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BAP!! > Protein Production > Flashcards

Protein Production Flashcards

1
Q

How does bioindustry work?

A

The aim is to sell your product for more than it costs to make it. There’s 2 methods to do this:
-Make a high-value added product and sell a little bit of it
-Make a low-value added product and sell a lot of it.
This depends a lot on the type of product you are making e.g. enzymes for detergents and for use in industry processes are cheap but proteins as drugs cost a lot more to produce.

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2
Q

What are the costs for industry?

A 
Raw materials
Catalysts
Purification
Packaging
Shipping
Storage
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3
Q

What are the raw materials costs?

A 
Maintaining conditions
Media
Cells
Substrates
Growth factors
Sterilisation
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4
Q

What are the catalyst costs?

A

Cells –> fairly cheap
Cell line development –> expensive in time and resources
Enzymes –> Cheap but need replacing frequently

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5
Q

What are the purification costs?

A

Resins
Conditions
Depends on the amount of steps and the purity that is required.

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6
Q

What are the packaging/shipping/storage costs?

A

Sterility, cold chain, needs to be stable, depends on how long it can be stored for.

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7
Q

What are the 3 main applications?

A

Protein Therapeutics
Biocatalysis
Drug Discovery

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8
Q

Describe protein therapeutics.

A

The aim is to prevent, treat or cure diseases. Currently there is a surge in biosimilars and biobetters and it’s related to diagnostics. Current products include: insulin, growth factors, monoclonal antibodies, enzymes and vaccines. There were $130 billion sales in 2013 and they produce around 1-2kg/year.

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9
Q

Describe biocatalysis.

A

The aim is to use enzymes to perform chemical synthesis. It is used in many industries: food, biofuels, science, textiles, greener processes. Its produced on medium-large scale and can often cut purification costs as its not essential that its pure. $4.2 billion sales in 2014 so not a huge market

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10
Q

Describe drug discovery

A

The aim is to discover new drugs that is a costly process as 1 in 12 makes it clinical trials. The benefits are worthwhile so pharma companies put lots of money into it. Bioinformatics and high-throughput techniques are used to decrease costs, (customer doesn’t always see this reduction)

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11
Q

What is mass balance?

Why is it useful?

A

Mass balance is a system used to calculate the conservation of matter. It’s useful to see how effective parts of the process are and where it needs to be improved.

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12
Q

What’s the main mass balance equation?

A

Accumulation = In - Out + Generation - Consumption

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13
Q

Define System

A

A defined entity with entry and exit boundaries

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14
Q

Define Process

A

Events within a system

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15
Q

Define Upstream and Downstream

A

Upstream - events before or during product formation

Downstream - events after product formation

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16
Q

Define Steady-state

A

System where the variables are not changing in time

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17
Q

Define lab scale, pilot scale, scale up & scale down

A

Lab scale - Small scale done in the discovery phase <10L
Pilot scale - Small scale of the full plant, used to optimise the process 10-100L
Scale up - Increase production level
Scale-down - Decrease production level to trouble-shoot or develop

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18
Q

Define commodity

A

Low cost product made on a large scale

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19
Q

Define high-value added and low-value added

A

High-value added - Product sells for much more than it costs to produce
Low-value added - Product sells for a little more than it costs to produce

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20
Q

What is a bioreactor and what are the 4 possible forms of it?

A

A bioreactor is a vessel in which a reaction takes place in.
Batch - Materials are put in, operates, operation finishes and the product removed.
Semi- batch - Material is added or removed during the operation
Fed- batch - Material is added during operation
Continuous - Material is added and removed during operation

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21
Q

Define yield

A

The amount of product produced as a concentration or percentage conversion

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22
Q

Define productivity

A

The yield with time taken into account

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23
Q

Define specific

A

Used with other terms to indicate per cell

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24
Q

What are some of the mass balance assumptions?

A

Steady-state - Accumulation = 0
Non-reacting System = Gen = 0, Con = 0
Basis - choose a number to enter the system 1,10,100,1000
Dry/Liquid - Define the % efficiency of purification steps

25
Q

What are the cell phases of growth?

A

Lag phase
Exponential/growth phase
Stationary phase
Death phase

26
Q

What is the equation for generation and how does this occur in specific cell growth phases?

