biotechnology Flashcards
What is biotechnology?
the use of biological systems to generate useful products
uses endogenous biochemical pathways to drive product formation
can use genetic engineering and gene transfer technologies to augment biochemical pathways (to up/down regulate gene expression)
biotechnology and pharmacy
the rising use of biologics
% of biologics on the market
- 12% in 2004
- 45% today
- 60% by 2024
What is the central dogma of molecular biology?
- DNA in nucleus with genetic material
- transcription creates a copy of a gene in the form of RNA
- RNA read by ribosomes and the therapeutic protein is made
What is a promoter?
it switches on genes
What switches off genes?
promoter at the 5’ end
What is removed when mRNA is made in eukaryotic cells?
introns
-> not in prokaryotic cells
How are proteins transported inside the cell?
in vesicles
What cuts DNA?
restriction enzymes
What do ligase enzymes do?
stick pieces of DNA back together
What do restriction enymes do?
cut DNA
What sticks DNA back together?
ligase enzymes
What is transformation in genetic engineering?
process of putting a piece of DNA inside a cell and cells grow and produce protein
What allows cells to replicate in the expression vector?
origin of replication
benefits of using prokaryotyes
- high growth rates
- can be grown relatively cheaply
- can be modified through genetic engineering easily
- can generate a lot of product
What are eukaryotes used for?
to make more specalised/complex products like mAb
eukaryotic expression systems that can be used
- CHO (Chinese hamster ovary cell)
- yeasts
- plants/tobacco
eukaryotic expression systems that can be used
- CHO (Chinese hamster ovary cell
- yeasts
- plants/tobacco
When are CHO cells used?
mammalian proteins
benefits with using yeast cells
can generate >500 g/L of culture
What are plant/tobacco cells used for?
generate simple proteins (vaccines)
genetics and high expression is a problem
What is removed from pro-hormone insulin (pre-pro-insulin) to give active insulin?
signal peptide (AA 1-24) C peptide (AA 57-87)
chains in active insulin
A and B chain
What part of insulin is removed in the ER?
signal peptide
gives pro-insulin
Where are the 3 disulfide bonds in insulin?
2 holding A and B chain together
1 internal on A chain
How is insulin formulated?
as a hexamer binded by Zn ion
-> 6 insulin monomers
steps to purify insulin
- shake flask
- production fermenter
- cell harvest - centrifugation
- cell breakage - homogenisation
- chromotographic separation
- concentration and formuation
- final product
insulin processng complexities
- expression in host
- expression format
- expression level
- expression strength
- impurities - host cell proteins/lipids/DNA/carbs
- removal of C peptide
- what is the dosage?
- formulation buffer?
- stability of formulated material
problems with liquid formulations
there can be some insoluble/soluble intermediates generated before or during formulation
considerations for biological formulations
pH
temperature
antimicrobial requirements
bioavailability modulation
process for formulation of liquid proteins
untrafiltration and diafiltration
UF/DF
ultrafiltration (UF/DF)
- insulin solution flows into filter
- filter allows molecules of a certain size through
- into permeate solution
- the membrane has a sepcific Mr cut off measured in kDa
- anything too big to pass through filter is put back into the starting material
- volume of starting material will dec but the protein conc increases
ultrafiltration (UF/DF)
insulin solution flows into filter
filter
diafiltration (UF/DF)
- volume of container (with protein) is kept the same
- buffer is fed in at the same vol it goes out
- 5-7 diavolumes of buffer exchanges (5-7x more of permeate)
formulation of buffer during DV in inuslin formation
- phosphate buffer pH 7.4
- glycerol
- m-cresol (excipient, anti-microbial and stabilises the protein)
problems in the expression/fermentation stage
amino acid substitution
misfolds
truncates - half the protein produced
problems in the purification process
pass through of impurities into the final formulation
-> buffers, resins, detergents, enzymes
What is used to allow AA to be swapped/changes in insulin formulations?
recombinant DNA technology
What controls the bioavailability of insulin?
the stability of the dimer association
-> 3 dimers of insulin make up the hexamer
lispro
less stable dimer association -> disassociates easier
conformations of the N terminus of the B chain of insulin
R state - relaxed, helix is fully extended by residues B1-B8
T state - taut, residues B1-B8 are are in an extended conformation, conserves alpha-helix of residues B9-B19
I state - intermediate between T and R states
-> in the hexamer dimers can be T6, R6 or T3R3
wild type vs Lispro
WT - 4 H bonds stabilise T:Rf dimers, van der Waals interactions also, shallow cleft betwee the B chains, tight association
Lispro - swap of Lys and Pro removes some van der Waal interactions, cleft is slightly wider between the 2 monomers, less association between the monomers so it will quickly disassociate in blood with the pH change
learning from early biological therapeutical development
- biologics are very specific for biological targets if they’re generated correctly
- systems used to generate biologics needs to be controlled/characterised to ensure correct product formulation
- recombinant DNA technologies allow generation of complex biological therapies
- once DNA sequence controlled, have control over protein structure, 1st gen to 2nd gen shorter time
- biological systems give product variability and product related impurities
- protein based drugs are stable if treated correctly, storage/use
- biologics can induce an immune response in a patient - wanted/unwanted
