Micro

Question 1

Can you draw + describe a typical water system up to and including WFI?

Answer 1

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

Question 2

What is the specification for purified water?

Answer 2

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

Question 3

What is the specification for Water for Injection?

Answer 3

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

Question 4

What would the micro specification for a solid dose API be?

Answer 4

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>

Question 5

What is the CFU limit for non-sterile products?

Answer 5

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

Question 6

What are the ideal properties of a preservative?

Answer 6

1. broad spectrum
2. rapid antimicrobial action
3. chemically stable & effective under all pH
4. Compatible with excipients & packaging
5. Physically undetectable
6. safe to use
7. Cost effective

Question 7

What type of preservatives can be used?

Answer 7

1. Physical Preservatives (e.g. Aw, pH, Temperature, Excipients resistent to degradation.
2. Chemical preservatives (e.g. essential oils/ perfumes, enzymes & proteins)
3. 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)

Question 8

What is a PET test and how do you complete it?

Answer 8

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.

Question 9

Where would you find the micro specification for a specific product /dosage form?

Answer 9

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

Question 10

What media would you use for environmental monitoring and water testing? What incubation conditions would you use?

Answer 10

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

Question 11

Where would you place settle plates in a room? How would you choose locations (and give some example locations)

Answer 11

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

Question 12

What are bacterial spores?

Answer 12

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.

Question 13

What are common bacteria which may be found in pharmaceutical manufacturing environment?

Answer 13

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)

Question 14

What do you know about yeasts and moulds?

Answer 14

*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.

Question 15

What types of micro media are used?

Answer 15

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

Question 16

What types of organism are used for growth promotion/ fertility testing of every batch of media?

Answer 16

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

Question 17

What should be considered for micro method validation?

Answer 17

Qualifiction, not Validation. Verify compendial methods.

1. 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)
2. Contamination Recovery (counting technique and sample issues. => inoculat a sample with a known # of ‘spiked’ organisms and compare results
3. Operator validation <validate></validate>
4. Data Integrity
5. Test Interpretation <pharmacopoeia></pharmacopoeia>

Question 18

What are major sources of pyrogens? How can they be reduced and what tests are used for pyrogens?

Answer 18

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

Question 19

What are potential concerns when using settle plates?

Answer 19

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.

Question 20

How would you set up an EM programme for a solid dose facility

Answer 20

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)

Question 21

What are main sources of micro contamination within a manufacturing facility and how can you reduce the risk potential.

Answer 21

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

Question 22

What does disinfection efficacy depend on? Give sime types and ideal propeties

Answer 22

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

Question 23

Talk through how you would introduce a new disinfectant

Answer 23

• 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

Question 24

How would you qualify a cleanroom?

Answer 24

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)

Question 25

What are practical considerations for performing media fill?

Answer 25

Also refer to guidance in Eudralex vol 4 Annex 1: Sterile Medicinal Products. Also FDA Guidance. Summary of considerations:
- Simulate total process (including set up)
- Batch record/ Protocol
- Include permitted intrusions and interventions
- Replace product with broad spectrum media (e.g. TSB- Tryptic Soy Broth)
- Fill sufficient containers <to> # depends on batch size. refer to Annex 1.
- Retain details of container filling order
- 100% reconcilliation of vials
- Incubate for growth of wide range organisms at temp suitable for recovery of bioburden and environmental isolates.
(e.g. 20-35oC for not less than 14 days. If two temperatures are used, the units should be incubated for at least 7 days at each temperature (starting with the lower temperature).
- 3 x consecutive acceptable runs from start up
- NO POSITIVES!!!!
- At least 2 media fills per year thereafter
- Review data for operational state during validation runs
- Set micro monitoring sites, frequencies & limits
- Commence routine production
- Keep records of interventions for future challenge.</to>

*Plan carefully! *Need knowledge of process and cleanroom design *cover all operators & steps, including shifts
*Don’t do it until process is perfect! (equipment, operators, simulation runs, fully qualified cleanroom)
* Include your setting up of equipment for monitoring.

