Insulin structure and manufacturing

Question 1

What is the primary structure of insulin?

Answer 1

Insulin consists of 51 amino acids:
2 peptide chains:
A= 21 aa
B= 30 aa
These chains are linked via disulphide bridges between A7 + B7 and A20 + B19
There is also an internal bridge in chain a between a6 and a11

Question 2

What is the only difference between the amino acid sequence of human and porcine insulin?

Answer 2

In Human insulin, the amino acid at B30 is Threonine but in porcine it is alanine

Question 3

What is the tertiary structure of insulin?

Answer 3

Disulphide bridges between chains a and b

Question 4

What is the quaternary structure of insulin?

Answer 4

• Anti-parallel beta sheet formed between B23 and B30 which is stabilised between hydrogen bonding involving B24 and B26
• consists of insulin hexamers- 3 insulin molecules and 2 zinc ions

Question 5

What is the source and manufacture of natural insulin?

Answer 5

• This is porcine and bovine insulin which is taken from the pancreas of a pig or cow
• it is important this insulin is suitable for human consumption
• must be free from transmissible spongiform encephalopathies (TSE)
• Had a long purification process

Question 6

What is the source and manufacture of semi-synthetic insulin?

Answer 6

• Is clinically identical to human insulin
• involves generating porcine insulin as normal and then enzymatically modifying it to change the amino acid at B30 from Alanine to threonine
• label must state “emp”-

Question 7

What is the source and manufacture of biosynthetic insulin?

Answer 7

• Chemically identical to human insulin
• Recombinant DNA technology which involves the following procedure:
  Identify the gene that codes for insulin in humans
  Insert this gene into a plasmid of a bacterial or fungal vector (e.g. Escherichia coli or Saccheromyces cerevisiae)
  Grow this modified bacterium or fungus
  Harvest and purify the human insulin
  -label must state method of preparation: “prb”= purified recombinant bacterial or “pyr” = purified yeast recombinant

Question 8

What is the unit of insulin?

Answer 8

International units (IU)= equal to around 0.034 mg

Question 9

What is the method of injecting insulin?

Answer 9

• Usually subcutaneously
• May be given as an IV in an emergency

Question 10

Which sites can insulin be given in, and which has the fastest absorption?

Answer 10

Abdominal wall, upper thigh, upper arm or buttock
- Absorption is fastest from the abdominal wall

Question 11

What factors can effect the rate of absorption of insulin?

Answer 11

• Heat
• Excercise

Question 12

What are the biological importance of physiochemical properties necessary for insulin to work well?

Answer 12

• Insulin molecules must be in solution and in the monomer form to have a biological action in the body
• Physical straw will effect dissolution from solid into solution
• Amorphous form will act faster than a crystalline form
• The dissociation rate from hexamer or dimer to monomer will effect the rate of action
• Presence of additives to the insulin mixture effect dissolution rate- e.g. the addition of Zinc ions and Protamine, decrease dissolution rate

Question 13

What is an example of a new human insulin analogue and how is the analogue form beneficial?

Answer 13

Insulin Aspart (Novorapid)
- This insulin is produced using recombinant yeast (saccheromyces cereviliae) in which the amino acid at B28 is changed from Proline in human insulin to aspartic acid in the analogue.
This changes causes:
- Aspartic acid is negatively charges at physiological pH whereas proline is non-ionisable so;
- increased charge repulsion between neighbouring molecules causes a decrease in self-aggregation, the molecules act more independently, the molecules will diffuse quicker and therefore have a faster clinical effect (approx 10-20 minutes onset action and peak at 1-3 hours)