A 
Generation = (mu)X
exponential phase (mu) = (mu)max
lag/stationary phase (mu) = 0
mu - specific growth rate
X - no. of cells
27
Q

How do you find the specific growth rate (mu)?

A

Monod kinetics are used which state that growth depends on a growth-limiting substrate and follows saturation kinetics S.
The equation is ((mu)max * S)/(Ks + S)
This is similar to Michaelis-Menton so a lineweaver-burk -like plot can be used to determine parameters.
y-intercept - 1/(mu)max
x-intercept - -1/Ks
gradient - Ks/(mu)max

28
Q

What is the cell death term? How does this fit with cell growth phases?

A

Consumption = KdX
Lag/exponential phase - neglect cell death
Death/stationary phase - KdX

29
Q

How do constitutive and inducible promoters affect protein production?

A

Constitutive promoters are often from central metabolism or other common promoters so are often correlated to cell growth. They are known as growth-associated products
Inducible promoters only have expression when they are induced. They are known as non-growth associated products.
In reality both promoters produce mixed results so you get both terms.

30
Q

What is the term for product generation?

A

Generation = alpha (mu) X + beta X
The first term is growth-associated and the second term is non-growth associated. Both terms refer to mixed promoters which is what normally occurs due to leakiness and expression changes over time.

31
Q

What 3 places are substrates consumed and what terms describe them?

A

Cell growth = 1/(Yx/s) * (((mu)max *S)/(Ks + S )) * X
Protein production = 1/(Yp/s) * LPkinetics
Maintenance = MsX

32
Q

What equation describes batch bioreactors?

A

Acc=Gen-Con

33
Q

What equation describes fed-batch bioreactors?

A

Acc = In+Gen - Con

34
Q

What equation describes continuous bioreactors?

A

O= In-Out+Gen-Con

35
Q

What equation describes a bioreactor that involves recycling?

A

Acc= In+ Recycle-Out-Purge +Gen - Con

36
Q

How do reactors in series get calculated?

A

They can be calculated separately with the Out of the previous reactor being the In of the next one.

37
Q

What mode of operation is the bioreactor for an airlift bioreactor?

A

Batch

38
Q

What mode of operation is the bioreactor that uses wave bags?

A

Batch

39
Q

What equation describes the no. of cells in the exponential growth phase/ are important for batch bioreactors?

A 
X = Xo*e^(mu)max*t
Treaction = ln(X/Xo) * 1/(mu)max
40
Q

What equation describes the dilution rate?

A

Dilution rate = flow rate in/ volume of reaction

41
Q

What equations are important when dealing with continuous bioreactors?

A 
mu = D = ((mu)max*Sss)*(Ks +Sss)
Sss = (Ks*D)/(mu)max - D
Xss = Yx/s(S-Sss)
Pss = Yp/s(S-Sss)
42
Q

What equations are important when dealing with a fed-batch bioreactor?

A 
The quasi-steady state assumption is used.
dX/dt ~ 0
mu ~ D
S ~ (D*Ks)/(mu)max - D
Xtotal = Xinitial + (Yx/s*Si*F)tfb
Ptotal = Pinitial + (Yp/s*Si*F)tfb
43
Q

Name some parts of the bioreactor.

A 
Motor
Exhaust/ air filter
Condenser
Impeller
Inlet air filter
Sparger
Cooling water inlet/outlet
Baffles
Cooling jacket
44
Q

What are the 4 key parts to fluid behaviour?

A

Boundary layer
Viscosity
Shear stress
Turbulence

45
Q

What is the boundary layer?

A

In a pipe the majority of fluid moves with the bulk. Molecules that are next to the wall experience friction and molecules near these molecules experience friction from them. This dissipates towards the centre of the pipe. The boundary layer is the layers of molecules that feel friction.

46
Q

How does the boundary layer relate to viscosity and shear stress?

A

Viscosity is the stickiness of a fluid. If there’s two plates with a fluid between them and you push down, the fluid will attempt to remain stationary but will eventually move, this is shear stress. Shear stress (tau) is proportional to the pushing force applied.
The depth of the penetration of the pushing force is the boundary layer (gamma).
More force, more the plate moves, bigger boundary layer.
Viscosity (mu) = shear stress (tau)/boundary layer (gamma)

47
Q

Describe viscosities and how they can be different in terms of ideal and non-ideal fluids.

