Lecture 16 - microbiology

Question 1

are are types of infective agents?

Answer 1

Prions

Viruses/Viroids

Bacteria (Unicellular, prokaryotic cells)
- Eubacteria
Gram (+)ve/(-)ve
Aerobic/Anaerobic/Facultative Anaerobes
- Archaea (extremophiles, no pharma interest)

Fungi (Eukaryotic cells)
Yeasts (unicellular)
Moulds (form mycelium or filaments

Question 2

describe the new terms sued in microbiology

Answer 2

Growth environment
- Saprophyte organisms – live on dead material
- Parasites – live in a host
- Pathogenic – live in, and damage, a host

Other common terms
- Obligate – ‘by necessity’
- Opportunistic pathogen – can ‘break out’
- Spores (spore forming) – ‘seeds’

Naming – Staphylococcus (S.) aureus

Question 3

what is the definition os sterile?

Answer 3

‘not able to produce offspring, crops or ideas’
‘clean’ (sterilizing wipes!)

Free from bacteria or other living organisms.

Complete absence of viable microorganisms

Question 4

what are sterile products?

Answer 4

Where you break a barrier, or treat a sensitive membrane
Drug products
- IV fluids (large and small)
- Wound, bladder irrigation
- Eye/ophthalmic prep

Medical devices
- Joints, pacemakers, sutures
- Needles

Others
- Surgical gloves
- Gowns etc

Question 5

what does the production of non sterile drug products require?

Answer 5

Production
- Clean and monitored environment and equipment.

Testing
- Growth of specific (pathogenic) organisms
- General counting/enumeration

Storage and Distibution
- Clean and secure environment (pest etc)

Question 6

what its the process of sterilisation to get to the patient to ensure it is sterile?

Answer 6

1. product is contaminated
2. the product is sterilised either by filtration etc
3. post sterilisation production the product is sealed
4. material is shipped in the sealed state
5. uncontaminated product reaches patient

Question 7

what can go wrong in sterilisation or products which makes it contaminated?

Answer 7

product not fully sterilised.

there’s a high number of bacteria bioburden, so when sterilisation takes place, all the bacteria are not killed as there are too many.

environmental controls post sterilisation are not in place so product gets contaminated

inappropriate partial selling so contaminants can get into product

Question 8

what is the requirement of steroid production in full process system?

Answer 8

the bioburden of the raw materials to be controlled, the sterilization process to be effective, the post sterilization environment to be controlled until we can seal the product, and then we need the integrity of the container to be assured so that there are no issues on distribution.

Question 9

what are sterilisation methods?

Answer 9

Heat (dry - oven)

Heat (steam/wet - autoclave)

Radiation (gamma rays/high energy electrons)

Gases (ethylene oxide, hydrogen peroxide, formaldehyde)

Filtration

Question 10

descried sterilisation by most heat (steam or autoclaving)

Answer 10

1. Kills micro-organisms by transfer of heat energy of SATURATED STEAM
2. 121oC, 30 apsi, for >15 min
3. Temp/pressure, steam quality, atmospheric gases critical.
4. Cycle and load validation (problems of ‘penetration in tubes and porous loads

Question 11

describe sterilisation by dry heat

Answer 11

1) Kills micro-organisms by oxidation.
2) 160o for 2 hours, 170oC for 1 hour.
3) Depyrogenation at 220oC for >4 hours (overkill)

Question 12

describe sterilisation by filtration

Answer 12

1) Removes micro-organisms using filters with 0.2 micrometre pore sizes.
2) ‘Capsule filters’: often using 2 in sequence for redundancy.
3) Filters are tested by ‘bubble point’ after use for integrity.

Question 13

sterilisation maths

Answer 13

A sterile product has a contamination of <1:106.
For heat: log reduction with time.
For filtration: log reduction.
…therefore, microbial load of a ‘sterilized solution’ depends on the bioburden (load) of the pre-sterilisation solution.

Question 14

what are quality control tests for sterile products?

Answer 14

physical tests;
appearance and closure integrity tests

chemical tests; assays nd impurities, content infirmity, visible and subvisisble particles, pH/ osmolarity and other product dependant tests

microbiological tests; sterility and endotoxins

Question 15

what is the requirement for closurifn integrity testing post sterilisation production?

Answer 15

Containers should be closed by appropriately validated methods. Containers closed by fusion, e.g. glass or plastic ampoules should be subject to 100% integrity testing. Samples of other containers should be checked for integrity according to appropriate procedures”

Different methods available (gas detection, vacuum decay, electrical conductivity), most commonly used is dye ingress test.

Question 16

describe sterility testing

Answer 16

Filtration
Drug solution filtered through two 0.2 mcm membranes to retain bacteria, and broth added

Direct inoculation
Sample of drug solution added directly to broth.

Growth media added
Fluid thioglycollate (anaerobic and aerobic bacteria media)(30-35oC)
Trypic soya broth (aerobic bacteria and fungi)
Incubated for 14 days and checked for growth. (No visible growth!!)
Inoculated for 3 days (bacteria) and 5 days (fungi) as a growth promotion test.
Inactivation of anti-microbial may be required, tested and validated.

Question 17

what are pyrogens and how are the detected ?

Answer 17

Pyrogens are substances that can cause fever when introduced into the body. Gram-negative bacteria’s cell wall components, such as lipopolysaccharides, act as exogenous pyrogens, triggering high temperatures. Notably, viable bacteria are not necessary for pyrogenicity, as even sterile products can elicit a pyrogenic response. These bacterial lipopolysaccharides activate an immune cascade that involves the release of endogenous pyrogens.

Current methods for testing pyrogenicity include the Limulus Amebocyte Lysate (LAL) test, which relies on clotting reactions in the blood of horseshoe crabs. The Rabbit Pyrogen Test, while reliable, is slow, expensive, and invasive, often considered a last resort. The Monocyte Activation Test (MAT), an ELISA-based assay measuring cytokine release in vitro, represents a more modern approach to detect pyrogens effectively.