Micro basics Flashcards
Microbiological Troubleshooting
Who – was present at time of recovery, what was going on?
What – identify organism – morphology? pathogen?
Where – assess risk depending on location and identity of organism
When – be sure to contain contamination with proper chemical sanitization and include all impacted lots in your scope.
How – were procedures followed? Is process robust?
What are the common types of micro-organisms?
Gram negatives - Pseudomonas aeruginosa, gram positives staph aureus, bacillus species, yeasts candida albicans, moulds aspergillus
What are the risks involved in manuf. with a mammalian cell line?
Virus/Mycoplasma, microbial spoilage
How is virus removed? How would you validate this?
Irradiation, heat, formaldehyde, pH
How is Bioburden, Endotoxin and Sterility controlled as part of a manuf. process?
Aseptic processing, facility design, quality of water, autoclaving, washing, depyrogentation etc
How are stoppers de-pyrogenated?
washed and steam sterilized in a processor, wash step is critical as you cannot apply the same temp as de-pyrogenation conditions.
What are the common sources of microbial contaminants?
People, water, raw materials and packaging materials
What is the source of :
Bacillus bacterium
Pseudomonas
Staphs
environment, soil
water
people
Why do gram negative bacteria pose a special risk to patients
endotoxins - cause sepsis, toxic shock, death. Produced from the breakdown of the cell membrane of gram negative bacteria when killed. Endotoxin is not living so cannot be killed so must be inactivated
How do you test for endotoxin?
LAL test using the blood from horseshoe crab. Also some new rapid methods being developed
Why would it not contain a preservative (multi-dose/single-dose)?
Water levels, single use
What tests would you perform on a preservative?
Preservative efficacy testing - spiked with test organisms including gram negative, gram positive, yeast and mould also any environmental isolates and reading taken over number of days
What is the statistical importance of the sterility test?
statistically poor at detecting anything other than gross contamination
A count of Staph is found at point of fill – what would you do, what is staph, where does it originate, what type of organism is it, is it pathogenic?
Staph is the genus and which is gram positive and typically sourced from humans (skin / respiratory tract)
Yes it is pathogenic S.Aureus is of particular concern
Point of fill will be grade A so will have a specification of < 1 cfu so this would be an OOS and a deviation will need to be raised to investigate.
Pseudomonas found in suite – what does this mean, what type of organism is it?
Pseud is the genus name and is a gram negative typically sourced from water or soil it is an opportunistic pathogen . it is objectionable – raise devo
Days later a leak is reported from the plant room above the suite, what do you do?
Raise a deviation
Stop production
Quarantine stock that may be implicated – including determination of location of stock.
Initial risk assessment to determine scope of leak / areas impacted
Potentially perform some additional environmental monitoring
Take any immediate corrections to stop leak and stop contamination spreading further.
Root cause investigation in to what caused the leak
CAPA’s
Potential batch rejections if Grade A has been compromised
Where would you find the micro spec for a liquid product and what is it?
Ph.Eur 5.1.4, BP or USP and is
Tablets – TAMC 103 CFU/g, TYMC 102 CFU/g; TYMC - 100cfu / 1ml, TAMC – 1000cfu / 1ml absence of E-coli in 1 g
Liquids (aqueous) (Oral use) – TAMC 102 CFU/ml, TYMC 101 CFU/ml; TYMC - 10cfu / 1ml, TAMC – 100cfu / 1ml absence of E-coli in 1 ml
Products Non-Oral (Not incl Rectal) (oromucosal, gingival, cutaneous, nasal, auricular) – TAMC 102 CFU/g or ml, TYMC 101 CFUg or /ml TYMC – 10cfu / ml TAMC – 100cfu / ml absence of Stap / Pseud in 1 g or ml (also for transdermal patches but per 1 patch)
Rectal: TAMC 103 CFU/g or ml, TYMC 102 CFU/g or ml
Vaginal: TAMC 102 CFU/ml, TYMC 101 CFU/ml, absence of Pseu / Staph / Candida in 1 g or ml
MDI (inhalation use) – TAMC 102 CFU/ml, TYMC 101 CFU/ml TYMC – 10cfu / ml TAMC – 100cfu / ml absence of Stap / Pseud / bile tolerant gram neg (BTGNO) in 1 g or ml
need to know HERBALS: Criteria C: TAMC 105 CFU/g or ml, TYMC 104 CFU/g or ml; BTGNO 104 CFU/g or ml, absence of E.coli in 1 g or ml, absence of Salmonella in 25g or ml
Rapid micro methods, what is:
Celsis rapid sterility
Oceloscope
Malditoft
Celsis rapid sterility - exactly like traditional test
Day 7 run a sample and compare ATP vs broth collaborator - good for mould and bacteria. If ATP lighter than broth positive sample
Use same canister material same as filter material used in the process
Some products not suitable for rapid methods
Add different reagents react with ATP
Oceloscope - rapid bioburden
Malditoft - # peaks matches with library - time it takes to hit the sample and come back up
Tell me about facilities control for a sterile manuf. process.
