Lecture 8 Flashcards Preview

Molecular Biotechnology > Lecture 8 > Flashcards

Flashcards in Lecture 8 Deck (26):
1

what parameters influence design of processing system at all scales

temp
pH
rate and type of mixing
oxygen demand
colour jk
sterility and containment
SA:V

2

With what increase in size do optimal conditions normally change

10 fold increase

3

Describe the general scheme for large scale fermentation process

formulate growth medium
sterilise equipment
grow a stock culture up to 1l
inoculate seed fermenter then finally production fermenter
seperate cells from culture medium once finished - centrifugation or filtration

4

When separating cells form culture medium what must considered

if protein is secreted or not - if yes can destroy cells if not must somehow get out of cells

5

What is the most common design for a fermentor and it's main principle

Stirred tank - growing microbes under defined conditions

6

What are the features of a stirred tank reactor

parameters are controllable
cooling jacket for temp
stirrers for aeration
sensors to measure pH

7

Name the 2 growing modes fermentation can accommodate, describing each

Batch
Closed system, fixed volume of medium inoculated, culture grows, medium changed as result of microbe growth

Fed batch
media added to vessel as process proceed
fermentation stopped when vessel is full
means you can extend exponential phase by adding more medium, diluting culture as you go along so you;re also diluting TOXINS

8

Give 2 examples where fed batch was better and what expression was controlled by

IFNgamma
initially produced from ecoli in batch - expression controlled by pl and cI (you get temp sensitive cI mutants, so denture this therefore get more expression cos less control)
Now in fed batch system, grown till late exponential then growth medium added at high temp.
20xgreater yield

Monoclonal AB fragments - fab
greatly imporved in fed
use lacI so cheap lactose as inducer. Cheap yay.

9

if you have multicopy plasmids, is radnom diffusion sufficient to stabilise them and pass on desired genes?

Yup.

10

If you are not a high copy plasmid microbe (give an example) how can the problem of plasmids not being passed on to some duaghters be solved

Plasmid based toxin-anti-toxin systems

11

What's the example of the toxin anti toxin system and which plasmid is it on in ecoli?

Hok/soc system, on R1 plasmid

12

What encodes a toxin in the h/s system?

hok

13

What's occuring if hok/sok plasmid is present

mok/hok transcript is made
also an anti-sense sok transcript is made
sok transcript INHIBITS TRANSLATION of mok/hok.
hok protein not made, cells ok.

14

What's occuring if no hok/soc plasmid made it to daughter cell

Sok (which is encoded by something else, so is present anyway) is an sRNA so degrades faster than the Hok mRNA.
Therefore the hok protein is translated and cell dies.
Ensures you;re only left with cells whihc have a specific plasmid present

15

How are mammalian cells normally cultured? What growth levels achieved over 3-4 days

stiirred tank reactor, batch culture
10^6cells/ml

16

What's the issue with growing mammallian cells in batch

glycosylation pattterns can change as sugars are depleted .
supply of N and pH also affect glycosylation

17

What is the solution to the batch problems for mammalian cells? and what growth levels are achieved

Fed batch cultured
>10^7cells/ml

18

what is downstream processing

recovering and purifying fermenation products

19

what do you base your choice of recovery method on

location of product - intra or extracellular
conc of porduct
physical and chemical properties of desired product
intended product use
min. acceptable purity
cost
what impurities need to be removed

20

3 methods of cell harvesting and what cells each type is used for. And general features why not

sedimentation
large particles settle in static culture]
centrifugation
cells that won't settle with gravity
force required depends on cell type
high energy and costs
Filtration
cheap but effective
plate frame filter commonly used-reusable

21

what is cell disruption and what are the sub types

when you break open cells to release cytoplasmic or periplasmic proteins
mechanical
non mechanical (for when not totally inside)

22

list mechanical cell disruptions

french press (just high pressure)
bead mills - physically ground
sonication - ultrasonic vibrations

23

list non-mechanical cell disruptions

osmotic shock - degrade wall with lysozyme, equilibriate with sucrose then move back to water. For periplasmic
Adding detergents
precipitate using ammonium sulphate (post release i guess)

24

What is the complete upstream process of mAB production

grown from cell storage bank - stable cell lines with selectable markers
fed batch growth of CHO cells
use defined serum free media to get reproducable hihger yields
harvest using centrigugation and filtration

25

What is the complete downstream process for mAB production

3 variations but generally:
use protein A for 1st affinity chromatography step
tight binding of Fc region on mAB to prot A
elute with low pH
98% purification in step 1
same matrix used multiple times

26

what sort of steps must downstream process of mAB use?

non-affinity polishing steps