Question 26

Give some examples of how you might apply routine EM monitoring

Answer 26

Follow requirements within : Eudralex volume 4: Annex 1- Manufacture of Sterile Medicinal Products and ISO 14644 ‘Cleanrooms and associated controlled environments’. Some examples of how this could be implemented in sterile facility:

General (weekly): Air/ Hard Surface/ Liquid sampling (change room, air lock, hatches, prep areas. Include worst case, high traffic/ low air flow, air extract returns, stepovers, sinks, door handles. Be realistic!)

Specific (each work session): Reflect process flow pattern and ‘follow’ the product.- exposure points, operator gloves, air (active & passive), operators, liquid sampling.

• Risk aManagement!! FMEA/ HACCAP : Structured monitoring

Sample for moulds weekly (SAB) Anareobes every 12-16 weeks <use pre-reduced swabs & media>

• Approved sampling plan
• Approved sampling procedures & records
• Trained samplers
• Approved cultivation & enumeration procedure
• Set limits! <action level = target level +/- 3 standard deviations>
• Clear identification policy.

Question 27

How should EM data be used in assessments?

Answer 27

A single poor result may prove little information, change in trend is key.
Monitoring cannot be used to justify bad practice, poor behaviour or inapprorpriate procedures.

Question 28

What would you do in the event of a micro EM failure?

Answer 28

1. Identify (quick test eg. gram stain?)
2. Consider likely source
3. Consider # organisms present
4. Consider type of sample
5. Assess if any other ‘out of linit’ results on day of sampling or since
6. Look for links to any real time physical data
7. Consider activities at the time of sampling
8. GO AND LOOK AT THE AREA!
9. Trend data

• Notify plant immediately. Quarantine Product. Investigate sampling operation & related procedures. Look at compliance, aseptoc practice, fingerdabs, stress, equipment problems, motivation, management issues, talk to teams, retraining needed? concentrate on the trend, if a persistent problem then a cause exists!

Question 29

What Indicator organisms could demonstrate sterilisation?

Answer 29

Biological Indicators:
Geobacillus Stearothermophilus (steam/autoclave),
Sporse of Bacillus Atrophaeus (EO/dry heat),
Spores of Bacillus Pumilus (radiation)
Bacillus diminuta (filtration)

Question 30

Describe the measures to prevent cross-contamination, including references to guidelines

Answer 30

Guidelines: Chapter 3 & 5, Guideline on setting health based exposure limits for use in risk identification in the manufacture of different medicinal products in shared facilities
Sources: uncontrolled release of dust, gases, vapours, aerosols, genetic material or organisms from active substances, other starting materials, and products in process, from residues on equipment, and from operators’ clothing.
Note: Pesticides/herbicides not permitted. Sensitising agents unlikely to be permitted in shared facilitie
Risk assessment to be complete to determine risk and mitigation measures Summary of measures (in Chapter 5):
1. Technical measures:
i. Dedicated manufacturing facility (premises and equipment);
ii. Self-contained production areas having separate processing equipment and separate heating, ventilation and air-conditioning (HVAC)
iii. Design of manufacturing process, premises and equipment to minimize opportunities for cross-contamination
iv. Use of “closed systems” for processing and material/product transfer between equipment;
v. Use of physical barrier systems, including isolators, as containment measures;
vi. Controlled removal of dust close to source of the contaminant e.g. through localised extraction;
vii. Dedication of equipment, dedication of product contact parts or dedication of selected parts which are harder to clean (e.g. filters), dedication of maintenance tools; viii. Use of single use disposable technologies; ix. Use of equipment designed for ease of cleaning;
x. Appropriate use of air-locks and pressure cascade to confine potential airborne contaminant within a specified area;
xi. Minimising the risk of contamination caused by recirculation or re-entry of untreated or insufficiently treated air;
xii. Use of automatic clean in place systems of validated effectiveness;
xiii. For common general wash areas, separation of equipment washing, drying and storage areas.
2. Organisational measures
i. Dedicating the whole manufacturing facility or a self contained production area on a campaign basis (
ii. Keeping specific protective clothing inside areas where products with high risk of cross-contamination are processed;
iii. Cleaning verification after each product campaign should be considered <QRM>
iv. Verification of cleaning of non product contact surfaces and monitoring of air
v. Specific measures for waste handling, contaminated rinsing water and soiled gowning;
vi. Recording of spills, accidental events or deviations from procedures;
vii. Design of cleaning processes for premises and equipment so as not to present a cross-contamination risk;
viii. Design of detailed records for cleaning processes to assure completion of cleaning -approved procedures and use of cleaning status labels
ix. Use of common general wash areas on a campaign basis;
x. Supervision of working behaviour to ensure training effectiveness and compliance with the relevant procedural controls.</QRM>