A

In terms of bioreactors the viscosity determines how much energy is required to stir the mixture. Ideal fluids have a constant viscosity. However sometimes a force is required before the viscosity if constant, known as yield stress. Other times the relationship is not linear.
Shear thin - viscosity decreases at high stirring
Shear thicken - viscosity increases at high stirring
Power law describes this : Tau = K(gamma)^n

48
Q

Describe turbulence

A

Ordered flow is called laminar flow. If the order breaks then turbulence occurs. You can determine turbulence using the reynolds number.
Reynolds number = densityspeedlength/visosity
Re<2100 –> laminar flow
Re > 10,000 (sometimes 4000) –> turbulence
Re in the middle –> transition region

49
Q

Describe mixing including the different levels of mixing

A

Mixing is important to ensure nutrients are distributed evenly so cells have equal access and there are no gradients. Most models assume adequate mixing.
Diffusion - Evens out gradients on a very small scale
Dispersion - Local mixing based on fluid movements.
Distribution - Moving parts around the reactor, done using an impeller.
You can use dye experiments to test it, the aim is full mix in 100 seconds.

50
Q

Describe the different types of impeller and mixing zones

A

Radial - straight blades, 4 mixing zones, good for gas retention. Rushton impeller is a 6 radial blade and is very popular.
Axial - pitched blades, 2 mixing zones, good for thermal mixing and keeping solids suspended.

51
Q

Describe the O2 transfer to cells process

A
  • O2 moves to the gas bubble edge
  • Gas moves to the liquid phase through the boundary layer of the liquid
  • Moves through the bulk liquid aided by mixing
  • Moves across cell/cell clump boundary layer
  • Crosses cell interface
  • Moves to correct area of the cell
52
Q

Why is aeration important?

A

Cells need oxygen. O2 has a low solubility (0.03g/kg) but there is a high demand for it. Bioreactors are sparged to increase oxygenation.

53
Q

What equations are relevant to aeration?

A

Qo = qoX (O2 demand =O2 uptake per cell no. of cells)
No2=KlA(Co2max -Co2) –> O2 transfer = mass transfer coefficient surface areadriving force
Determine the max no. of cells that can be supported by setting O2 transfer rate and oxygen demand equal.
Xmax= KlA*Co2max/qo

54
Q

How can you measure the mass transfer coefficent?

A

Oxygen balance method –> Use a mass balance at steady state to determine it. In-Out = Amount dissolved

  • reliable, single measurement
  • needs to be accurate with sophisticated machines

Dynamic method –> Cut off the air, reconnect it. Measure O2 throughout. Choose 2 points and find kl on the slope, using ln(Co2final - Co21)/(Co2final - Co22)

  • easy experiment, low cost
  • less accurate, uses artificial conditions
55
Q

How does the surface area affect aeration?

A

A is the surface area term which depends on the size of the bubbles. Breaking a bubble in half increases the surface area by 26%. Bubbles can be broken by mixing which also keeps oxygen bubbles in the reactor for longer.

56
Q

What is downstream processing? Give an example

A

Downstream processing is the process of purifying a product. The aim is the highest yield in the fewest steps, in the purest form necessary.
Monoclonal Antibodies (Normally secreted)
-Protein A chromatography
-low pH - to deactivate viruses
-cation exchange - to remove nucleic acids
-filtration - to remove virus particles
-ultrafiiltration
-formulation

57
Q

Describe some methods of downstream processing

A

Removing medium: Centrifugation - speed depends on host cell; Flitration - normal or tangential flow, positive pressure or vacuum; Settling
Cell lysis: HIgh pressure, sound waves, grinding
Inclusion Bodies: Denature and refold correctly
Precipitation: ammonium sulphate precipitation, removes protein or contaiminants
Chromatography: size exclusion, ion exchange, hydrophobic interaction, affinity chromatography.

58
Q

Why do we scale up/down and what are the problems?

A

We scale up to start producing the product to sell, but we don’t normally know how the product/ cells etc will react.
Volumes scale but factors like aeration and mixing need to be tested and controlled.
We scale down to trouble shoot, but again we don’t really know what we are changing, but it’s a hell of a lot cheaper.

BAP!! (8 decks)

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