biotechnology recap

Question 1

what is biotechnology?

Answer 1

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

Question 2

what is biotransormation?

Answer 2

substrate uses a cell

metabolic reactions, seconary metabolites and enzyme formation occur within the cell to give a product

Question 3

what is exogeneous product formation?

Answer 3

genetic engineering in the cell to give product

Question 4

what is engineered cell generation?

Answer 4

genetic engineering within the cell

cell division occurs

Question 5

how do genes get switched on/off?

Answer 5

Genes need to be told to switch-on (at promoters) and sometimes when to switch-off (at promoters and 3’-end elements)

Question 6

what is different to eukaryotic genes in genes and gene products?

Answer 6

Eukaryotic genes need to have the introns spliced-out

Question 7

where are proteins packaged and transported?

Answer 7

Proteins are packaged and transported around the cell in vesicles

Question 8

what mechanism is in place in vessicles so that a vessicle is able to dock with a receiving membrane?

Answer 8

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

Question 9

what is the 4 step process that summaries genetic engineering?

Answer 9

1-restriction enzymes
2-ligase enzymes
3-transformation
4-selection of clones

Question 10

what are the benefits of using prokaryotics for expression strains?

Answer 10

• 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

Question 11

what are the most used viral protomers for eukaryotes?

Answer 11

CMV/SV40 for strong expression

Question 12

what types of viral promoters are used for lower level of expression or for cell-specific expresion patterns

Answer 12

More specific promoters will be used for lower expression levels (control) or for cell-specific expression patterns (mammalian expression)

Question 13

what is the usual expression host for eukaryotes?

Answer 13

•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

Question 14

what is native insulin?

Answer 14

Native insulin is expressed as a pro-hormone that
has a leader (signal) peptide attached
= pre-pro-Insulin

Question 15

what happens to insulin when the pro-insulin is folded?

Answer 15

The signal peptide is removed in the ER as the

pro-insulin is folded

Question 16

what happens in diabetes to the insulin structure?

Answer 16

endogenous mutations that are implicated in diabetes

Question 17

how does the purification of insulin occur?

Answer 17

shake flask
production fermenter
cell harvest
cell breakage
chromatographic separation
concentration and formulation
final product

Question 18

what is the best suited formulation of insulin?

Answer 18

For most therapeutic proteins – liquid formulation is required
for administration

Question 19

what biological formulation considerations would you have during the formulation process of insulin?

Answer 19

Structure/conformation preservation: pH/temperature effects
need for product-stabilising excipients
Antimicrobial requirements
Bioavailability modulation

Question 20

what is uf/df

Answer 20

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

Question 21

during uf/df what is the rate of fluid pumped? what pressure?

Answer 21

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.

Question 22

does a more or less concentrated form of the molecule exist after uf/df filtration?

Answer 22

The liquid permeates through the membrane and the feed emerges in a more concentrated
form at the exit of the module.

Question 23

what happens in diafultration?

Answer 23

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)

Question 24

what are the subtleties to be taken into consideration ?

Answer 24

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

Question 25

what is the insulin lispro MOA?

Answer 25

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

Question 26

what are the two alternative that the N terminus of the B chain of insulin lispro may adapt to?

Answer 26

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

Question 27

what is the I-state?

Answer 27

(I-state: intermediate between the T and

the R states)

Question 28

what are insulin dimers stabilised by?

Answer 28

by hydrogen bonds, formed between the B-chain β-strands of

adjacent monomers.

Question 29

what is wild type insulin?

Answer 29

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

Question 30

how does lispro stabilise?

Answer 30

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