Micro Flashcards
Can you draw + describe a typical water system up to and including WFI?
Potable water –> Storage tank + chlorine –> pre-filtration –> Carbon bed –> water softener –> reverse osmosis –> deionisation –> purified water storage tank
Purified water –> UV –> user points –> return loop to storage tank
Purified water –> multi-effect distillation –> WFI storage tank –> user points hot OR Heat exchanger and user points cold
- Feed Water
- Assess quality (seasonality),
- Water Pre-Treatment: Remove Hardness <calcium> & organics
Chlorination: Improves microbial control
Sand/ Media Filtration: Remove large particles
Carbon Beds: Removes organics & chlorine
Water Softener: Remove Calcium & Magnesium cations- prevent scale (resin regeneration)
Organic Scavengers: Remove large mol weight organics
Filtration Screen & Depth Filters: Reduce bioburden & large particulates</calcium> - Water Purification
o Purified Water generation
-Reverse Osmosis OR Chemical Regenerable Deionisation Systems OR
Twin bed & mixed bed systems and Service Deionisation OR Continuous Electrodeionisation (CEDI)
o WFI generation - Singe Effect OR Multi Effect OR Vapour Compression OR RO- (USP and from From April 2017 PhEUr- Annex 1 doesn’t permit yet)
- Storage tanks (capacity at peak demand)
o Totally draining, Sterilising vent filter, Sanitary bursting disc & level switching/ control
o MOC (316L SS for WFI, polypropylene perhaps earlier), surface finish & treatment <e.g.></e.g.> - Distribution Pipework
o Zero deadlegs, Sized for ultimate velocity (1-3m/sec), Drainable, Sanitary instrumentation & pump design
o NO FILTRATION, Welding- orbital welds, pit & crevice free, Valves & pumps- sanitary design
o UV or Ozone sanitisation
o MOC (316L SS for WFI, polypropylene perhaps earlier), surface finish & treatment
What is the specification for purified water?
TOC <500pb
Conductivity <4.3 us/cm at 20degC
Nitrates <0.2ppm
Heavy metals <0.1ppm
Aerobic bacteria (TVC) <100cfu/ml
Endotoxins <0.25 iu/ml
What is the specification for Water for Injection?
TOC <500pb
Conductivity <1.1 us/cm at 20degC
Nitrates <0.2ppm
Heavy metals <0.1ppm
Aerobic bacteria (TVC) <10cfu/ml
Endotoxins <0.25 iu/ml
What would the micro specification for a solid dose API be?
ICH Q6A Specifications: Test Procedures and Acceptance Criteria for New Drug Substances and New Drug Products: Chemical Substances .
ICH Q6B: Test Procedures and Acceptance Criteria for biotechnological/ biological products
There may be a need to specify the total count of aerobic microorganisms, the total count of yeasts and molds, and the absence of specific objectionable bacteria (e.g., Staphylococcus aureus, Escherichia coli, Salmonella, Pseudomonas aeruginosa). Any micro specification should be suitably determined using pharmacopoeial procedures.
The type of microbial test(s) and acceptance criteria should be based on the nature of the drug substance, method of manufacture, and the intended use of the drug product.
With acceptable scientific justification, it could be possible to propose no microbial limit testing for a solid oral dosage form API
«<whereas>>></whereas>
What is the CFU limit for non-sterile products?
Ph Eur 5.14 ‘microbiological quality of non sterile products for pharmaceutical use’
Non aqueous:
Total Aerobic Microbial Count TAMC: 10e3 cfu/ml or g
Total Combined Yeasts 7 Moulds Count TYMC: 10e2 cfu/ml or g
Aqueous:
Total Aerobic Microbial Count TAMC: 10^2 cfu/ml or g
Total Combined Yeasts & Moulds Count TYMC: 1061 cfu/ml or g
What are the ideal properties of a preservative?
- broad spectrum
- rapid antimicrobial action
- chemically stable & effective under all pH
- Compatible with excipients & packaging
- Physically undetectable
- safe to use
- Cost effective
What type of preservatives can be used?
