folding of complex proteins and inclusion bodies.
• Bacteria lack post-translational modifications and may produce endotoxins.
• Yeast allows for post-translational modifications that differ from mammalian cells and can degrade proteins.
• Insect and mammalian cells require laborious construction of over-expressing strains and expensive media.
• Insect and mammalian cells have low growth rates and can be difficult to scale up.
• Transgenic plants and animals have long developmental cycles and are subject to contamination problems, including animal viruses and prions.

Choice of Host Cells

• The choice depends on the characteristics and properties of the protein of interest and how big it is.
• The amount of protein needed, whether it needs to be secreted extracellularly, and endotoxin presence must be considered.
• The need for post-translational modifications like glycosylation also influences the host cell type.
• Escherichia coli (E. coli) and Chinese hamster ovary (CHO) are the most commonly used.
• E. coli are prokaryotic cells, while CHO cells are eukaryotic cells.

Prokaryotic vs. Eukaryotic Systems

• Eukaryotic cells have a defined nucleus, organelles (rough ER, smooth ER, mitochondrion, Golgi apparatus, lysosome, etc), a cytoskeleton and ribosomes.
• Eukaryotic ribosomes are found in both free form in the cytoplasm and membrane-bound form in the rough ER.
• Prokaryotic cells do not have a nucleus, organelles, or a cytoskeleton.

Host Cells – Bacteria (E. coli)

• E. coli is a common microbial species used for biopharmaceutical production; K12 and B strains are commonly used strains.
• E. coli are Gram-negative bacteria.
• The first recombinant therapeutic protein produced in E. coli cells was insulin.

Advantages of E. coli

• The molecular biology of E. coli is well-characterized, which facilitates genetic manipulation.
• E. coli can give high expression levels of recombinant protein, up to 30% of total cellular protein.
• E. coli grows rapidly, with a doubling time of approximately 20 minutes, on simple and inexpensive media.

Disadvantages of E. coli

• Recombinant proteins accumulate intracellularly which requires additional processing steps to purify proteins from rest of host cell proteins.
• Proteins synthesized rapidly and in high levels may form insoluble aggregates called inclusion bodies.
• Isolated insoluble proteins need refolding to be active.
• E. coli lacks the ability to perform post-translational modifications, such as glycosylation.
• Lipopolysaccharides (LPS) are present on its surface, acting as pyrogens.

Problems with Inclusion Bodies

• Incorrectly folded recombinant protein accumulates as inclusion bodies in E. coli cells.
• Inclusion bodies have to be extracted from the cells and isolated and then have to be solubilized and denatured.
• Refolding of the protein takes place outside the cell with the addition of enzymes and factors.
• Correctly folded protein is then separated from incorrectly folded protein (which forms aggregates).

Host Cells – Mammalian Cells

• Primary cultures originate from normal animal tissues, but are maintainable in vitro for a limited number of generations of cultures.
• Primary cell types are suspension cells derived from blood and adherent cells (e.g., fibroblasts from connective tissues).
• Continuous cell lines are immortalized cell lines.
• Transformed cell lines are from human fibroblasts immortalized by simian virus 40 (SV40) infection.
• Continuous cells can be cultured for an infinite number of generations and may suffer phenotypic drift and lose in vivo characteristics after being subcultured.
• Examples of adherent cell lines include Vero (monkey kidney), stem cells, HeLa (cervical carcinoma), and CHO-K1 (Chinese hamster ovary).
• Examples of suspension cell lines are HEK293 (human embryonic kidney) cells and CHO-K1.

Mammalian Cell Growth Factors

• Doubling time is between 12 and 24 hours, depending on cell type, pH, temperature, gas supply, medium and routine maintenance.
• Mammalian cell division occurs via mitosis; the cycle consists of 4 phases.
• Primary cell lines are highly regulated and more complex when manipulating cell growth
• Transformed cell lines contain multiple mutations in genes that helps them regulate the cell cycle.
• Transformed cell lines are less dependent on growth factors, and in vitro cell growth occurs in less complex medium.
• Mammalian cells do not re-enter a cell cycle after completing one, it can remain in the pre-G1 phase

Chinese Hamster Ovary (CHO) Cells

• CHO cells are the preferred mammalian cell line for protein synthesis; they grow in suspension culture, are safe to use and less viruses grow in them.
• CHO cells grow in serum-free and chemically defined media, ensuring reproducibility between cell cultures.
• Glycosylation of glycoproteins produced by CHO cells are more bioactive and compatible in humans.
• Genetic engineering techniques can also manipulate a higher yield of proteins from CHO cells.
• The first recombinant therapeutic protein produced from a CHO cell was Tissue plasminogen activator (r-tPA).
• Today, approximately 70% of all recombinant protein therapeutics are produced using CHO cells.

Biopharmaceutical Production – Comparison between Expression Systems

• E. coli and yeast uses simple media types, insect cells are similar but mammalian cells require complex compounds.
• E. coli and yeast have higher level of expression than insect and mammalian cells.
• E. coli, yeast and insect cells all require hours, whereas mammalian cells require days or longer.
• E. coli has the shortest doubling time, whereas yeast has the longest.
• All of the expression systems show variable levels of protein proteolytic processing whereas glycosylation, fatty acid acylation, and amidation, sulfation and y-carboxylation is lacking in E. coli.
• E.Coli and Insect cells have a protein proteolytic processing of (+/-), both Yeast and Mammalian cells are (+) in comparision