The Biopharmaceutical Revolution Flashcards
What is Recombinant DNA technology
a series of techniques to manipulate and isolate DNA segments of interest
what 4 areas does the rDNA market deal with
- blood products
- immunotherapy products
- infectious disease combatants
- growth factors for mammalian cells
examples of rDNA blood products
thrombolytics
clotting agents
septic shock drugs
examples of rDNA immunotherapy products
interferons
interleukins
colony stimulating factors
examples of rDNA infectious disease combatants
hep B & AIDS vaccines
examples of rDNA growth factors for mammalian cells (6)
epidermal
insulin-like
fibroblast
transforming
platelet-derived
growth hormone releasing factors
what are the different types of recombinant hosts
- bacterial (E. coli)
- yeast (Pichia pastoris)
- insect cells
- mammalian cells
selection of recombinant host depends on what factors?
- protein complexity
- required modifications
- scalability
- cost
advantages of bacteria (E. coli) as recombinant host (10)
- easy to quickly and precisely modify genome
- rapid growth
- simple/cheap/flexible culture conditions
- easy to reduce protease activity
- possible to avoid incorporation of amino acids analogues
- simple promoter control
- easy to alter plasmid copy
- easy to alter metabolic carbon flow
- formation of intracellular disulphide bonds is easy
- reproducible performance
disadvantages of bacteria (E. coli) as recombinant host (4)
- formation of inclusion bodies
- no post-translation modification (issues with solubility, function and resemblance)
- endotoxin contamination
- downstream processing can be quite involved
advantages of yeast (pichia pastoris) as recombinant host (6)
- low cost
- rapid expression
- easy to scale up (can grow high cell densities)
- high levels protein production
- some post-translational modifications (glycosylation & phosphorylation)
- secrete system facilitates purification
disadvantages of yeast as recombinant host (5)
- not suitable for many human proteins (only those whose function is unaffected by glycosylation patterns)
- host strain must be carefully selected
- presence of mannose cause immunological rejection in mammals
- different post-translational processing from mammals
- low authenticity & bioactivity
advantages of insect cells as recombinant host (6)
- post-translational modification
- high cell density
- slightly more complex
- use baculovirus-infected cells (eukaryotic protein processing capabilities)
- can fold, modify, transport and assemble new polypeptides
- highly authentic, soluble end products
disadvantages of insect cells as recombinant host (3)
- costly
- different protein processing pathways than in higher eukaryotes (partial glycosylation)
- over expression may result in IBs
advantages of mammalian (CHO & HEK293) cells as recombinant host (6)
- similar post-translational modification
- produce versatile human-compatible proteins
- mammalian proteins more similar to native proteins
- transport to correct destinations
- optimal folding
- optimal bioactivity preservations
