Discuss Pharmaceutical Products
What Tests are done to test for pyrogens?
- Sterile products contain no micro-organisms
- Must have lower than specified amount of pyrogens
- Pyrogen Tests:
- Rabbit test
- LAL test
- Pyrogen Tests:
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?
- 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
Define D Value
Rate at which bacteria are killed (quoted at temp. e.g. D121)
Define Inactivation Factor (IF)
What fraction of the population is killed
Define Z Value
Number of °C temp. change required to achieve 10 fold change in D value
What is the importance of starting with a low bioburden?
- Shorter autoclaving times
- Reduce energy cost
- Shorter heat exposure
- Reduce degradation of active ingredient
- Fewer dead cells
- Reduce risk of product failing pyrogen test
What are the Methods of Sterilisation?
- Heat
- Moist heat
- Dry heat
- Radiation
- Filter Sterilisation
- Non-ionisation radiation
Describe Moist Heat Under Pressure as a method for sterilisation
- Causes denaturation, coagulation and hydrolysis of protein
- 121°C/15min/15psi
- Doesn’t destroy pyrogens
Describe Sterilisation with Dry Heat
- >160°C, <250°C
- Nonaqueous-based products: oils, powders
- Solid items: metal and glassware
- Dry materials unaffected by high temperatures
Describe the efficiency of sterilisation of moist heat using biological indicators
- 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
Describe the efficiency of sterilisation of dry heat using biological indicators
- Envelope contains a strip of filter paper with spores of B. Subtilus
What two tests are used to test for pyrogens?
- Rabbit Test
- LAL Test
Describe the Rabbit Test
- Involves rise in temperature when rabbit is injected with pyrogen containing solution
- 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
- 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
- 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
What are the Advantages and Disadvantages of the Rabbit Test?
- Advantages:
- Broader range of pyrogens detected
- Disadvantages:
- Less sensitive than LAL test
- Consumes rabbits
Describe the LAL Test (Limulus Amebocyte Lysate)
- In vitro assay for the detection and quantitation of bacterial endotoxin in injectable drugs or solutions for parenteral administration
Describe Gamma Radiation Sterilisation
- 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
- Indirectly via free radicals generated from aqueous medium within or around cell
- Micro-organisms can exhibit different levels of resistance to radiation
- Resistant species have the ability to repair breaks in DNA
- Spores more resistant
Compare Gamma Radiation and Electron Beam Radiation
- 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
What are the Advantages of Ionising Radiation (Gamma and Electron Beam)
- 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
What are the Disadvantages of Ionising Radiation (Gamma and Electron Bean)?
- 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
What are the types of Non-ionisation Radiation?
- UV Sterilisation
- Gas Sterilisation
- Filter Sterilisation
What are the Advantages and Disadvantages of Gas Sterilisation and what is its MOA?
- 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
Describe Filter Sterilisation and Filtration by Sieving
What does Filter Sterilisation require?
- 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>
- Filtration by sieving
- Membrane filtration
- Well-defined pore structure
- Pore size ratings from 0.04micrometre up to 8micrometre
- Requires aseptic conditions
