Producing proteins on a large scale 3 Flashcards
What is the cooling jacket of a bioreactor filled with?
Water - used to control temperature of the cells
What is the purpose of the impeller?
To give homogeneity to cells
What is the ‘boundary layer’?
The distance that the retardation of molecules by friction with the edge of pipes and retarded molecules penetrates.
What property of the fluid is the boundary layer related to?
The viscosity of the fluid
What is the equation for Tau?
F/A
hence tau is proportional to force
What is the symbol for viscosity?
mu
what is du/dy a measure of?
velocity gradient which is a measure of the border layer
what is a newtonian fluid?
A fluid that has a constant viscosity (mu) regardless of the pressure applied
what is a non-newtonian fluid?
A fluid without a constant viscosity
Is a bingham plastic newtonian?
No - bingham plastic has a yield stress and then after that behaves as a perfect fluid (a newtonian liquid)
What is shear thickening and thinning?
Shear thinking and shear thinning – viscosity gets higher or lower as you apply more force
These never behave as a perfect fluid even after a threshold
what is the relationship between tau and gamma in newtonian fluids?
constant relationship - i.e. constant viscosity
If line on the gamma v tau graph doesn’t go through the origin then the fluid is not newtonian
What is the Reynolds number threshold for laminar flow?
< 2100
What are the units of the reynolds number?
unitless
Is turbulent or laminar flow better for mixing cells?
turbulent (assuming cells are not very sensitive to sheer stress)
What is reynolds number for?
determining whether flow is turbulent or laminar
You are a biochemical engineer. To increase the reynolds number in your bioreactor what should you do?
Increase the speed of propellor (or diameter of propellor)
Unlikely to be able to change the viscosity of the fluid
Why do we mix bioreactors?
For homogeneity (nutrients, oxygen, temperature) Ensure all cells in same condition and hence results are reliable and reproducible and can be modelled
Radial blades are at a X˚ angle to the shaft.
What is X
90
What is a rushton impellor?
The most common impellor. It is radial
What is an axial propellor?
Blades are at a slant and so pushes medium to the bottom of the reactor
Why is an radial impellor better for aeration?
Creates smaller current beneath the larger current which means that oxygen bubbles exist in the medium for longer
Is axial or radial flow better for temperature homogeneity?
Axial flow
Is vortex mixing bad or good?
Bad - it is too ordered
How might you get an estimate of mixing time?
inject a dye (tracer) a measure how long it takes to mix in
- normally 4x the circulation time
Why do you want to minimise steps downstream?
you lose a bit of protein at each step
How are cells separated from the medium?
centrifugation (batch or continuous)
filtration
What is the purpose of ultrafugation?
Used to concentrate proteins
- water allowed through pores but not protein
Both yeast and bacteria are strong resistant cells. Which requirer the higher speed centrifugation?
Bacteria - because they are small
yeast are larger cells
Why are Filamentous fungi cells not good candidates for separation from medium by centrifugation?
Mycelial (chains of cells) and have have high water retention in the pellet
What centrifugation speed are mammalian cells limited to ?
800rpm
What is the difference between vacuum filtration and positive pressure filtration?
Vacuum - pulled through by flowing water
Positive pressure - pushed through filter
Why is tangential flow preferable to normal flow filtration?
tangential flow prevents build up of substance on the filter paper
Name 3 general methods of cell lysis?
sonification
pressure
grinding
How would you use pressure to lyse a cell?
french press - fired against ‘wall’
can be continous
What is an inclusion body?
intracellular aggregates of misfolded proteins
Why are inclusion bodies not always a bad thing?
You can collect them and then you have very pure samples of desired protein
However is only useful if you are able to refold the protein
What are the advantages of precipitation for protein purification?
It scales easily and can often give a very good level of purification
What are the disadvantages of precipitation for protein purification?
Optimised on a case-by-case basis
The salt need to then be removed later
What are common substances used for precipitation?
ammonium sulphate
PEG
What are the four main types of chromatography?
Size exclusion
Ion exchange
Hydrophobic interaction
Affinity
What is size exclusion chromatography?
Resin with has big holes in - big protein can’t fit in holes and hence move down faster, smaller retained longer because fit in hole and are slowed down
(GRAVITY)
What is ion exchange chromatography?
Resin has a charge and proteins interact
eluted using buffer at PI of protein
What is hydrophobic interaction chromatography?
Interaction between hydrophobic surface and hydrophobic substance
What is affinity chromatography?
Specific binding between protein of interest and resin
What is IMAC?
Immobilised metal affinity chromatography
Using a metal in the resin that binds something specific
What is viral inactivation?
Kill Virus’ so that the product is safe as biopharmaceutical even if it is a virus that is typically unable to infect human cells
What is viral inactivation achieved?
A low pH
What is nanofiltration?
Filtration to remove the dead virus.
Why is scale up a problem?
You can never completely reproduce physical and chemical conditions when you change the scales. You can only ever keep one condition the same.
You cannot not keep the power requirement and the air flow rate in proportion for example
What is scale down used for?
Use the inverse process of ‘scaling down’ to try and replicate the conditions of our big bioreactor on the small scale so that we can trouble shoot more easily and cheaply
