biotechnology recap Flashcards
what is biotechnology?
Is the use of biological systems to generate useful products
•Uses endogenous biochemical pathways to drive product formation
•Can utilise genetic engineering and gene transfer technologies to augment biochemical pathways – this can be to up- or down-regulate gene expression
what is biotransormation?
substrate uses a cell
metabolic reactions, seconary metabolites and enzyme formation occur within the cell to give a product
what is exogeneous product formation?
genetic engineering in the cell to give product
what is engineered cell generation?
genetic engineering within the cell
cell division occurs
how do genes get switched on/off?
Genes need to be told to switch-on (at promoters) and sometimes when to switch-off (at promoters and 3’-end elements)
what is different to eukaryotic genes in genes and gene products?
Eukaryotic genes need to have the introns spliced-out
where are proteins packaged and transported?
Proteins are packaged and transported around the cell in vesicles
what mechanism is in place in vessicles so that a vessicle is able to dock with a receiving membrane?
Membrane budding generates a vesicle that is then able to “dock” with a receiving
membrane – specific proteins are sued to ensure that the vesicle docks with the
correct membrane
what is the 4 step process that summaries genetic engineering?
1-restriction enzymes
2-ligase enzymes
3-transformation
4-selection of clones
what are the benefits of using prokaryotics for expression strains?
- they usually have high growth rates
- can be grown relatively cheaply
- can be modified through genetic engineering quite easily
- Can generate quite a lot of product
what are the most used viral protomers for eukaryotes?
CMV/SV40 for strong expression
what types of viral promoters are used for lower level of expression or for cell-specific expresion patterns
More specific promoters will be used for lower expression levels (control) or for cell-specific expression patterns (mammalian expression)
what is the usual expression host for eukaryotes?
•The Chinese hamster ovary (CHO) cell for mammalian proteins **used in most cases ** Some yeasts (Pichia pastoris) can be used to generate > 500 g/L of culture
what is native insulin?
Native insulin is expressed as a pro-hormone that
has a leader (signal) peptide attached
= pre-pro-Insulin
what happens to insulin when the pro-insulin is folded?
The signal peptide is removed in the ER as the
pro-insulin is folded
what happens in diabetes to the insulin structure?
endogenous mutations that are implicated in diabetes
how does the purification of insulin occur?
shake flask production fermenter cell harvest cell breakage chromatographic separation concentration and formulation final product
what is the best suited formulation of insulin?
For most therapeutic proteins – liquid formulation is required
for administration
what biological formulation considerations would you have during the formulation process of insulin?
Structure/conformation preservation: pH/temperature effects
need for product-stabilising excipients
Antimicrobial requirements
Bioavailability modulation
what is uf/df
UF/DF is basically “filtration” but using a membrane that will allow molecules of a specified size pass through,
whilst retaining/recycling molecules of a larger size
The membrane has a specific MW “cut-off” measured in kDa
during uf/df what is the rate of fluid pumped? what pressure?
The particle-containing fluid to be filtered is pumped at a velocity in the range 1–8 m/s
parallel to the face of the membrane and with a pressure difference of 0.1–0.5 MN/m2 (MPa)
across the membrane.
does a more or less concentrated form of the molecule exist after uf/df filtration?
The liquid permeates through the membrane and the feed emerges in a more concentrated
form at the exit of the module.
what happens in diafultration?
Formulation of proteins is carried out by slow introduction of the formulation buffer
to the protein
The volume stays the same (Rate buffer OUT = Rate Buffer IN) and so the [protein]
stays constant so the buffer is exchanged slowly (5-7 Diavolumes = full buffer
exchanges)
what are the subtleties to be taken into consideration ?
Fermentation/Expression stage:
•The expression system used can dictate the quality of the product formed
•Amino acid substitutions/misfolds/truncates
Purification:
•Can be pass-through of process-related impurities (buffers, resins,
detergents, enzymes) into the final formulation
•Biologics can be quite heterogeneous – need to reduce this in the
purification process (charge and hydrophobicity variants are a pain!)
Formulation:
•The formulation is at the “mercy” of the fermentation and the
purification: rubbish in = rubbish out!
•The structure of the protein has to be maintained – even after storage for
a long period of time
what is the insulin lispro MOA?
Insulin is formulated as a hexameric structure (6 x insulin monomers) – coordinated by a zinc ion Insulin can be thought of as being a trimer of of dimers – the stability of the dimer association (bioavailability) Lispro has less stable dimeric association = dissociates more easily
what are the two alternative that the N terminus of the B chain of insulin lispro may adapt to?
T-state (taut): residues B1–B8 are in an extended conformation, which is followed by the central, structurally conserved α-helix of residues B9–B19 R-state (relaxed): the helix is further fully extended by residues B1–B8 Rf– frayed variant of the R-state
what is the I-state?
(I-state: intermediate between the T and
the R states)
what are insulin dimers stabilised by?
by hydrogen bonds, formed between the B-chain β-strands of
adjacent monomers.
what is wild type insulin?
our hydrogen bonds stabilize the T:Rf dimers
van der Waals interactions between the Pro28/Lys29 of one monomer and the GlyB20-GlyB23
loop of the adjacent monomer also contribute to dimer formation
how does lispro stabilise?
Inversion of the prolyl and lysyl residues near the B-chain C-terminus changes path of the protein
backbone and eliminates some van der Waals interactions between insulin monomers at the
dimer interface
