Controlling contamination

Which medicinal products have to be completely sterile?

Ophthalmic

Parenteral

Which medicinal products can be non-sterile?

Oral

Rectal

Topical

Inhaled

What are the regulations around non-sterile medicinal products?

Limits on the number of contaminants - 10²-10³ CFU/ml for bacteria, 10¹ for fungi

Absence of specific microbes e.g E.coli for oral, S.aureus and P.aeruginosa for topical

What are the risks which determine the sterility necessary of a product?

Aqueous products - microbes like water so oily products are less likely to be contaminated

Use of product - hazard can vary according to route

Nature of product - it may support microbial growth well e.g parental nutrition

Intended recipient - risk differs for neonates compared to a healthy adult

Presence of disease, wounds or organ damage

List the resistance of sterilising agents that organisms have from least to most

Multicellular organisms

Vegetative (dividing) bacteria, fungi, algae, protozoa, large viruses

Fungal spores

Small viruses

Bacterial endospores

Prions

Are bacteria prokaryotes or eukaryotes?

Prokaryotes

Describe the structure of bacteria

No membrane enclosed organelles

Divided into gram negative and gram positive

Contains: cell wall, cell membrane, plasmids, ribosomes, nucleoid, cytoplasm

Describe the cell wall of gram positive bacteria

Thick peptidoglycan layer

Lipoteichoic acid (spans whole membrane)

Teichoic acid (intrinsic)

Describe the cell wall of gram negative bacteria

Thin peptidoglycan layer

Contains porins which restrict the size of molecules entering/ exiting the cell

Lipopolysaccharides

Where will biofilms grow?

Wound/ organ infections

Indwelling medical devices

Growth on surfaces e.g in hospitals

Air/ water handling systems

Biological liquid systems (dialysis equipment)

Describe the minimum inhibitory concentration test

Puts the same concentration and species of bacteria in different concentrations of an antibiotic

Once the solution is clear that demonstrates no bacterial growth

The MIC is the lowest concentration that’ll clear the solution

Do gram positive or gram negative bacteria sporulate?

Gram positive

Why do bacteria sporulate?

It’s a survival strategy for when nutrients become exhausted

What are spores resistant to? Give an example

Heat, radiation, desiccation and chemical agents

C.diff

Describe the process of sporulation

1. An asymmetrical septum begins to form on the bacterium

2. The bacterium then engulfs the smaller bit after the septum - this forms a forspore

3. Cortex forms around the spore

4. A spore coat then forms

5. At this point its very dense with a very low water concentration

6. The mother cell undergoes lysis and the spore is released

Are fungi eukaryotic or prokaryotic?

Eukaryotic

What are the 3 types of fungi?

Yeasts

Macroscopic filamentous moulds

Multicellular filamentous moulds

Describe the cell wall structure of fungi

From the membrane building out: chitin, beta-glucan, mannan

Very different cell wall structure to bacteria, hence why antibiotics don’t work

How are non-pathogenic species of fungi useful?

Penicillium species can produce penicillin

What are the rarer contaminants?

Viruses

Protozoa

Prions

What are the consequences of microbial contamination?

Health hazard - organisms/ toxins but also depends on person, product and route of administration

Spoilage - breakdown of active component / another component, might lose aesthetic appeal and you lose compliance, might also have financial implications

What are the precautions in manufacture of sterile products used to reduce likelihood of contamination?

single use sterile packs OR manufacture in a clean room

What is the purpose of preservatives?

to decrease the risk of microbial contamination throughout product shelf life

they should not be used to mask a poor manufacturing process, product must be to standard sterility before use

What are the ideal properties of a preservative?

Broad spectrum

Shouldn’t interact with API or excipients

Shouldn’t be toxic

Rapid antimicrobial action

Chemically stable

Effective at product pH

Compatible with formulation

Physically undetectable

Constitute a small proportion of the product

Cost effective

Active at low concentration

Long acting

What are three examples of preservatives in the UK?

Organic acids - benzoic acid

Parabens - propyl paraben

Aromatic alcohols

Substituted alcohols

Biguanides

Substituted phenolic agents - chlorocresol

Describe the properties of benzoic acid as a preservative

limited by pH dependance

work up to pH values of pKa (<4.2)

oral products, tablets and capsules

Describe the properties of parabens as a preservative

overcome pH dependence of organic acids

oral products, tablets and capsules

stop growth but won’t kill so used in combination

What is the test to evaluate preservatives?

Challenge test

Describe the challenge test

Assesses activity with the product in its final container

Inoculate preserved product with 105 - 106 organisms per ml/gram of product

Incubate at a specific temperature and sample over 28 days

Assess viability by viable count

Preservative is adequate if the decrease in viability meets the criteria of acceptance

In the criteria of acceptance, what does NI mean?

less than ½ log increase from the last value given for the viable count

In the criteria of acceptance, what does NR mean?

less than 100 organisms per gram or ml of product

Why do a suspension test?

To determine how long it takes for an antimicrobial to kill the bacteria

Describe the suspension test

Microorganism is added to antimicrobial in aqueous solution

Samples are taken at specific time points and inoculated in an inactivator broth

When solution is no longer cloudy, there’s not much bacteria left or plate to count surviving bacteria

What are the factors affecting the choice of preservative?

Intended application - product type (sterile/non-sterile), route of administration, moisture content, susceptibility to attack - nutrients?

No. and type of microorganisms present - what are the most likely contaminating microorganisms

Safety - non-toxic in relation to administration, safe to handle in manufacture

Cost - must not constitute a large cost

Stability - not affected by other products in formulation, effective over a range of pHs, effective over product shelf- life

Micro-environment - preservative available, moisture content as low as possible, storage temperature adequate

Properties of chemical agent - pH of the product and concentration/ dilution of the preservative