Grade A – Limits for Micro/Particulates, Air changes, Clean-up rate etc
What are the requirements for HVAC?
ISO14644, DP, velocity, air change rates, Separate MALs and PALs
What types of organism would you expect to find if building work is going on?
Environmental such as bacillus, moulds such as aspergillus
In a clean room environment what are the 3 common bacteria and where do they come from?
Gram negative - Pseudomonas aeruginosa from water system, Gram positive - Staph aureus from people, Mould - aspergillus from material packaging.
What does an elevated level of Ps.A indicate?
How does it differ from others?
When killed what is left?
What happens when it is injected?
Contamination from the water system (possibly biofilm) or water ingress.
Its gram positive there risk of endotoxin. Endotoxin.
Toxic shock, death
What are the limits for particles in Grade A/B/C environments?
Grade Air Settle Contact FD
A 0 0 0 0
B 10 5 5 5
C 100 50 25 0
D 200 100 50 0
What is the warehouse temperature range?
15-25° C
Cold Chain Distribution – you have a 5metre sq room and the layout is changing, what do you need to consider?
Change of hot and cold spots / air circulation due to change in location
How would you validate a cold store?
IQ, OQ, PQ including empty and full chamber temperature mapping - worst case to see hot and cold spots, any areas of concern, where to locate continuous monitoring probes, seasonal variation should also be mapped
One of your products is stored in an external warehouse, the temperature storage
requirement is 20°C. You get a call from the warehouse manager that he has been made
aware the temperature is fluctuating between 15 and 25°C and has been for two months.
You have product in the warehouse and in the market place
What would you do?
What are the ICH stability storage conditions?
Quarantine all stock within your control
What stored in the warehouse and whats the temperature required - is that actually a problem?
Determination of impact to product as a result of exposure to higher / lower temperature – look at accelerated storage condition data for product.
Look at mapping / Trending – duration of excursion and range
Call engineering to investigate and resolve excursion ASAP
25/30 degrees c, 65% H and accelerated 40 degrees C, 70% H
Your engineer tells you that they have had to change two HEPA filters in your facility as they were blocked, what are your concerns? It’s a liquid product
You are told that there is external work going on outside which is causing the build up
Raise a deviation
Annex 1 and ISO14644 – Clean room classification limits
Has the area classification is maintained – likely to be Grade C/D depending on open or closed system
Environmental monitoring will be less frequent due to lower risk
What are they blocked with ?
Frequency of Change of HEPA filters – What is the preventative maintenance schedule is it sufficient.
Potential impact to product – quarantine stock
Review EM data, Air Flow and Change rates, Air pressures, - anything that could identify the point of cause.
How long has work been ongoing?
Filter Integrity Test
Smoke Test
Tell me about EM action and alerts
Cleanrooms should be monitored during processing, and also outside of production operations. Monitoring should be performed using principles of risk management to determine the locations and frequency of monitoring required. This rationale should be documented within an environmental monitoring programme. This programme should be set up in all sterile manufacturing environments, documenting the state of control of the facility and assessing the effectiveness of cleaning and aseptic processes. One key factor of the programme is the setting of appropriate action and alert limits.
Action and alert limits should be controlled and specified in standard operating procedures. Action limits are generally set through regulatory guidelines such as Annex 1, the USP or ISO standards, whereas alert levels are specific to the facility and should be set based on the results of performance qualification (PQ) tests and trends in historical data.
There are several different approaches that can be taken to set alert limits. As long as these are justified in the environmental monitoring plan and the contamination control strategy, it does not matter which method is used. Once each alert limit has been set based on the results of environmental monitoring PQ tests, there should be a procedure in place to track and trend these and re-evaluate the values if necessary.
Three of the most commonly used approaches to setting alert limits are the standard deviation approach, the cut-off value approach, or the non-parametric tolerance limit approach.