Question 31

How can you get rid of a biofilm?

Answer 31

Depends upon the complexity of the system and the severity of the biofilm formation.
Thermal inactivation - (>75oC) will typically require repeated and/or extended elevation of temperature compared to the routine sanitisation cycle. While the utilisation of a hot water flush through systems is considered acceptable in order to minimise the planktonic contaminants and biofilm existing within a system, it is known not to be fully effective in the removal of biofilm mass.
chemical sanitising agents - effective method for biofilm removal but introduces the risk of residual chemicals remaining in the water system. Therefore, monitoring should take place after chemical sanitisation to ensure that the chemicals have been removed from the system. ideal mode of action of chemical sanitising agents in the context of biofilm is to both penetrate and provide the appropriate kill to the organisms in question. Appropriate velocity during flushing will aide in the removal of debris and chemicals.

Ensure that the systems recirculating or flowing and the sanitising agents utilised are not introduced into a system to exert their mode of action in a passive mechanism.
Any physical removal approaches should be used with caution because of the high potential of damaging the surface leading to higher risk of recolonization sites and/or corrosive attack (rouging in the case of stainless steel).
Appropriate removal of cellular debris should also be considered, as excessive debris can result in increased levels of endotoxin/exotoxin etc. existing within the system e.g. by emptying and refilling the entire system with fresh water.

Frequent, rotation of disinfectants and detergents and inclusion of sporicidal agents should beconsidered as part of a robust strategy.
It should be noted that once a biofilm has been established it may be difficult to remove even using the methods above. Any biofilm removal should be followed by a period of intense monitoring before returning the system to use to ensure that the biofilm has been effectively removed and water quality is consistent with the specification.

A robust preventative maintenance programme is essential in order to maintain equipment and premises to a standard that will not add significant risk from a contamination viewpoint. Consider regular inspection of utilities, process equipment and transfer lines for obvious signs of deterioration – O-rings, gaskets, seals – regular inspection and replacement.

Agents: hypochlorite, hydrogen peroxide/peracetic acid solutions, sodium hydroxide. Appropriate contact times established.
Ozonation should be also considered for loop and distribution systems for WFI water. The destruction of ozone can be done by UV irradiation/treatment.

Question 32

What type of microorganisms would contaminate solid dosages? Any worries if present?

Answer 32

Micooganisms which have require very small amounts of available water (given the fact it is so low in a solid dosage form).
Proliferation does depend on composition of product (nutritional factors, water, storage temp, pH, Pack design). Also the contamination source(s); raw materials (particularly if natural origin), water quality and content, storage/ handling, environment, packaging, etc._)

Most gram-positive bacteria, such as Enterococcus spp. (including Vancomycin resistant enterococci), Staphylococcus aureus (including MRSA), or Streptococcus pyogenes, survive for months on dry surfaces. Many gram-negative species, such as Acinetobacter spp., Escherichia coli, Klebsiella spp., Pseudomonas aeruginosa, Serratia marcescens, or Shigella spp., can also survive for months. Potential also for surface mold and yeast contamination.

Particular concern is enterobacteriae (e.g. E.Coli), also any particularly resistant to antibiotics. Also any drug given to particularly immunosupressed patients.

Question 33

What are the changes to Annex 1? What is Contamination control strategy?

Answer 33

The revised “Scope” and “Principle” sections now clearly reference the importance of applying the principles of Quality Risk Management (QRM).

The importance of non-conformity investigation related to sterility or microbiological load is emphasized.

