Lecture 7: Sources Of Recombinant Proteins

Question 0

Describe the different types of expression systems.

Answer 0

There are a variety of expression systems which are used, or could potentially be used for the production of recombinant biopharmaceuticals. Examples include:
E. coli and additional prokaryotic systems, eg bacilli.
Yeast, eg S. Cerevisiae
Fungi, particularly Aspergilli
Animal cell culture (particularly CHO and BHK)
Transgenic animals (focus on sheep and goats)
Plant-based expression systems
Insect cell culture systems

Question 1

Describe the manufacture of biopharmaceuticals.

Answer 1

Highly regulated and rigorously controlled to ensure a safe and effective product (compliance with safety and quality standards).
Most biopharmaceuticals currently available are produced by genetic engineering, using various recombinant expression systems.
Product purity is essential, for example thalidomide:
- R-thalidomide : sedative used for the control of morning sickness in pregnant women.
-S-thalidomide: teratogenic (causes malformations). inserts into GC-rich DNA to affect promotor regions of genes (affects limb, eye and blood vessel development)

Question 2

What considerations should be made when choosing an expression system?

Answer 2

Cost
Speed
Post-translational modification (eg pattern of glycosylation)
Protein folding
Safety
Scalability

Question 3

Discuss the advantages and disadvantages of bacteria as an expression system.

Answer 3

ADVANTAGES
Well characterised
High level of protein expression =/<30% of total cellular protein
Rapid and cheap
DISADVANTAGES
heterologous proteins accumulate intracellularly. This complicates downstream processing (requires homogenisation, removal of cellular debris and extensive purification)
No post-translational modification (glycosylation)
Presence of LPS on surface (LPS is pyrogenic)

Question 4

Give an example of the use of a bacterial expression system.

Answer 4

E. coli is considered a satisfactory expression system for interleukin-2.
IL-2 is a t-cell growth factor. It is produced by T-cells in response to an antigen, and activates many aspects of the immune response.
It is a glycoprotein: o-linked glycosylation sugars on Thr3. Single chain polypeptide of 133aa.
Because the unglycosylated form displays the same biological activity to the native glycosylated form, E. coli is a satisfactory production system for IL-2.

Question 5

Discuss the advantages and disadvantages of yeasts as an expression system.

Answer 5

ADVANTAGES
well characterised
Generally regarded as safe
Grows relatively quickly in inexpensive media
Tough outer wall protects cells from physical damage
Can undergo industrial-scale fermentation
Has post-translational modification
DISADVANTAGES
glycosylation pattern differs from native protein (usually high mannose, which triggers rapid clearance for the blood stream leading to short half-life of protein). Some yeast sugar motifs can be immunogenic
Expression levels are low (=/<5% total cellular protein)

Question 6

Give an example of the use of a yeast expression system.

Answer 6

Yeast has been used to produce HPV capsid proteins for use in the Gardasil vaccine.
Genes encoding HPV capsid proteins of HPV virus types 6, 11, 16 and 18 are inserted into a yeast plasmid vector, taken up by yeast cells and cultured. The HPV capsid proteins are then purified and incorporated into the vaccine.
Gardasil is approved for the prevention of cervical, vulvar and vaginal cancers caused by HPV types 16 and 18.

Question 7

Describe the advantages and disadvantages of insect cells as expression systems (Baculovirus).

Answer 7

ADVANTAGES
high-level Intracellular protein expression
Rapid and inexpensive culture
Free of human pathogens
DISADVANTAGES
targeted extra cellular production leads to the low level accumulation of the desired protein (mg/L range)
Post translational modifications can be incomplete, and vary from native form (especially glycosylation).

Question 8

Describe the use of Baculovirus as an expression system.

Answer 8

Ther cervarix cervical cancer vaccine was produced in a Baculovirus expression system using cells derived from the insect Trichoplasma ni.
It has been approved for prevention of cervical, vulvar and vaginal cancers caused by HPV types 16 and 18.