- Physical Preservatives (e.g. Aw, pH, Temperature, Excipients resistent to degradation.
- Chemical preservatives (e.g. essential oils/ perfumes, enzymes & proteins)
- Synthetic Preservatives (e.g. Acids/ Salts, Alcohols, hydrobenzoates, phenols, Quarternary ammonium compounds).
Examples:
Oral/Topical (Acid, hydrobenzoate) Parenteral (Sulphites, phenol, benzyl alcohol) Opthalmic (phenylethanol, chlorobutanol)
What is a PET test and how do you complete it?
In the case of products packaged in multiple-dose containers, antimicrobial preservatives are added to inhibit the growth of microorganisms that may be introduced from repeatedly withdrawing individual use doses.
Preservative efficacy test (PET) is performed on these products to determine the type and minimum effective concentration of preservative required for satisfactory multi-dose pharmaceutical formulation. Dosage forms include ophthalmic, oral liquid, ear preparations and topical preparations.
Methods of assessment are defined within Ph. Eur, BP, USP and the JP.
The test consists of challenging the preparation with live organisms at a series of time points over an in use period of up to 28 days. At each time point analysis is performed to establish if organisms are present or not and therefore if the preservative is effective.
Where would you find the micro specification for a specific product /dosage form?
Within pharmacopoeia.
Typical requirements are harmonised:
- Non sterile Non-Aqueous
TAMC 10^3,
TYMC 10^2
- Non sterile Aqueous:
TAMC 10^2,
TYMC 10^1
What media would you use for environmental monitoring and water testing? What incubation conditions would you use?
Facility EM: TSA, SAB & R2A Agar
Water:
High-nutrient media such as Plate Count Agar (PCA) for the isolation of heterotrophic bacteria and/or low-nutrient media such as R2A for the isolation of slow growing “oligotrophic” bacteria.
- Routine & in-process EM: Settle plates, Active air plates & Finger Dabs 20-25°C for 3-5 days then 30-35°C for 2-3 days
- EM during testing: Settle plates & finger dabs 30-35°C for 3-5 days
- Sub-culture of isolates for identification, propagation of wild strain organisms from stocks for use in testing & inoculation controls during growth promotion/ bioburden suitability testing. Variable, usually 30-35° for 18-24 hours for sub-culture, inoculation controls to match testing
- TSA Milliflex cassettes (Bioburden testing of all in-process and BDS samples, equipment flush bioburdens) 30-35°C for 3-5 days or 3 days minimum depending on method
- SDA Milliflex cassettes ( Bioburden testing of in-process and BDS samples for TYMC, when required) 20-25°C for 5-7 days or 5 days minimum, depending on method
- R2A Milliflex cassettes (Bioburden testing of WFI and water samples) 30-35°C for 5 days minimum
Where would you place settle plates in a room? How would you choose locations (and give some example locations)
Settle plates should be placed in areas of high risk of product contamination- as close to activities as possible without causing obstruction of activities or contamination by the plates themselves. Assess the process, risks and decide ‘worst case’ locations based upon risk assessment.
- Within filling zone (e.g. RABS/ Isolator)
- Where any open manipulations take place- e.g. charge/ discharge activities
- Where any aseptic connections are made
Note: Settle plates placed in areas of high airflow, turbulence, or where high temperature or low humidity conditions exist may dry out or otherwise change their properties so that bacteria or fungi that previously settled on the plates die, as do newly captured ones. Validation studies should be conducted to determine how long a settle plate can be left under the specific conditions of use and still retain full growth promotion for the microorganisms of interest.
background:
Passive monitoring uses “settle plates”, which are standard Petri dishes containing culture media, which are exposed to the air for a given time in order to collect biological particles which “sediment” out and are then incubated. Results are expressed in CFU/plate/time or in CFU/m2/hour
What are bacterial spores?
Gram +ve bacilli- when conditions are tough form a spore. Thick wall, highly resistant, low metabolic activity, require low water content. When conditions are better can germinate and return to vegitative form.