The standard deviation approach should only be used where historical data includes high counts, and when data is normally distributed. To calculate these values, a mean and standard deviation should be calculated; alert levels are set at the mean plus 2 standard deviations.
However, data collected from cleanrooms often contain many counts of zero, with a dispersion of counts which are not often normally distributed. Therefore, alternative approaches may also be considered.
The cut-off value approach takes into account the 95th percentile of historical data and sets the alert limit at this level. The non-parametric tolerance limit approach similarly is used for samples which are not normally distributed. This takes into consideration the 95th percent confidence intervals are used, where 95% of the samples will pass the established alert limit.
Environmental monitoring data should be analysed regularly to assess whether any trends can be identified in the data. Routine review of environmental monitoring data provides confidence in the control of a facility and allows rapid identification of any adverse trends which may be forming. A trend may indicate a potential problem with the environmental control systems in place within a facility. A single breach may indicate an issue at the time, however multiple breaches, or trends, may indicate a loss of control which must be addressed.
Environmental monitoring trend reports should be generated regularly to assess and identify trends in data. These reports should assess any excursions which had occurred and identify any trends in excursion rate or critical recovery rate over a period of time. Any Out of Trend or Out of Specification results should be investigated.
As part of these reports, alert and action limits should be reviewed to confirm that they are appropriate, and these may be altered to reflect changes within the facility. These may require alteration due to process changes or advances in technology. All assessments of excursions and trends, and changes to limits, should be subject to management review, to ensure that appropriate action is being taken and control is being maintained.
Procedures should be in place to define the actions to be taken following limit breaches or trend identification. These will often include the raising of investigations to assess the impact of the event and to determine a root cause. CAPA should be implemented to prevent reoccurrence, and assessment of the return to expected operating parameters should be provided.
Any abnormally high number of recoveries, or significant excursions, should have specific investigations performed to determine whether this incident is an isolated occurrence or can be correlated with other recoveries. An assessment of prior recovery rates should be made to indicate any unusual patterns which may be developing.
Breaching an alert limit may not require immediate investigation and corrective action; however, some form of follow-up action is usually required to assess whether the affected area may be involved in part of an adverse trend, and to ensure that the affected area has returned to within normal operating parameters. Subsequent alert limit breaches may indicate a trend, and these would warrant the same investigation as an action limit breach.
If an action limit is breached, immediate investigation should be performed to assess the impact of the excursion, determine a root cause of the excursion, and initiate subsequent CAPA. This investigation may determine that the root cause was laboratory error and there was no true failure. However, if a true failure is identified, further investigation should be performed, with identification of recoveries and review of additional samples all used to attempt to determine the root cause and aid in the assessment of impact. Follow-up monitoring may be performed, and remedial actions such as additional cleaning or retraining may be required. An assessment may also be required for previously filled batches.
Limit breaches provide a rough outline of the cleanliness of the monitoring location and can provide confidence that manufacture has occurred in a clean environment. However, they can only provide a snapshot of information, they cannot provide a full picture of the environment. A single viable sample with no growth does not guarantee control, and conversely, a single excursion does not indicate loss of control.
Therefore, as well as looking at breaches, it is important to supplement these with additional data, such as trending and contamination rates.
What are the basic nutritional requirements for life with the (microorganism’s) single goal of cell division:
- Energy – sunlight, inorganics, organics
- Nitrogen – gas, ammonia, nitrate/nitrite or a nitrogenous organic compound
- Carbon – Carbon dioxide or monoxide, methane or complex organic material
- Minerals – Phosphorous, Sulfur, Magnesium, Potassium, Sodium and Calcium
- Water
- Trace Elements – Iron, Zinc, Cobalt, etc.
Micro-organism temperature requirements
- Psychrophiles grow best at temperatures below 10°C or 50°F.
- Mesophiles grow best between 20° and 45°C or 68° and 113°F.
- Thermophiles grow best between 50°and 80ºC or 122° and 176°F.
- Extreme Thermophiles grow best above 80°C or 176°F.
- Heat Resistant Organisms are normally mesophiles that have sporulated and; therefore, protected from heat.
Micro-organism Respiration requirements
- Obligate Aerobic Bacteria ‐ Absolutely require oxygen.
- Microaerophilic Bacteria ‐ Require oxygen but uncomfortable at high concentrations.
- Facultative Anaerobic Bacteria ‐ Utilise oxygen when available, but can metabolise in the absence of oxygen equally well.
- Aerotolerant Anaerobic Bacteria ‐ Show no preference to aerobic respiration if O2 is present. Essentially anaerobic
- bacteria.
