Recombinant Pharmaceuticals 1 and 2 Flashcards
Define the following terms in Pharmaceutical Biotechnology
A) Biotechnology
B) Processes
C) Biopharmaceuticals
D) Pharmaceutical biotechnology
A)
- the use of living things, especially cells and bacteria, in industrial processes
B)
- genetic engineering
C)
- medicine and drugs that are produced using biotechnology
D)
- biotechnological manufacturing processes of pharmaceutical products
What are the two types of cells?
- Prokaryotic cells
- Eukaryotic cells
How are products of rDNA technology produced? Describe what are the ways. How is the desired product recovered?
Produced by genetic modification in which DNA coding for the required product is introduced
- by means of a plasmid or a viral vector into a suitable micro-organism or cell line, in which that DNA is expressed and translated into protein
- The desired product is then recovered by extraction and purification.
- The cell or micro-organism before harbouring the vector is referred to as the host cell, and the stable association of the two used in the manufacturing process is referred to as the host-vector system.
Define what a vector and plasmid is.
Vector: A vehicle (e.g. a plasmid) used to transfer the genetic material such as DNA sequences from the donor organism to the target cell of the recipient organism.
Plasmid: Plasmids are fragments of double-stranded DNA that typically carry genes and can replicate independently from chromosomal DNA
What are the importance of plasmids?
- Easy to work with
- Self-replicating
- Stable
- Functional in many species and can be useful for a diverse set of applications
How does human insulin production occur? (HUMULIN R)
HUMULIN R is produced by recombinant DNA technology utilising a non-pathogenic laboratory strain of Escherichia coli (E.coli)
- Human insulin producing gene taken out of DNA
- Plasmid DNA taken out of E.coli bacteria
- Bacterial DNA and human gene combined
- Plasmid is reintroduced into bacteria
- Engineered bacteria multiply producing insulin
- Insulin is separated and purified to produce human insulin
What has to be considered when producing proteins for therapeutic use? What specifications have to be met?
A number of issues must be considered related to the manufacturing, purification and characterisation of the products
- Biotechnological products for therapeutic use have to meet strict specifications, especially when used via the parenteral route
What are the two systems used to produce biotech products (proteins of therapeutic interest)? Give examples.
Expression systems
- Transgenic animals
- plants
Cultivation systems
- Medium –> contaminants
Describe the process of how transgenic animals are used to get proteins of therapeutic interest.
- Foreign genes introduced into animals
- Desired protein is expressed in the milk
Advantage: Cheap method to produce in vast quantities
Disadvantage: Animal health
Describe the process of how plants are used to get proteins of therapeutic interest.
- Human albumin has been expressed in potatoes
Issues: stability and phenolic oxidases
Potential: expressed protein in edible seeds eg rice
For cultivation systems: medium;
A) What are the factors that have to be considered for the optimal growth of cells?
B) What else has to be considered?
C) How to prepare the final medium?
A)
- pH, oxygen tension, and temperature are chosen and controlled appropriately
B)
- medium with the proper nutrients is provided
C)
- components are dissolved in purified water before sterile filtration
For cultivation systems: contaminants
A) What are examples of host-related contaminants?
B) What are examples of product-related contaminants?
C) What are examples of process-related contaminants?
A)
- Viruses
- Host derived proteins acid DNA
- Endotoxins (from gram-negative bacterial hosts)
B)
- Amino acid substitution and deletion
- Denatured protein
- Protein fragments
- Dimers and aggregates
C)
- Growth medium components
- Metals
What is downstream processing? How does the purification cost compare to the upstream part of the production process?
Series of operations required to take biological materials such as cells, tissue culture fluid, or plant tissues, and derive from them a pure and homogeneous protein product.
- Recovering a biological reagent from a cell culture supernatant is one of the critical parts of the manufacturing procedure for biotech products and purification costs typically outweigh those of the upstream part of the production process
What are the separation techniques involved in downstream processing? THREE different reasons.
Filtration/centrifugation
- Products from biotechnological industry must be separated from biological systems that contain suspended particulate material, including whole cells, lysed cell material, and fragments of broken cells
- the expense and effectiveness of such methods is highly dependent on the physical nature of the particulate material and of the product.
Precipitation
- The solubility of a particular protein depends on the physico-chemical environment, for example pH, ionic species and ionic strength of the solution
Chromatography
- Primarily separated based on differences in distribution between two phases, one which is the stationary phase (mostly a solid phase) and the other which moves
- Adsorption, Ion-exchange, immunoaffinity, hydrophobic interaction chromatographies
What are the FOUR factors for formulating biotech products?
- Excipients used in parenteral formulations of biotech products
- Microbiological considerations
- Shelf life of protein-based pharmaceuticals
- Route of administration
What are the routes of administration for the formulation of biotech products?
- Parenteral route
- Oral
> protein degradation in the gastrointestinal (GI) tract and
> poor permeability of the wall of the GI tract in case of a passive transport process
> Oral vaccines
- Alternative routes (see attached image) –> nasal, pulmonary, rectal, buccal, transdermal
Describe what live attenuated vaccines and non-living vaccines are. Give examples of each vaccine.
Live attenuated vaccines
- a first step to improved live vaccines was the attenuation of virulent microorganisms by serial passage and selection of mutant strains with reduced virulence or toxicity
- Examples are vaccine strains for oral polio vaccine, measles-mumps-rubella (MMR) combination vaccine, and tuberculosis vaccine consisting of bacille Calmette-Gue´rin (BCG)
Non-living vaccines (two types)
- Inactivation of whole bacteria or viruses
> A number of reagents (e.g., formaldehyde, glutaraldehyde) and heat are commonly used for inactivation
- Subunit vaccines: eg Diphtheria and tetanus toxoids