What are common bacteria which may be found in pharmaceutical manufacturing environment?
Gram +ve cocci: Staphylococcus (e.g. epidermis <human> Aureus <less common, nasal passages)</human>
Gram +ve bacilli: Bacillus (e.g. Atropheus <environment, soil, water, atmosphere, cardboard, paper, wood> can form spores
Gram -ve bacilli: Psudomonas (e.g. Aeuruginosa <stagnant/ static water, drains, ubends, deadlegs, borehole, mophead/bucket, wet equipment- musty smell. can be resistant to disinfectants/ preservatives)
What do you know about yeasts and moulds?
*Moulds
- Produce spores. Simple nutritional requirements (don’t need much water), longer incubation period for growth, specialised methods of staining / identification. Widespread in environment, soil, decaying vegetation, exposed plaster, damaged pipework lagging, 2nd most common cleanroom contaminant after bacteria.
*Yeasts
On staining: large gram +ve spheres/ovoids, Can produce resistant spores but more commonly produce less resistant ‘buds’, require high levels of nutrition and moisture. Rare contaminant of cleanrooms but can cause spoilage of pharma products that contain high levels of water & carbohydrate.
What types of micro media are used?
1) Broad Spectrum General Purpose Media: e.g. TSA & NA <for>
2) Enhanced Media: e.g. CBA, MA <specific>
3) Selective Media: e.g. Centrimide agar <to>
4) Differential Media: e.g. Baird Parker or Macconkey agar <to>
5) Enrichment Media: e.g. Buffered Peptone Water, Selenite Cysteine Broth <enhance target organism & inhibity unwanted organisms></to></to></specific></for>
Anaerobic media (remove dissolved o2): e.g. Sodium thioglycolate, thioglycolate broth
What types of organism are used for growth promotion/ fertility testing of every batch of media?
A range of organisms maintained under precise conditions (to avoid subculture mutations), including those which are:
- easy to grow
- difficult to grow
- organisms to be inhibited
- laboratory isolated strains appropriate to the facility
Pharmacopoeial organisms
(Agar):
TSA: S.aureus, Ps. Aeuriginosa, B.subtilis (inoculate <100cfu/ml, <=3 days at 30-35oC)
TSA + SDA: C.albicans, A.brasiliensis (inoculate <100cfu/ml, <=5 days at 20-25oC)
(Broth): S.aureus, Ps. Aeuriginosa, B.subtilis
What should be considered for micro method validation?
Qualifiction, not Validation. Verify compendial methods.
- Product Toxicity (antimicrobial/ bacteristatic quality of sample- need to neutralise to stop inhibition. Note: membrane filtration overcomes this since it removes the sample from the test, but still common to use a neuralising rinse as part of test)
- Contamination Recovery (counting technique and sample issues. => inoculat a sample with a known # of ‘spiked’ organisms and compare results
- Operator validation <validate></validate>
- Data Integrity
- Test Interpretation <pharmacopoeia></pharmacopoeia>
What are major sources of pyrogens? How can they be reduced and what tests are used for pyrogens?
Pyrogens = non bacteria or bacterial origin chemical entities that produce fever. e.g. LPS from bacterial cell wall
Sources: 1) raw materials (especially animal/ plant origin), 2) Water (gram -ve organisms e.g. pseudomonas) 3) people (gram -ve organisms can colonise humans but greater threat is humans as a vehicle for transmission e.g. moving bugs around during cleaning activities).
Reduction: a) inactivation (hydrolysis, oxidation, dry heat) b) Removal (rinsing, distillation, ultrafiltration, RO, Activated carbon, charged media or hydrophobic attraction)
Testing: a) Rabbit Pyrogen b) LAL- gel clot, turbidometric or chromogenic
What are potential concerns when using settle plates?
Can potentially dry out or be contaminated by activities/ personnel/ splashing of cleaning fluids etc- therefore wouldn’t provide a result which is representative of the environment itself.
How would you set up an EM programme for a solid dose facility
Talk through Annex 1 as providing guidance and limits for processing related to sterile products.
