The industrial use of microorganisms

Question 1

What are the three stages of the production process?

Answer 1

• Stage 1; Upstream processing - preparation of liquid medium, separation of particulate and inhibitory chemicals from the medium, sterilisation, air purification
• Stage 2; Fermentation - conversion of substrate to desired product by bacteria, yeast or mammalian cells
• Stage 3; Downstream processing - separation of cells from fermentation broth, purification and concentration of desired product and waste disposal or recycling

Question 2

In upstream processing, there is medium preparation. What happens here?

Answer 2

• Medium is formulated and optimised for host to get optimum product yield at minimum cost.
• Separation of particulate and inhibitory chemicals from medium
• Medium sterilisation and air purification
• Sterilisation of bioreactor so that you don’t contaminate the reactor vessels
• Adequate supply of producer cells (cell banking and maintenance).

Question 3

How do cell banking systems work? How are the cultures of newly constructed production cell line aliquoted? First tier is called?
Second tier is called?

Answer 3

The production cell lines are stored in a two tiered cell bank. This ensures long term availability of producer cell lines.
Aliquoted into 5 ampoules.
Master cell bank
Working cell bank

Question 4

Stage 2; Fermentation - How is the volume of medium increased?

Answer 4

Start of with contents of single ampule of working cell bank.
Next is lab scale starter culture; where there are few 100ml of medium.
Next is production scale starter culture; where there is a small bioreactor with several litres/ tens of litres of medium.
Lastly there is production scale bioreactor; several thousands/ tens of thousands of litres of medium
PROGRESSIVELY, LARGER VOLUMES OF MEDIUM ARE INOCULATED.

Question 5

Name some properties and function of bioreactors.

Answer 5

• high grade stainless steel, fully sterilisable
• jacketed for temp control
• impeller ensures even distribution of nutrients and cells
• Air/gas sparger (supplies oxygen)
• Ports contain: probes for pH, temp. Ports also allow pH adjustment and addition of nutrients,

Question 6

What are some other types of bioreactor?

Answer 6

airlift, fluidised bed, packed bed, hollow fibre and membrane bioreactors

Question 7

What does the impeller do?

Answer 7

Impeller blades smash bubbles to very small size, increasing dissolution rate of oxygen.
Bubbles rise to surface but have tortuous flow due to blades - increasing residence time.

Question 8

Dissolved oxygen is important for what kind of bacteria? Oxygen is very insoluble in _____ systems. Solubility decreases with ______ in temp and dissolved solutes.

Answer 8

Aerobic
aqueous
increased

Question 9

What component of bioreactor provides oxygen?

Answer 9

Air/ Gas sparger

Question 10

Dissolution of oxygen depends on

Answer 10

Size of air bubbles

Residence time in fermenter

Question 11

What sterilisers the bioreactor vessels?

Answer 11

Steam

Question 12

Which type of bioreactor does not have an impeller?

Answer 12

Air lift. It only has a sparger

Question 13

Which type of bioreactor does not have an liquid medium?

Answer 13

Fluidised bed

Question 14

Fermentations can only be carried out in cell types that grow in _______. i.e. most _______, some simple ______ (e.g yeasts).
Only a few mammalian cells can grow in suspension, most are “______-________” i.e. they need to grow attached to solid surfaces. Mammalian cells are wall-less and more _______ (weak).

Answer 14

suspension
prokaryotes
eukaryotes
anchorage-dependent 
fragile

Question 15

Mammalian cell culture reactors use? Are these cheap?

Answer 15

Airlift

EXPENSIVE!

Question 16

Expand on downstream processing.

Answer 16

Normally carried out in a pharmaceutical clean room.
Operators use Grade A laminar flow.
Filling process is automated to prevent contamination between operator and product.

Question 17

What are the three downstream processing steps?

Answer 17

1. Cell harvesting and recovery of crude product
2. (Concentration if necessary and) Purification
3. Final formulation

Question 18

During cell harvesting and recovery of crude product:

Once fermented, if protein is expressed intracellularly..

Answer 18

• Recovery of producer cells (centrifugation or filtration)
• Cellular disruption (homogenisation)
• Removal of cellular debris (centrifugation or filtration)

Question 19

During cell harvesting and recovery of crude product:

Once fermented, if protein is expressed extracellularly..

Answer 19

Removal of cells from media (centrifugation or filtration)

Question 20

Following fermentation, if protein is expressed intracellularly, the product needs to be recovered from?

Answer 20

producer cells

Question 21

Following fermentation, if protein is expressed extracellularly, the product needs to be recovered from?

Answer 21

culture medium

Question 22

During purification: if protein is expressed intracellularly:

Answer 22

Initial purification/ concentration (ultrafiltration/precipitation) AND THEN chromatographic purification (usually 2-4 chromatographic steps)

Question 23

During purification: if protein is expressed extracellularly:

Answer 23

concentration (ultrafiltration/precipitation) AND THEN chromatographic purification (usually 2-4 chromatographic steps)

Question 24

During final product formulation: What happens after 2-4 chromatographic steps?

Answer 24

Adjustment of potency and addition of excipients
Sterile filtration and aseptic filling
THEN either:
(freeze drying –> powder preparation –> sealing of final product container, labelling and packaging)
OR
(liquid preparation –> sealing of final product container, labelling and packaging)

Question 25

Analysis of final product. 
Presence of following impurities may have what medical consequences?
a) microorganisms
b) viral particles
c) pyrogenic substances
d) DNA
e) contaminating proteins

Answer 25

a) microbial infection, septicaemia
b) viral infection
c) fever
d) possible immune reaction
e) immune reaction/ unwanted biological activity