Producing proteins on a large scale 3

Question 1

What is the cooling jacket of a bioreactor filled with?

Answer 1

Water - used to control temperature of the cells

Question 2

What is the purpose of the impeller?

Answer 2

To give homogeneity to cells

Question 3

What is the ‘boundary layer’?

Answer 3

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

Question 4

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

Answer 4

The viscosity of the fluid

Question 5

What is the equation for Tau?

Answer 5

F/A

hence tau is proportional to force

Question 6

What is the symbol for viscosity?

Answer 6

mu

Question 7

what is du/dy a measure of?

Answer 7

velocity gradient which is a measure of the border layer

Question 8

what is a newtonian fluid?

Answer 8

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

Question 9

what is a non-newtonian fluid?

Answer 9

A fluid without a constant viscosity

Question 10

Is a bingham plastic newtonian?

Answer 10

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

Question 11

What is shear thickening and thinning?

Answer 11

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

Question 12

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

Answer 12

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

Question 13

What is the Reynolds number threshold for laminar flow?

Answer 13

< 2100

Question 14

What are the units of the reynolds number?

Answer 14

unitless

Question 15

Is turbulent or laminar flow better for mixing cells?

Answer 15

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

Question 16

What is reynolds number for?

Answer 16

determining whether flow is turbulent or laminar

Question 17

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

Answer 17

Increase the speed of propellor (or diameter of propellor)

Unlikely to be able to change the viscosity of the fluid

Question 18

Why do we mix bioreactors?

Answer 18

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

Question 19

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

What is X

Answer 19

90

Question 20

What is a rushton impellor?

Answer 20

The most common impellor. It is radial

Question 21

What is an axial propellor?

Answer 21

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

Question 22

Why is an radial impellor better for aeration?

Answer 22

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

Question 23

Is axial or radial flow better for temperature homogeneity?

Answer 23

Axial flow

Question 24

Is vortex mixing bad or good?

Answer 24

Bad - it is too ordered

Question 25

How might you get an estimate of mixing time?

Answer 25

inject a dye (tracer) a measure how long it takes to mix in | - normally 4x the circulation time

Question 26

Why do you want to minimise steps downstream?

Answer 26

you lose a bit of protein at each step

Question 27

How are cells separated from the medium?

Answer 27

centrifugation (batch or continuous) | filtration

Question 28

What is the purpose of ultrafugation?

Answer 28

Used to concentrate proteins | - water allowed through pores but not protein

Question 29

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

Answer 29

Bacteria - because they are small | yeast are larger cells

Question 30

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

Answer 30

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

Question 31

What centrifugation speed are mammalian cells limited to ?

Answer 31

800rpm

Question 32

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

Answer 32

Vacuum - pulled through by flowing water | Positive pressure - pushed through filter

Question 33

Why is tangential flow preferable to normal flow filtration?

Answer 33

tangential flow prevents build up of substance on the filter paper

Question 34

Name 3 general methods of cell lysis?

Answer 34

sonification pressure grinding

Question 35

How would you use pressure to lyse a cell?

Answer 35

french press - fired against 'wall' | can be continous

Question 36

What is an inclusion body?

Answer 36

intracellular aggregates of misfolded proteins

Question 37

Why are inclusion bodies not always a bad thing?

Answer 37

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

Question 38

What are the advantages of precipitation for protein purification?

Answer 38

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

Question 39

What are the disadvantages of precipitation for protein purification?

Answer 39

Optimised on a case-by-case basis | The salt need to then be removed later

Question 40

What are common substances used for precipitation?

Answer 40

ammonium sulphate | PEG

Question 41

What are the four main types of chromatography?

Answer 41

Size exclusion Ion exchange Hydrophobic interaction Affinity

Question 42

What is size exclusion chromatography?

Answer 42

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)

Question 43

What is ion exchange chromatography?

Answer 43

Resin has a charge and proteins interact | eluted using buffer at PI of protein

Question 44

What is hydrophobic interaction chromatography?

Answer 44

Interaction between hydrophobic surface and hydrophobic substance

Question 45

What is affinity chromatography?

Answer 45

Specific binding between protein of interest and resin

Question 46

What is IMAC?

Answer 46

Immobilised metal affinity chromatography | Using a metal in the resin that binds something specific

Question 47

What is viral inactivation?

Answer 47

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

Question 48

What is viral inactivation achieved?

Answer 48

A low pH

Question 49

What is nanofiltration?

Answer 49

Filtration to remove the dead virus.

Question 50

Why is scale up a problem?

Answer 50

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

Question 51

What is scale down used for?

Answer 51

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