Ben Forbes - All IPM COPY Flashcards
what are the standards for sterile products
- Sterility test
- Pyrogens test
- Particle-free
- Preservative Efficacy Test
what is a serial dilution? what is it based on, and what are the pros.
its a form of viable counting. Based on most probable number (MPN). It is a statically based assay
- A series of dilutions are made and each dilution is inoculated into several tubes of broth
- Incubate 30 - 35°C for 5 days , then observe for growth OR no growth.
- Reference to tables -> to estimate MPN
- Good method if # of microbes is unknown
- Good for a wide range of microbes
what mechanisms affect drugs staying on eyes?
- Tear flow = washes away drugs
- Blink frequency = eyelids wash away the fluid coating surface of the eye
- Drug properties: If Irritant = this leads to increased tear flow, and an increased blink frequency ∴ this reduces retention time
Why do we have preservatives
Protection against:
- Residual contamination not removed by GMP in non-sterile product
- Contamination introduced during use
e.g. creams, eye drops
Name examples of preservative and what pH they are effective at
Benzoic and Scorbic acid as a pka of 4.2-5, and are active at lower pH’s
Phenolics are only affective above pH 9
Cationic preservatives are inactive below pH 4-5 e.g Chlorhexidine
advantages and disadvantages of light blockage method
Pros:
- Rapid
- Accurate
Cons:
- Affected by shape and transparency
- If particle is transparent then the result is biased
- Variation b/w commercially available instruments
what ingredient can dilate the pupil? what constricts?
Mydriatics dilate (e.g atropine) and miotics constrict (e.g pilocarpine)
Explain this equation
equation showing Effect of dilution on preservative
- t1 and t2 = time required to reduce a viable population to the same extent using the preservative at different concentration; C1 and C2
- indicates reduction in preservative activity due to dilution
- e.g. Phenol activity = 6, when diluted 4-foldà activity reduces by 46 = 4096 times
How does rabbit testing work
inject rabit with formulations and see if temperature rises. If 3 rabbits have a combined increase of temperature > 2.65oC, then product fails.
Advantages
The febrile response of the rabbits are similar to human (so humans have the same reactions)
Disadvantages
- Expensive
- Time consuming
- It’s NOT easy to measure the temperature of a rabbit
- Ethics lol
Explain how the light blockage method works
A high intensity light source is used shone on the particle as it passes through the detection chamber. The particle passes through the light source (typically a laser or halogen light) and scatters the light which is detected by a photo detector.
Detects
Particles > 2um:
Particles > 5um:
what is special about water for injection B.P
water for injection BP is a specialist product that is sterile and pyrogen free- the water is distilled in specialist distillation equipment that prevents any carry over of water droplets. Heat to 80oC to prevent any recontamination and use within 4 hrs
Explain the visual inspection pros and cons, and state the GMP guidelines here, if any
Advantages
- Detects large contamination + incompatibilities in admixtures e.g. CaCl2, K2PO4
- No need to destroy product
Disadvantages
- Only LARGE particles are detected by human eye
- Subjective
GMP requires each container of a batch to be examined by trained personnel
e.g. look through polarised light
what do we pyrogen test?
Tests are applied to ALL injections > 15 ml and powders for reconstitution, especially infusions that are large volume, direct IV and in seriously ill patients
what are particulates, and what is the issue with defining them?
Particles -> mobile undissolved substances unintentionally present in parenteral solutions. Particles can be living and non-living. It must be removed to produce a sterile product
There is no single standard of particle to match against
Only a certain type of product needs Preservative efficacy testing name two types
Oral mixtures (to check for absence of E.Coli) and high in sugar products, to check for absence of Saccharomyces
physical properties of pyrogens
They are non-living (so you cannot use preservatives to kill them; no antibiotic; chemical strategies can be used)
They are thermostable- can survive in temperature of up 250 OC; so they cannot be destroyed via heat.
They are water soluble ∴ during filtration, passes through the filter paper easily
They are non-volatile -> use this as a target
Limitations of Preservative Efficacy Testing
The 4 organisms (or the organisms) inoculated with sample are not representative of all possible contaminants Lab strains of bacteria have different patterns of resistance to the wild stains contaminating the product during use or manufacture.
