Unit 3 - How to manufacture a therapeutic protein

Question 1

What are the first four steps to produce a therapeutic protein?

Answer 1

Transformation
- double stranded recombinant plasmid DNA introduced into bacterial cell
Selection
- isolate bacteria containing target DNA
Propagation
- cell culture produces hundreds of millions of new bacteria
Transfer to large scale expression host
- many copies of purified plasmid isolated from lysed bacteria

Question 2

What are the stages in the production phase of production a therapeutic protein?

Answer 2

Transfer to large scale expression host
Fermentation
- allows large scale production
Harvesting
- cell lysis
- soluble/insoluble proteins
- refolding
Purification
- remove everything other than the protein
Characterisation
- to make sure it has properties which are required for it to be biologically active
Activity assays, interaction analysis
- to make sure it works

Question 3

What are the three summarised steps in the manufacturing process of therapeutic proteins?

Answer 3

Growth
Lyse
Purify

Question 4

What factors need to be considered when developing a large scale production process?

Answer 4

Recombinant protein expression system
- seed stock
- provenance of starting material
Production media
Downstream processing
Production facilities
- dedicated production facilities
- bio-security
Costs

Question 5

How can recombinant proteins be harvested from bacterial host?

Answer 5

Secreted proteins
- easy to purify
Soluble proteins trapped inside cell
- release by cell lysis
Proteins can form insoluble inclusion bodies
- release by cell lysis
- refold as soluble protein

Question 6

What can be used to lyse bacterial cells?

Answer 6

Enzymes
Sonication
French press

Question 7

What are inclusion bodies?

Answer 7

Over expression of recombinant proteins in E.coli can lead to the formation of insoluble structures

Question 8

How can therapeutic proteins be recovered from inclusion bodies in bacteria?

Answer 8

Denature the protein

- 6M urea

Question 9

What process is carried out for protein solubilisation and refolding?

Answer 9

Dialysis

Question 10

How can chromatography be used to purify therapeutic proteins?

Answer 10

Several steps are typically required
Combined approaches most powerful
- ion exchange
- size exclusion
- hydrophobic interaction
- protein A
- removal of LPS-pyogen

Question 11

What considerations need to be taken into account during purification of therapeutic proteins using chromatography?

Answer 11

Each step increases purity or removes contaminant

- 10 fold loss of material

Question 12

How can the therapeutic protein be physically classified?

Answer 12

SDS-PAGE
Reversed phase HPLC
Amino acid analysis
n-terminal sequencing
Sugar analysis
- if glycosylated

Question 13

How can the therapeutic protein be biologically classified?

Answer 13

Activity assays

• enzyme activity
• binding properties
• cellular response
• immunogenicity

Question 14

What is the formulation polishing step in making therapeutic proteins?

Answer 14

Protein is transferred to a final formulation

Question 15

What is the filling step in making therapeutic proteins?

Answer 15

Final format filling

- vial

Question 16

What is the finishing step in making therapeutic proteins?

Answer 16

Product must be inspected for colour, clarity as issue of acceptable for release
Sample is analysed to ensure it meets physical and biological specifications
- final product characterisation

Question 17

What characteristics are examined in therapeutic proteins in final product characterisation?

Answer 17

Proper protein content
pH
Excipient levels
Osmolality
Sterility
Lack of antigenicity
Physical appearance

Question 18

Why is stability of therapeutic proteins important to test during final product characterisation?

Answer 18

Determine acceptable storage/shelf life

Question 19

What are the three codes of practice in manufacturing therapeutic proteins?

Answer 19

Good Manufacturing Practices (GMP)
Good Laboratory Practice (GLP)
Good Clinical Practice (GCP)

Question 20

What is Good Manufacturing Practice?

Answer 20

Practices required in order to conform to the guidelines recommended by agencies that control the authorisation and licensing of the manufacture and sale of food and beverages, cosmetics and pharmaceutical products

Question 21

What is Good Laboratory Practice?

Answer 21

A set of principles that provides a framework within which laboratory studies are planned, performed, monitored, recorded, reported and archived