Biologics Flashcards
(20 cards)
What is a biologic drug?
A biologic is a drug derived from a living organism, often more complex than chemically synthesized drugs, and includes insulin, antibodies, and vaccine components.
What are the four main stages from gene to biologic product?
- Isolate the gene. 2. Insert the gene into a cloning vector. 3. Introduce the vector into an expression host. 4. Scale up production and purify the product.
What is a cloning vector and why is it important?
A cloning vector is a stable DNA molecule (usually a plasmid) into which a gene of interest is inserted. It allows replication and expression of the gene in host cells.
What are plasmids and why are they used in cloning?
Plasmids are small, circular, double-stranded DNA molecules found in bacteria that replicate independently of chromosomal DNA. They are ideal for gene cloning because they are stable and easily manipulated.
How is the gene of interest introduced into a plasmid?
The gene is inserted using restriction enzymes and ligases to create a chimeric plasmid. Modern commercial systems simplify this insertion process.
What is an expression host and what are common examples?
An expression host is an organism used to produce a protein from a cloned gene. Common examples include E. coli, yeast, and mammalian cells (e.g., CHO cells).
How is gene expression achieved in a host cell?
The host transcribes the inserted gene into mRNA, then translates it into protein, mimicking the central dogma of molecular biology (DNA → RNA → Protein).
How can you make sure only transformed cells grow?
The plasmid contains a selectable marker (e.g., antibiotic resistance gene), so only cells that took up the plasmid survive on selective media.
What methods are used to introduce plasmids into bacteria?
- Chemical transformation (e.g., CaCl2 and heat shock), 2. Electroporation (electric shock), 3. Liposome-mediated fusion.
Why is reverse transcriptase used before cloning eukaryotic genes?
Reverse transcriptase converts mRNA to cDNA, removing introns (non-coding regions), which is necessary since bacteria cannot process introns.
What are CHO cells and why are they used?
Chinese Hamster Ovary (CHO) cells are mammalian cells used to express complex proteins with human-like post-translational modifications, especially antibodies.
What determines the choice of expression system?
It depends on the complexity of the protein. Simple proteins may be expressed in bacteria; complex proteins requiring modifications need mammalian cells.
What are the three main types of expression systems?
- Bacterial (e.g., E. coli), 2. Yeast, 3. Mammalian cells (e.g., CHO).
Advantages and disadvantages of using E. coli as an expression host?
Advantages: fast growth, low cost, high yield. Disadvantages: no glycosylation, proteins may form inactive aggregates (inclusion bodies).
What is upstream processing?
Upstream processing involves preparing the cell culture and growth medium, sterilizing equipment, and culturing cells to be used for fermentation.
What is fermentation in biologic production?
Fermentation is the process of growing the expression host in large bioreactors to produce the target protein in bulk.
What is downstream processing?
It involves recovering, purifying, and formulating the protein product after fermentation, including removal of impurities and packaging.
Why are biologic drugs expensive?
Due to complexity of production, need for sterile processing, post-translational modifications, and significant product loss during purification.
What are wave bags and why are they used?
Wave bags are disposable, sterile bioreactors that reduce contamination risk and simplify cleanup compared to traditional stainless steel bioreactors.
What is a master cell bank and why is it important?
A master cell bank is a collection of genetically identical cells stored for consistent production of biologics, ensuring reproducibility and quality control.
