Sterilisation

Question 1

Discuss Pharmaceutical Products
What Tests are done to test for pyrogens?

Answer 1

• Sterile products contain no micro-organisms
• Must have lower than specified amount of pyrogens
  
  • Pyrogen Tests:
    
    • Rabbit test
    • LAL test

Question 2

Discuss Pyrogens (lipopolysaccharides)

What are the most common pyrogens?

Where are lipopolysaccharides (LPS) found?

Where are pyrogens produced from?

What is the immune system’s role in Pyrogens?

Answer 2

• Any substance that causes a rise in body temperature when injected in mamal
• Endotoxins from Gram-negative bacteria
• LPS found in outer membrane
• Produced from death and break-down of bacteria
• Immune system
  
  • Recognises LPS as foreign
  • Immune response develops, leading to increase in temperature

Question 3

Define D Value

Answer 3

Rate at which bacteria are killed (quoted at temp. e.g. D121)

Question 4

Define Inactivation Factor (IF)

Answer 4

What fraction of the population is killed

Question 5

Define Z Value

Answer 5

Number of °C temp. change required to achieve 10 fold change in D value

Question 6

What is the importance of starting with a low bioburden?

Answer 6

• Shorter autoclaving times
  
  • Reduce energy cost
• Shorter heat exposure
  
  • Reduce degradation of active ingredient
• Fewer dead cells
  
  • Reduce risk of product failing pyrogen test

Question 7

What are the Methods of Sterilisation?

Answer 7

• Heat
  
  • Moist heat
  • Dry heat
• Radiation
• Filter Sterilisation
• Non-ionisation radiation

Question 8

Describe Moist Heat Under Pressure as a method for sterilisation

Answer 8

• Causes denaturation, coagulation and hydrolysis of protein
• 121°C/15min/15psi
• Doesn’t destroy pyrogens

Question 9

Describe Sterilisation with Dry Heat

Answer 9

• >160°C, <250°C
• Nonaqueous-based products: oils, powders
• Solid items: metal and glassware
• Dry materials unaffected by high temperatures

Question 10

Describe the efficiency of sterilisation of moist heat using biological indicators

Answer 10

• Ampoules containing nutrient broth, CHO, pH indicator and spores of B. Stearothermophilus
• After sterilisation, ampoules are incubated @ 60°C for 24-48hr
  
  • If effective, ampoules remain red-violet
  • If not effective, growth will turn liquid turbid and yellow

Question 11

Describe the efficiency of sterilisation of dry heat using biological indicators

Answer 11

• Envelope contains a strip of filter paper with spores of B. Subtilus

Question 12

What two tests are used to test for pyrogens?

Answer 12

• Rabbit Test
• LAL Test

Question 13

Describe the Rabbit Test

Answer 13

• Involves rise in temperature when rabbit is injected with pyrogen containing solution

1. If the sum of responses isn’t greater than 1.4°C and any rabbit shows the response less than 0.6°C, the product passes the test
2. If the sum of responses is >1°C or any rabbit shows the reponse 0.6°C or greater, continue the test using 3 rabbits
3. If test is done using 5 rabbits, then if sum of responses of all 5 rabbits is greater than 3.7°C and the individual response of not more than 3 rabbits is greater than 0.6°C, the product passes the test

Question 14

What are the Advantages and Disadvantages of the Rabbit Test?

Answer 14

• Advantages:
  
  • Broader range of pyrogens detected
• Disadvantages:
  
  • Less sensitive than LAL test
  • Consumes rabbits

Question 15

Describe the LAL Test (Limulus Amebocyte Lysate)

Answer 15

• In vitro assay for the detection and quantitation of bacterial endotoxin in injectable drugs or solutions for parenteral administration

Question 16

Describe Gamma Radiation Sterilisation

Answer 16

• Affects DNA in organism directly via free radical generation
  
  • Indirectly via free radicals generated from aqueous medium within or around cell
    
    • Hydroxyl radical most effective
• Micro-organisms can exhibit different levels of resistance to radiation
  
  • Resistant species have the ability to repair breaks in DNA
  • Spores more resistant

Question 17

Compare Gamma Radiation and Electron Beam Radiation

Answer 17

• Gamma Radiation
  
  • ​Cannot be switched off
  • Slow (require hours of exposure)
  • Deep penetration - can sterilise large items (80cm)
  • Long exposure can cause product damage
• Electron Beam
  
  • ​Can be switched off
  • Fast - minutes/seconds
  • Unsuitable for dense materials (8cm)
  • Less damage than with gamma

Question 18

What are the Advantages of Ionising Radiation (Gamma and Electron Beam)

Answer 18

• High efficiency
• Performed at room temperature
• Insignificant rise in temperature of the material
• Good penetration, dependent on thickness
• Solids, solutions, devices, containers
• Terminal and continuous operation

Question 19

What are the Disadvantages of Ionising Radiation (Gamma and Electron Bean)?

Answer 19

• Expensive
• Access to radiation source
• Potential chemical damage to the product/active ingredients/excipients
• Damage more likely with aqueous systems than solids
• Stability impact during storage

Question 20

What are the types of Non-ionisation Radiation?

Answer 20

• UV Sterilisation
• Gas Sterilisation
• Filter Sterilisation

Question 21

What are the Advantages and Disadvantages of Gas Sterilisation and what is its MOA?

Answer 21

• Similar MOA to gamma radiation
• Effective for surfaces but there are risks with its use
• Advantages:
  
  • ​Highly reactive towards micro-organisms
  • Good permeability
• Disadvantages:
  
  • ​Toxicity
  • Highly reactive towards surfaces
  • Explosive
  • Long desorption times

Question 22

Describe Filter Sterilisation and Filtration by Sieving

What does Filter Sterilisation require?

Answer 22

• Physical removal of microbes by passing a gas or liquid through filter
  
  • Pores of filter large enough for liquid but too small for microbes (<micrometre></micrometre>
  </micrometre>
• Filtration by sieving
  
  • Membrane filtration
  • Well-defined pore structure
  • Pore size ratings from 0.04micrometre up to 8micrometre
• Requires aseptic conditions