28 days cannot be extrapolated to an expected shelf life of 2-3 years (which is the shelf life of API) so will the preservative function for this long time period? Sample of 1 ml will be unable to detect <1 organism/ml
What are pyrogens?
Pyrogens are not removed by filtration. They are
- Defined by their action: They are fever-inducing substances (so anything you inject that will induce a fever )
- They do NOT have a chemical structure
- Most potent ones are from Gram -VE bacteria
- They have a high MW
- They are lipids but their potency is enhanced by protein/polysaccharide
What tool do we use to define the paramateters of microbial contamination?
In-house guideleines for hospitals and industry, and the BP:
Pharmacopeia
>states the regulatory controls
- Works within limits
- Total viable counts changes for FORM of meds
- Oral, inhalation
- Herbal meds have higher limits than synthetic meds
- Absence of specific organism
- S. auerus and pseudomonas affects inhalation products
- If microbe present = product fails
how do we label eye drops for expiry purposes?
- Theatre (minims = SINGLE use) - 24hrs à discard after use
- Hospital- 1 week (due to risk of Hospital acquired infection once the eye drop bottle is opened)
- Community – 28 days
- > Label for EACH eye, esp. in infections
what tests are there for Microbial Quality Assurance?
- Test for microbial contamination
- Preservative efficacy test
- Sterility test – a count test, where the no. (Of microbes) is 0, so not too different to test for microbial contamination.
How to detect specific organisms
Talk about preservative interactions with different container types
Aqueous phase concentration can be reduced by adsorption to the container.
Examples:
- Glass container -> fairly inert
- Rubber closures -> liquid soluble preservatives adsorbed e.g. 60% loss of chlorocresol
- Plastic containers -> loss of thiomersal and chlorbutol (preservatives) from contact lens solutions packed in plastic
how can i eliminate pyrogens in water?
If we distill the water (evaporate the water and condense it) any pyrogenic material will be left behind.
what chelating agents should we add to opthalamic products
EDTA
Explain the Test for Microbial Contamination
Work towards the BP limits and European pharmacopeia limits. The route of administration determines what limits permitted and not permitted.
E.g. things for the skin (topical applications) must be free from opportunistic skin pathogens. So things such as S.aureus and pseudomonas and for oral preparation, such as tablets, E.coli
Principles of viable counting techniques
Generally bacteria are faster growing than fungi
- So the bacteria have a different type of agar that they grow in compared to fungi
- For BACTERIA: Casein soya bean digest agar
- For FUNGI: Sabouraud Glucose Agar with Abx [to stop bacteria growing]
why must we adjust the pH in an IV injection?
increases stability on injection
- reduces pain, irritation, necrosis
- reduce growth of microbes
- increase physiological activity
- buffers -> increases shelf-life by stopping degradation
-> withstands sterilization
Discuss how to do membrane filtration for testing the presence of microbes
For 10-100 cfu/ml
The organisms need to be retained on the surface of a filter.
- 10ml sample filtered through 0.45µm pore size filter
- Filter washed with 3 x 100ml buffer -> to ensure no false results
- Filter transferred to agar plate to encourage growth
State the different methods of detection
Visual inspection
Optical microscopes
Electrical Sensing Zone method
Light blockage method
principle requirements for eye drops
- Chemically stable
- Particle free
- Preserved (multiple dose containers)
- Sterile when issued
State the two types of Pyrogen testing
- Rabbit test
- LAL test
what are key features for ‘water for injection’
- DISTILLED. Used and autoclaved within 4 hrs or for later use autoclaved immediately or held at 8oC
- Sterile
- Particle- free, pyrogen-free
- CO2-free (avoid precipitates; boil for 10mins)
- Air-free i.e. O2-free (use N2 to prevent oxidation)
Origins of particulates
Raw ingredients: Drugs / Solvent
The FINAL container:
- Material NOT removed during rinsing PRIOR to filling
Environmental: Skin flakes/ dandruff/ cellulose
Container + Closure: Deposition of closure components during sterilisation. E.g ZnO, Clay
What is a Preservative Efficacy Test?
a 28 day est to verify whether or not the preservative strategy is effective for the product
LAL Test. Explain it
IN-vitro test and very sensitive at detecting bacteria ENDOtoxins. Based on defence mechanism of horseshoe crab, that has enzymes to recognise endotoxins and form a gel.
