Producing proteins on a large scale 2

Question 1

What are the main stages of cell culture growth?

Answer 1

Lag phase, Exponential phase, stationary phase, decline phase

Question 2

Cell growth is considered a ‘what’ order reaction

Answer 2

first order reaction

Question 3

In the equation

Generation = muX what do the variables represent?

Answer 3

mu = Specific cell growth rate
X = the number of cells

Question 4

What is the Monod kinetic formula?

Answer 4

mu = (mumax*X)/(Ks+S)

Question 5

What is mumax?

Answer 5

maximum specific growth rate – represents the cells in exponential phase

Question 6

What is Ks?

Answer 6

The growth- limiting substrate concentrations to give half the µmax

Question 7

What are the axis of the double reciprocal linear graph used to show monod kinetics?

Answer 7

x - 1/S

Y - 1/mu

Question 8

In the lineweaver burk plot what are the intercepts?

Answer 8

X - -1/Ks

y - 1/mumax

Question 9

In the context of cells what is consumption?

Answer 9

Cell death
comsumption = KdX
no cell death is assumed in exponential or lag phase (in batch)
and through continuous reactor

Question 10

In the context of cell what is Kd?

Answer 10

The death constant

Question 11

If your product is under a constitutive promoter is the product production growth associated?

Answer 11

Yes

Question 12

If your product is under a inducible promoter is the product production growth associated?

Answer 12

no - non-growth associated promoter

Question 13

What is the Leudeking-piret formula?

Answer 13

Generation = alphamuX + betaX

Question 14

With a constitutive promoter what equals zero:

a) alphamuX
b) betaX

Answer 14

betaX

- growth associated

Question 15

How do you determine Yx/s, Yp/s and Ms?

Answer 15

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

Question 16

What is Qs?

Answer 16

specific utilisation of substrate

Question 17

What scale production might you use a batch reactor for?

Answer 17

Small Scale Production

Question 18

What are the limitations of a batch reactor?

Answer 18

Expensive because of downtime

Inhibition due to high conc of things cells produce e.g. lactate

Question 19

What cells are often cultured in fed (semi-) batch reactors?

Answer 19

Mammalian cells

Question 20

In fed batch the amount of what stays roughly constant?

Answer 20

Substrate

Question 21

In continuous stirred tank reactors what are the benefits?

Answer 21

Constant productivity
Steady state physiology
Control cell growth rate via dilution

Question 22

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?

Answer 22

Continuous stirred tank reactor

Question 23

What is the principle of an airlift reactor?

Answer 23

Uses airflow to promote mixing.

Air dissolves in medium which get less dense, rises, bubbles burst, gets more dense, sinks

Question 24

What is the benefit of a airlift reactor?

Answer 24

No moving parts
Easy to maintain sterility
Allows a large volume 
Low energy input
Low shear

Question 25

What is a labile substrate?

Answer 25

Prone to degradation

Question 26

What are the drawbacks of an airlift reactor?

Answer 26

Large size necessary
Hard to maintain homogeneity
Needs a lot of air
Foaming occurs

Question 27

It airlift reactor batch or continuous?

Answer 27

Batch and occasionally fed-batch

Question 28

What is a wave bag?

Answer 28

A bag ( ~5L) on agitator tipped back and forth to mix medium

Question 29

What is the difficulty of wave bags?

Answer 29

Hard to scale up

Question 30

In exponential phase batch growth what is mu equal to?

Answer 30

mu = mumax

Question 31

What is Kd at exponential phase?

Answer 31

Negliable

Question 32

What assumptions are necessary before exponential phase batch growth?

Answer 32

mu = mumax

Negliable

Question 33

What are the assumptions in a continuous reactor calculation?

Answer 33

Kd=zero

No cells enter the reactor (Xi=0)

Question 34

What is the maximum dilution rate?

Answer 34

The point at which D = mumax

Question 35

What are the Quashi steady state assumptions?

Answer 35

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

Question 36

What what point do you start counting time in a fed batch?

Answer 36

As the substrate is added