Solid dose products covered by pharmaceopoieal limits for TAMC/ TYMC but no specific classification requirements for cleanrooms.
- EM programme would be risk based depending on your background established levels.
Would monitor at an appropriate frequency based on these levels and the particular product requirements and look for OOT spikes.
Typically would also review on an annual basis and determine if any additional controls or change to monitoring/ frequency are required.
To elaborate in further detail how you would set up and do the monitoring- would talk through the Quality M’s (Man, Method, Machine, Material, Measurement, Milieu)
What are main sources of micro contamination within a manufacturing facility and how can you reduce the risk potential.
Micro Contamination Habitat Example Organisms How to reduce risk
- Atmosphere (Air & gases) Spore formers (Bacillus/ clostridium) Good HVAC design, air change (HEPA, if sterile)
Gram +ve (Staphyloccus) Temp & humidity control, surface disinfection
Sporing mould (aspergillus) Gowning, reduce exposed product/ material etc
Yeasts (Rodoturla) Dusts & contamination containment, pressure dif
- Terrestrial Wall/ Ceiling (moulds) Easy to clean,good finish, keep dry, avoid wood
floors & drains, equipment (bacteria) Dismantle, good cleaning, avoid hotspots
Pipelines (bacteria- e.g. pseudomonas) No deadlegs, surface finish, drain
Cleaning equipment change mops etc, make solutions on day, dry!
Raw materials (especially natural, typical Good suppliers, pre treatment,
plant origin contaminants include pseudomonas, Packaging, prevent moisture
bacillus, and potential human pathogens eg E coli Test on receipt
& salmonella) - Aquatic Water systems- e,g, pseudomonas Design, treatment, draining, temperature, sanitise
- People Skin (staph/ strep/ fungi etc) +Mouth (yeast) Closed systems, hygeine, clothing, ventillation
intestines- Gram -ve anaerobic rods Aseptic
What does disinfection efficacy depend on? Give sime types and ideal propeties
Temperature
Application
Concentration
Time
Types - e.g. QUATS, Alcohols, Chlorine Compounds
Ideal: no residue, soluble, quick kill, wide spectrum, low concentration, non toxic, stable, compatible with cleaning materials, low foam, not affected by water hardness, non toxic to environment/ people, easy to discard, economical, sporicidal).
Sporicides and sterilants for the removal of spores from hard surfaces. e.g. Spor-Klenz
Disinfectants for surface cleaning with broad spectrum antimicrobial efficacy e.g. Phenolic: Vesphene QUAT: HB+
Ready-to-use alcohols for the removal of process residues and routine glove decontamination
Talk through how you would introduce a new disinfectant
- Supplier tech data sheets
*Lab studies (dilution)
*Lab studies (surfaces)
*Facility based studies - Implementation considerations <talk through chapters: PQS, Personnel, Premises & Equipment, Documentation, Production, QC, Outsourced Activities, Complaints & Recall, Self Inspection
How would you qualify a cleanroom?
Follow guidance in Eudralex vol 4 Annex 1: Sterile Medicinal Products & Annex 15: Qualfication & Validation
<Qualification for EM, cannot validate as the environement is not static!> Check that you have:
a) Compliance with established standards.
b) Correlation with manufacturing (e.g. sterile medial fills)
c) Basis for a sound monitoringg plan ->maintenance/ housekeeping -> unexpected batch specific incident detected & evaluated
(Intensive EM - Proces Simulations - Routine Monitoring)
1. Train operators
2. Establish air system & key equipment
3. Qualify room for physical parameters
4. Monitor surfaces & equipment (corners etc. hot spots)
5. Clean & Disinfect (at least x 2)
6. Intensive Monitoring: Air (active & passive), surface sampling <standards for ‘at rest’, unmanned must be met
7. Select micro monitoring sites for operational state
8. Suitably gowned/trained operators perforrm physical & micro monitoring (surfaces & staff) during simulated activities
9. Confirm compliance (include clean up rate, retuen to ‘at rest’ conditions)
10. Perform process validation (i.e. media fills for aseptic products only)