Advantages
- Quick
- Sensitive (1 picogram/ml)
- Quantifiable
- Inexpensive
Disadvantages
- pH, cations etc. can affect results
What parameters define Microbial Contamination?
Hazards (product spoilage)
Limits (the design of ‘sterile’ and non-sterile priducts)
Control measures (GMP, Formulation and Preservatives)
A hazard depends on
Size of particles:
Large particles > 590 um block the needle
Particles > 8um lodge in the lung
Particles 3-5 um taken up the spleen + liver
Particles < 3um may aggregate
Size of blockage
Shape, surface characteristics of the particle
- Affects adherence
Nature of particle
Host response
why must opthalamic producs be sterile?
because the eye is a vulnerable, moist environment in which microbes flourish ∴ gets infected easily.
How do hazards occur?
Hazards occur by the product spoiling due to:
- Breakdown of the active agent by microbes (e.g Atropine eye-drops degraded from pseudomonas)
- Breakdown of the formulation (e.g polysaccharides)
- Reduced acceptability (due to microbes waste)
- Degredation of preservative (e.g phenol -> Pseudomonas
Overally an unfit for use by patient
Sources of pyrogens?
Solvents (i.e.water) – because pyrogens come from bacterial cell and bacteria likes to grow in moist environments.
e.g Large volume of parenteral have a lot of water in them. ∴ susceptible to high amounts of pyrogens
Why is preservative solubility important and how can we increase preservative solubility?
Preservatives must be soluble in aq phase of a formulation to ensure antimicrobial activity.
Co-solvents can increase solubility AND activity, in the order of:
Ethanol > propylene glycol > glycerol
when selecting a preservative, what main factors do we consider?
- Type of formulation
- Ingredients
- Pysico-chemical properties
- Route of admin
- Non-irritant
- Non-sensitizing
- Non- toxic
- Colourless
- No taste
- Inexpensive
what determines drug retention?
- Natural mechanisms
- Volume Normal eye drop drug volume = ~ 25-50 µL.
Ideal eye drop volume: 5-10 µL BUT highly costly > not supported by the NHS.
Real life: use excess volume as patients are bad at administering
- pH Severe tearing at a pH <5, but buffer the eye drop to pH 7.4 to aid retention
- Viscosity - increased visocity = increased retention time. e.g Hypermellose
- Anti-oxidants
- Preservatives
How do i do a plate count in “viable counteng techniques”?
50-300 cfu/ml
- 1ml sample added to molten agar OR spread onto the surface of an over dried agar plate.
- For BACTERIA: Casein soya bean digest agar- bacteria (incubate 30-35 °C – their optimum temperature for 5 days)
- For FUNGI: Sabouraud Glucose Agar with Abx [to stop bacteria growing] (incubate 20 -25 °C for 5 days)
What are the hazards of particulate matter
Vascular occlusion
Directly by particles > 7.2 um could block arterioles/ capillaries. Indirectly through formation of emboli
Inflammatory response
If particles are concentrated in an area. If persistent can lead to cancerous response!
Cancerous response
Antigenic response
Patient becomes sensitised to material and has an allergic response to material if reintroduced to the body later on.
Any solubility issues with preservatives? Examples?
Solubilisation (micellar systems)
- Preservative is solubilised above the critical micelle conc (CMC) and is not solubilised below this.
- preservatives will favourable partition into micelle as they are lipophilic, out of the aq phase.
- but the microbes prefer the aqueous phase
X causes reduction in preservative in aqueous phase, where bugs live!
Example: Parabens preservative
Formulation factors affecting Microbial Content
- Water content: Suspensions, aqueous meds are most susceptible
- Nutritional value
E.g. aromatic water such as peppermint have short T1/2
Pseudomonas can survive in distilled water with limited nutrition available
- pH - most bacteria optimal ~ 7
- Osmotic pressure: Decreases growth
- Surface tension
- Oxygen tension: Store products under Nitrogen to inhibit aerobic microbes
- Storage
- Temp
What are Parenterals, with examples?
Every route that is NOT oral:
Oily (lipophilic drugs)
Emulsion (o/w – lipophilic drugs)
Colloidal solutions
Mixed solvent systems (poorly soluble drugs)
Aqueous solutions
Suspensions (insoluble drugs)
Treatment of glaucoma?
Beta-blocker e.g Timolol
Miotics