Question 9

Discuss the advantages and disadvantages of mammalian cells as expression systems.

Answer 9

ADVANTAGE
Undergo post translational modification.
DISADVANTAGES
Complex nutritional requirements
Slow cell growth
Susceptible to physical damage
Increased production costs

Question 10

What are the components of mammalian expression systems?

Answer 10

The pCI-neo vector is one plasmid used in mammalian expression systems. It has numerous components, including:
SV40 enhancer/early promotor for plasmid replication in host
CMV: promiscuous enhancer/promotor which leads to constitutive expression of insert
MCS: unique restriction sites for cloning
Ampr: ampicillin resistance gene for selection of transfected cells
Neo: neomycin phosphotransferase gene- resistance to antibiotic G-418

Question 11

Describe the transformation (transfection) of mammalian cells.

Answer 11

Calcium phosphate forms a precipitate with the DNA. Precipitate complexes, and is taken into the cell by endocytosis.
Lipofection: cationic lipid-based system which forms micelles with DNA inside them. Micelles fuse with the plasma membrane.
Electroporation drives charged DNA into cell

Question 12

Describe Electroporation.

Answer 12

Cells are subjected to short, high-energy electric pulse. This leads to brief opening of the membrane of the cell, driving the charged DNA into the cell.

Question 13

Discuss the advantages and disadvantages of plant expression systems.

Answer 13

ADVANTAGES
cost of plant cultivation is low
Harvest methods are inexpensive and well established
Ease of scale-up
Proteins expressed in seeds are stable for a long time
Free of human pathogens
DISADVANTAGES
Low or variable expression levels
Potential of post-transcriptional gene silencing (sequence-specific mRNA degradation)
Glycosylation patterns differ for native human product (plant fly conforms are immunogenic)
Environmental/public concerns: escape of GM plants
Seasonal/geographical nature of plant growth

Question 14

Describe the use of a plant expression system.

Answer 14

CaroRx is a plantibody that is produced in tobacco, and approved for use in Europe.
It binds specifically to Streptococcus mutans which is the bacteria responsible for tooth decay, and prevents the bacteria from adhering to teeth effectively eliminating the bacteria for up to 2 years.
CaroRx alters the niche repopulation dynamics of competing microbes on tooth surface.

Question 15

Discuss the advantages and disadvantages of transgenic animals as expression systems.

Answer 15

ADVANTAGES
ease of harvesting crude product
Availability of commercial milking system, designed with maximum process hygeine.
High expression levels (1-60g/L)
Ongoing supply of product guaranteed by breeding
Milk proteins well characterised (rational development of downstream processing protocols)
DISADVANTAGES
Variability of expression levels
Characterisation of exact nature of post-translational modifications
Lag time between generation of transgenic embryo and commencement of routine product manufacture

Question 16

Describe the use of a transgenic animal as an expression system.

Answer 16

Atryn (anti-thrombin).
A human gene that produces the blood protein antithrombin is inserted into a short strand of goat DNA.
The modified DNA is injected into the nucleus of a fertilised goat egg, which is then implanted into a female.
Kids born from the modified eggs are tested for the presence of antithrombin in their milk. Promising kids are then bred normally to create a herd of transgenic goats.
Milk from the herd is filtered and purified. Annually, each goat can produce as much antithrombin as 90000 human blood donations.

Question 17

Describe how master and working cell banks are created.

Answer 17

A product-producing cell line is aliquotted and stored in liquid nitrogen to create the master cell bank (contains over 200 ampoules).
The master cell bank is expanded to create a working cell bank (working cell bank made from one ampoule of the master cell bank).

Question 18

Describe the upstream processing stages of cell banks.

Answer 18

The single ampoule of the working cell bank is removed from storage. The working cell bank is propagated to generate a lab-scale starter culture of a few hundred mL.
This starter culture is then up scaled to a small bioreactor of a few litres, and this is then upscaled to a production scale bioreactor of thousands of litres.