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Biotechnology > Producing proteins on a large scale 3 > Flashcards

Producing proteins on a large scale 3 Flashcards

1
Q

What is the cooling jacket of a bioreactor filled with?

A

Water - used to control temperature of the cells

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2
Q

What is the purpose of the impeller?

A

To give homogeneity to cells

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3
Q

What is the ‘boundary layer’?

A

The distance that the retardation of molecules by friction with the edge of pipes and retarded molecules penetrates.

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4
Q

What property of the fluid is the boundary layer related to?

A

The viscosity of the fluid

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5
Q

What is the equation for Tau?

A

F/A

hence tau is proportional to force

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6
Q

What is the symbol for viscosity?

A

mu

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7
Q

what is du/dy a measure of?

A

velocity gradient which is a measure of the border layer

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8
Q

what is a newtonian fluid?

A

A fluid that has a constant viscosity (mu) regardless of the pressure applied

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9
Q

what is a non-newtonian fluid?

A

A fluid without a constant viscosity

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10
Q

Is a bingham plastic newtonian?

A

No - bingham plastic has a yield stress and then after that behaves as a perfect fluid (a newtonian liquid)

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11
Q

What is shear thickening and thinning?

A

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

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12
Q

what is the relationship between tau and gamma in newtonian fluids?

A

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

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13
Q

What is the Reynolds number threshold for laminar flow?

A

< 2100

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14
Q

What are the units of the reynolds number?

A

unitless

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15
Q

Is turbulent or laminar flow better for mixing cells?

A

turbulent (assuming cells are not very sensitive to sheer stress)

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16
Q

What is reynolds number for?

A

determining whether flow is turbulent or laminar

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17
Q

You are a biochemical engineer. To increase the reynolds number in your bioreactor what should you do?

A

Increase the speed of propellor (or diameter of propellor)

Unlikely to be able to change the viscosity of the fluid

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18
Q

Why do we mix bioreactors?

A 
For homogeneity (nutrients, oxygen, temperature)
Ensure all cells in same condition and hence results are reliable and reproducible and can be modelled
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19
Q

Radial blades are at a X˚ angle to the shaft.

What is X

A

90

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20
Q

What is a rushton impellor?

A

The most common impellor. It is radial

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21
Q

What is an axial propellor?

A

Blades are at a slant and so pushes medium to the bottom of the reactor

22
Q

Why is an radial impellor better for aeration?

A

Creates smaller current beneath the larger current which means that oxygen bubbles exist in the medium for longer

23
Q

Is axial or radial flow better for temperature homogeneity?

A

Axial flow

24
Q

Is vortex mixing bad or good?

A

Bad - it is too ordered

25
Q

How might you get an estimate of mixing time?

A

inject a dye (tracer) a measure how long it takes to mix in

- normally 4x the circulation time

26
Q

Why do you want to minimise steps downstream?

A

you lose a bit of protein at each step

27
Q

How are cells separated from the medium?

A

centrifugation (batch or continuous)

filtration

28
Q

What is the purpose of ultrafugation?

A

Used to concentrate proteins

- water allowed through pores but not protein

29
Q

Both yeast and bacteria are strong resistant cells. Which requirer the higher speed centrifugation?

A

Bacteria - because they are small

yeast are larger cells

30
Q

Why are Filamentous fungi cells not good candidates for separation from medium by centrifugation?

A

Mycelial (chains of cells) and have have high water retention in the pellet

31
Q

What centrifugation speed are mammalian cells limited to ?

A

800rpm

32
Q

What is the difference between vacuum filtration and positive pressure filtration?

A

Vacuum - pulled through by flowing water

Positive pressure - pushed through filter

33
Q

Why is tangential flow preferable to normal flow filtration?

A

tangential flow prevents build up of substance on the filter paper

34
Q

Name 3 general methods of cell lysis?

A

sonification
pressure
grinding

35
Q

How would you use pressure to lyse a cell?

A

french press - fired against ‘wall’

can be continous

36
Q

What is an inclusion body?

A

intracellular aggregates of misfolded proteins

37
Q

Why are inclusion bodies not always a bad thing?

A

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

38
Q

What are the advantages of precipitation for protein purification?

A

It scales easily and can often give a very good level of purification

39
Q

What are the disadvantages of precipitation for protein purification?

A

Optimised on a case-by-case basis

The salt need to then be removed later

40
Q

What are common substances used for precipitation?

A

ammonium sulphate

PEG

41
Q

What are the four main types of chromatography?

A

Size exclusion
Ion exchange
Hydrophobic interaction
Affinity

42
Q

What is size exclusion chromatography?

A

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)

43
Q

What is ion exchange chromatography?

A

Resin has a charge and proteins interact

eluted using buffer at PI of protein

44
Q

What is hydrophobic interaction chromatography?

A

Interaction between hydrophobic surface and hydrophobic substance

45
Q

What is affinity chromatography?

A

Specific binding between protein of interest and resin

46
Q

What is IMAC?

A

Immobilised metal affinity chromatography

Using a metal in the resin that binds something specific

47
Q

What is viral inactivation?

A

Kill Virus’ so that the product is safe as biopharmaceutical even if it is a virus that is typically unable to infect human cells

48
Q

What is viral inactivation achieved?

A

A low pH

49
Q

What is nanofiltration?

A

Filtration to remove the dead virus.

50
Q

Why is scale up a problem?

A

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

51
Q

What is scale down used for?

A

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

Biotechnology (13 decks)

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