- Obligate Anaerobic Bacteria ‐ Only metabolise anaerobically, cannot survive in the presence of oxygen.
Micro-organism environment requirements
Range of pH for different bacteria is broad, from pH 2-12!
Gram positive cocci
- people, skin
- Staph epidermidis can cause lung infections from skin shedding into the air being the microbes method of transportation
- Staph Aureus is associated with wound infections, toxic shock syndrome, boils and pneumonia
Gram positive rods
- spore formers - dirt, environment
- Bacillus subtilis can cause eye infections and even meningitis in people with weakened immune systems
Gram negative rods
- water - pyrogenic response
Fungai
- Mould from cardboard, building fabric and exposed plaster - spore formers
- Yeast
Mycoplasma
- Mycoplasma has no cell wall so cannot be gram stained.
- Mycoplasma can escape sterile filtration.
- Associated with Arthritis and Pneumonia in immunocompromised patients
What dosage form is at higher risk of Micro contamination
Liquids pose the highest risk then creams, ointments, freeze drier product, tablets and capsules present the lowest risk - the more aqueous the product the more at risk from gram negative organisms
Why is streaking technique carried out?
Streaking done to obtain a pure culture of a microbe
What is the pH of Water, saliva, blood
all neutral pH approx pH 7
Types of microbiological growth media
Broad Spectrum General Purpose Media
Enhanced Media
Selective Media
Differential Media
Enrichment Media
Tell me about Broad Spectrum General Purpose Media
Highly nutritious media formulated to grow a broad spectrum of microorganisms from low numbers. Typically used for enumeration.
* Tryptone Soy Agar
* Nutrient Agar
Tell me about Enhanced Media
General purpose media supplemented with specific growth requirements in order to allow the growth of fastidious organisms with specific growth requirements.
* Chocolate Blood Agar
○ Contains Factors X and Y from blood essential for the growth of Neisseria sp
* Milk Agar
○ Contains dried milk powder to enhance the growth of dairy pathogens
Tell me about Selective Media
Media which contain growth inhibitors which exclude the growth of unwanted organisms but allow the growth of the target organisms. These inhibitors take advantage of differences in biochemistry between different organisms.
* Cetrimide Agar
○ Contains Cetrimide, a broad spectrum disinfectant with little activity against pseudomonas species which utilise cetrimide as a carbon source
○ Pseudomonas CFC Agar Contains Cephaloridine (a broad spectrum antibiotic), Fucidin (a gram negative antibiotic) and cetrimide, to select against all organisms except pseudomonads
Tell me about Differential Media
Media which have been formulated to allow the growth of a number of bacteria but which result in the ability to differentiate between the isolates. These media are often also selective media in order to reduce the target spectrum for differentiation.
* Baird Parker Agar
○ Formulated to differentiate between Staphylococcus aureus and other Staphylococci. The medium contains potassium tellurite and lithium chloride which inhibit most coliforms.
§ Staphylococci metabolise the tellurite to telluride turning the colonies black. The agar contains egg yolk emulsion.
§ S. aeureus contains lipase which clears the egg yolk around the colony, and lecithinase which precipitates lecithin close to the colony.
* MacConkey Agar
○ Contains Bile salts which inhibit all organisms except those which have adapted to the environment found in the mammalian gut. The agar also contains lactose and a pH indicator (neutral red). Any organisms that can tolerate bile and will ferment lactose to lactic acid will be visible as colonies with a yellow halo on a blue agar.
Tell me about Enrichment Media
Very often a target organism may be present in a sample in very low numbers compared with other organisms closely related to it. In order to be able to isolate the target organism its numbers must be increased in relation to other organisms. Enrichment media attempt to do this by enhancing the nutritive qualities appropriate to the target organism while introducing an inhibition to other unwanted organisms. Enrichment media are particularly useful if the target organism is sub‐lethally damaged.
* Selenite Cystine Broth
○ Sodium biselenite inhibits organisms other than salmonellae while L‐Cystine and sodium phosphate enhance the growth of salmoneallae. Incubation at 370C for 24 hrs will increase the growth of low numbers of salmonellae sufficiently to be detected on selective agars such as XLD and DCA.
* Buffered Peptone Water
○ The peptone in BPW provides all the nutrition and the absence of carbohydrates minimises acid production. The solution is well buffered which ensures a neutral pH. These conditions allow the recovery of low numbers of sub‐lethally damaged organisms without them being over‐run by other more aggressive organisms