The revised “Premises” chapter now clearly uses the term “cleanrooms” and outlines the different classes and requirements for classification.

design and use of airlocks, along with clear specifications for airflow patterns and air pressure requirements.

need to be able to observe Class A and B areas from the outside.

CCS should be
implemented across the facility in order to define all critical
control points and to assess the effectiveness of all the
controls (design, procedural, technical and organisational)
and monitoring measures employed to manage risks to
medicinal product quality and safety.

Question 34

Describe good sampling practice of purified water points? How frequent should you sample?

Answer 34

1. Pre-sanitise - IPA
2. Flush 2 mins
3. Sample

Container - Prep, Sterile, Pyrogen free, Labelling

Sampler training

Storage of sample

Question 35

What is the difference between disinfection and sanitization?

Answer 35

Disinfection destruction of microorganisms to reduce them (not sterilize). Not necessarily killing all microorganisms but reducing them to an acceptable level for a defined purpose.

Sanitization being the process of cleaning and disinfection in a single process (the agent used usually has surfactant properties making it surface active for cleaning) often associated with CIP processes.

Question 36

You are a QP, CEO wants a new sterile product manufactured, asked how I would perform EM qualification of the Suite.

Answer 36

Contamination control strategy
3 cleaning event
ISO14644
Worst case locations
Process simulation trails - stress test
EM - finger dab, surfaces
training
smoke studies
laminar flow controls
utilities
setting up alter limits

Wanted everything for this including Laminar flow controls, viable/nonviable limits for Grade A&B rooms, positions of the plates and air sampling within the Grade A (I said close to point of fil which led to discussion around risk of product hitting the plates). Spent a little time on utilities and WFI testing (QC and Micro).

Question 37

What considerations would you have for setting up an environmental monitoring programme in your cream manufacture suite?

What are you looking for?

Objectives?

How do you determine sampling sites?

Answer 37

Activities
type of product
air flow patterns
monitoring and gowning
containment of process
air changes
risk assessment
high traffic areas
alert and action limits

Air contamination - active

Air contamination - settle plates

Operator contamination - fingers

Surface contamination - contact plates

Product integrity - sterility test

Operator training

Process integrity - process simulation trial

Cleaning procedures validated

Question 38

What are the Annex 1, microbiological requirements within a clean room, use of agar plates, grades of area

Answer 38

Air Bourne

Grade (at rest, in operation 0.5um per m3)
A (3520, 2520)
B (3520, 352000)
C (352000, 2520000)
D (3520000, not defined)

micro contamination in operation

Grade (air sample (cfu/m3), settle plates (cfu in 4 hrs), contact plates (cfu/plate)
A (no growth)
B (10, 5, 5)
C (100, 50, 25)
D (200, 100, 50)

Question 39

Your micro department informs you that they have discovered a trend of Micrococcus luteus and Bacillus subtilis in the tablet compression room of your grade D facility. What are your concerns?

What type of micro are these?
Are they pathogenic?
Where is guidance found on cleanrooms?
Where is guidance found on micro for tablets?
Was originally above alert level, but now above action level. What action would you take?

Answer 39

Micrococcus luteus - skin, human flora
Bacillus subtilis - environment, carboard, apples

cleaning and gowning
Deviation
impact assessment
identify source
spore forming - could be an issue
cleaning regime

Question 40

You have a cream product that has failed TYMC (yeasts and moulds). You have retested and it passes, you therefore have one passing result and one failing result.

Answer 40

• sampling - retesting protocol
• preservative efficacy test
• testing errors - operators
• OOS - reason for retest, statistical relevance, how many tests
• PWP system and data
• species recovered

Question 41

Is Preservative Efficacy Testing a Pharmacopeial method or created in house?

Answer 41

Pharmacopeia

Question 42

How is PET performed?

Answer 42

a inoculum with a 10^6 for candida, pseudomonas, staphylococcus aureus and aspergillis is created.

inoculum added to the product

Tested at stipulated time points stated within BP (6hrs, 24hrs, 48 hrs, 7 days, 14 days, 28 days)

preservative neutralised when removed at sample test point and spread onto agar

results based on log reductions