Producing proteins on a large scale 2 Flashcards
What are the main stages of cell culture growth?
Lag phase, Exponential phase, stationary phase, decline phase
Cell growth is considered a ‘what’ order reaction
first order reaction
In the equation
Generation = muX what do the variables represent?
mu = Specific cell growth rate X = the number of cells
What is the Monod kinetic formula?
mu = (mumax*X)/(Ks+S)
What is mumax?
maximum specific growth rate – represents the cells in exponential phase
What is Ks?
The growth- limiting substrate concentrations to give half the µmax
What are the axis of the double reciprocal linear graph used to show monod kinetics?
x - 1/S
Y - 1/mu
In the lineweaver burk plot what are the intercepts?
X - -1/Ks
y - 1/mumax
In the context of cells what is consumption?
Cell death
comsumption = KdX
no cell death is assumed in exponential or lag phase (in batch)
and through continuous reactor
In the context of cell what is Kd?
The death constant
If your product is under a constitutive promoter is the product production growth associated?
Yes
If your product is under a inducible promoter is the product production growth associated?
no - non-growth associated promoter
What is the Leudeking-piret formula?
Generation = alphamuX + betaX
With a constitutive promoter what equals zero:
a) alphamuX
b) betaX
betaX
- growth associated
How do you determine Yx/s, Yp/s and Ms?
Determined by plotting graphs
Yx/s - gradient of X/S graph
Yp/s - gradient of P/S graph
Ms - Qs/mu graph extrapolated back to the y axis
What is Qs?
specific utilisation of substrate
What scale production might you use a batch reactor for?
Small Scale Production
What are the limitations of a batch reactor?
Expensive because of downtime
Inhibition due to high conc of things cells produce e.g. lactate
What cells are often cultured in fed (semi-) batch reactors?
Mammalian cells
In fed batch the amount of what stays roughly constant?
Substrate
In continuous stirred tank reactors what are the benefits?
Constant productivity
Steady state physiology
Control cell growth rate via dilution
In what type of bioreactor is there a sensors to ensure that the volume that enters the tank is the same as that leaves the tank?
Continuous stirred tank reactor
What is the principle of an airlift reactor?
Uses airflow to promote mixing.
Air dissolves in medium which get less dense, rises, bubbles burst, gets more dense, sinks
What is the benefit of a airlift reactor?
No moving parts Easy to maintain sterility Allows a large volume Low energy input Low shear
What is a labile substrate?
Prone to degradation
What are the drawbacks of an airlift reactor?
Large size necessary
Hard to maintain homogeneity
Needs a lot of air
Foaming occurs
It airlift reactor batch or continuous?
Batch and occasionally fed-batch
What is a wave bag?
A bag ( ~5L) on agitator tipped back and forth to mix medium
What is the difficulty of wave bags?
Hard to scale up
In exponential phase batch growth what is mu equal to?
mu = mumax
What is Kd at exponential phase?
Negliable
What assumptions are necessary before exponential phase batch growth?
mu = mumax
Negliable
What are the assumptions in a continuous reactor calculation?
Kd=zero
No cells enter the reactor (Xi=0)
What is the maximum dilution rate?
The point at which D = mumax
What are the Quashi steady state assumptions?
Cells grow a lot and starve before you add substrate – use substrate very quickly
M is negligible
Non-growth associated protein product is negligible
dx/dt = zero
dv/dt = zero
What what point do you start counting time in a fed batch?
As the substrate is added
